磷酸酶PHLPP对TLR及RIG-I触发的Ⅰ型干扰素产生的选择性调控作用及机制研究
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摘要
天然免疫应答是由能够识别病原相关分子模式(Pathogen-associated molecular patterns, PAMPs)的受体所介导的,这些受体统称为模式识别受体(Pattern recognition receptor, PRRs)。Toll样受体(Toll-like receptors, TLRs)是一类重要的模式识别受体,主要表达于巨噬细胞和树突状细胞(Dendritic cells, DCs)表面,特异性识别病原体中特定的分子结构,激活其下游MyD88或TRIF依赖的信号通路,激活MAPK、NFKB或者IRF3信号,最终激活天然免疫细胞产生炎性细胞因子和Ⅰ型干扰素,构成机体免疫系统抵御病原体入侵的第一道屏障。TLRs在各种生物体内高度保守,目前在小鼠中已发现12种TLRs。
另一类受到广泛关注的模式识别受体是维甲酸诱导的基因Ⅰ样受体(retinoid acid-inducible geneⅠ(RIG-I)-like receptors, RLRs),包括RIG-I和MDA5 (melanoma differentiation-associated gene 5),它们都可以识别胞浆中的病毒RNA。常用于活化RIG-I信号的RNA病毒有水泡口炎病毒(Vesicular stomatitis virus, VSV)、仙台病毒(Sendai virus, SeV)等。RIG-I/MDA5识别相应病毒RNA后,通过招募接头分子IPS-1 (Interferon-beta promoter stimulator 1)等激活下游信号,引起IRF-3以及NF-κB的核转位,最终促进Ⅰ型干扰素和炎性细胞因子产生,激发机体抗病毒反应。
TLR及RIG-I不仅启动天然免疫应答,控制炎症反应的性质、强度和持续时间,还可以调节获得性免疫应答的强度和类型,成为连接初始免疫应答和获得性免疫应答的桥梁。所以,TLR及RIG-I信号过度活化或活化不足会导致机体免疫功能异常和疾病的发生。许多其他信号通路参与对TLR及RIG-I信号的严密调控,然而到目前为止,对TLR和RIG-I信号通路调控的分子机制还没有完全研究清楚。因此,对TLR及RIG-I信号转导调控机制的深入研究具有重要的理论意义和应用价值。
PHLPP (PH domain leucine-rich repeat protein phosphatase)是一种具有PH(pleckstrin homology)结构域并且富含亮氨酸重复基序的丝/苏氨酸蛋白磷酸酶,它由N端的PH结构域、LRR (leucine-rich repeat)结构域、PP2C (protein phosphatase2C)样磷酸酶活性结构域以及C端的PDZ binding结构域组成,大鼠的同源物编码1687个氨基酸。以前的研究发现在大鼠脑组织细胞中,PHLPP能通过其LRR区域直接与K-Ras相结合,负向调控K-Ras和MAPK信号途径；PHLPP也能够抑制ERK通路活化,参与大鼠脑组织长时间记忆的形成。PHLPP在多种肿瘤细胞中低表达,能够通过PP2C样磷酸酶活性区特异性作用于Akt的473位丝氨酸,使其去磷酸化,从而抑制Akt依赖的肿瘤细胞增殖,促进肿瘤细胞凋亡。PHLPP还可以依赖其PH区域使PKC去磷酸化,从而调控细胞内PKC的水平。这些研究提示PHLPP可通过其不同的功能域参与多条信号转导通路的调控。
蛋白磷酸酶在免疫应答中的调控作用受到越来越多的重视。已经报道数个磷酸酶,包括SHP-1 (Src homology region 2 domain-containing phosphatase 1)、SHP-2、SHIP-1 (Src homology 2 domain containing inositol-5'-phosphatase-1)和MKP-1(MAPK phosphatase-1)等,分别通过不同的机制调控TLR或RIG-I触发的天然免疫应答中炎性细胞因子和/或Ⅰ型干扰素的产生。那么PHLPP作为另一重要的参与细胞信号转导调控的蛋白磷酸酶,是否参与天然免疫应答中TLR及RIG-I信号转导的调控尚不清楚。
本实验分三部分内容对PHLPP是否参与天然免疫的调控以及可能的相关机制等问题进行了研究。
一、PHLPP对TLR及RIG-I触发巨噬细胞产生Ⅰ型干扰素的选择性促进作用
我们研究发现PHLPP在小鼠脑组织、脾脏及淋巴结等组织和T细胞、B细胞、NK细胞、DC、腹腔巨噬细胞以及RAW264.7细胞株等免疫细胞中广泛表达。并且TLR配体LPS、poly(I:C)刺激以及RIG-I配体VSV感染可以诱导巨噬细胞表达PHLPP蛋白增加。
在小鼠巨噬细胞中,用靶向PHLPP的siRNA干扰PHLPP表达,发现可以显著抑制TLR4配体LPS、TLR3配体poly(I:C)以及RIG-I配体VSV诱导的Ⅰ型干扰素的产生,但对炎性细胞因子TNF-α、IL-6的产生无明显影响。另外,干扰PHLPP表达对CpG ODN诱导的Ⅰ型干扰素及炎性细胞因子产生均无显著影响。过表达PHLPP可以显著增强LPS、poly(I:C)以及VSV诱导的Ⅰ型干扰素的产生,而对炎性细胞因子TNF-α、IL-6的产生也无明显影响。
细胞因子报告基因结果显示,过表达PHLPP可以显著促进TRIF、组成性活化的RIG-I(RIG I-N)以及MDA5活化的IFN-β报告基因的活性,并且呈剂量依赖性；但对TRIF以及MyD88活化TNF-α报告基因的活性无明显影响,对MyD88活化IFN-β报告基因的活性也无明显影响。
通过PB转座子技术建立的PHLPP基因敲除小鼠,其腹腔巨噬细胞中PHLPP表达降低80%以上。实验发现PHLPP基因敲除小鼠的巨噬细胞在体外对LPS.poly(I:C)以及VSV诱导的IFN-α和IFN-β产生水平较野生型小鼠巨噬细胞明显下降,而炎性细胞因子TNF-α、IL-6的水平无明显差异。CpG ODN诱导的炎性细胞因子以及Ⅰ型干扰素的水平在两种细胞中均没有明显差异。在PHLPP基因敲除小鼠骨髓来源的DC中我们也得到了类似的结果。体内试验显示,PHLPP基因敲除小鼠及对照小鼠腹腔注射LPS.poly(I:C)或者VSV后,PHLPP基因敲除小鼠腹腔巨噬细胞中IFN-α和IFN-β的mRNA水平较对照小鼠均显著下降,而炎性细胞因子TNF-α和IL-6的mRNA水平无明显变化；PHLPP基因敲除小鼠血清中IFN-β水平较对照小鼠显著降低,而炎性细胞因子水平无明显变化。
以上研究结果显示PHLPP促进TLR配体诱导的TRIF依赖的以及RIG-I配体诱导的Ⅰ型干扰素的产生,而对其诱导的炎性细胞因子产生以及MyD88依赖的细胞因子产生无明显影响。
二、PHLPP选择性调控TLR及RIG-I触发Ⅰ型干扰素产生的分子机制研究
为明确PHLPP各个结构域的功能,我们根据PHLPP的分子结构构建了不同的PHLPP缺失突变载体。瞬时转染这些缺失突变载体后,检测对TRIF活化IFN-β报告基因的活性的影响。结果发现缺失LRR或者PP2C结构域的PHLPP突变体不能促进TRIF活化IFN-β报告基因的活性,而缺失PH或者PDZ结构域的PHLPP突变体仍然能够显著促进TRIF活化IFN-β报告基因的活性；单一表达PP2C结构域的PHLPP突变体不能促进TRIF活化IFN-β报告基因的活性,而表达LRR和PP2C结构域的PHLPP突变体能够显著促进TRIF活化IFN-β报告基因的活性。同时,我们发现在巨噬细胞中瞬时转染缺失LRR或者PP2C结构域的PHLPP突变体不能促进poly(I:C)诱导的Ⅰ型干扰素的产生。由此认为,PHLPP正向调控TLR及RIG-I触发的Ⅰ型干扰素的产生是依赖于其LRR结构域和磷酸酶活性结构域PP2C。
接着,我们观察了PHLPP对TLR及RIG-I信号活化的MAPKs、NF-κB的影响。发现PHLPP表达降低对poly(I:C)诱导的ERK、JNK、p38和IKKα/β的磷酸化没有明显影响。同时,报告基因结果显示过表达PHLPP对MyD88、TRIF或RIG-I活化的NF-κB报告基因活性也没有显著影响。因此,PHLPP不能促进TLR及RIG-I触发的MAPKs和NF-κB信号通路的活化,从而对炎性细胞因子产生没有显著影响。
那么PHLPP调控TLR及RIG-I信号触发的Ⅰ型干扰素产生的靶分子是什么呢?研究发现过表达PHLPP可以显著促进TBK1和IRF3活化的IFN-β报告基因的活性以及TRIF活化IRF3报告基因的活性,并且均具有剂量依赖性,提示PHLPP在Ⅰ型干扰素产生信号途径中作用在IRF3分子水平。
进一步免疫沉淀和GST-pull down实验表明PHLPP可以通过其LRR结构域直接与IRF3的C端相结合。共聚焦显微镜观察显示在巨噬细胞静息状态下PHLPP在胞浆、胞核中均有分布,而IRF3全部在胞浆中；poly(I:C)刺激或SeV感染后,PHLPP有往胞核中聚集的趋势,IRF3转位入核；并且PHLPP与IRF3在细胞核内产生共定位,且分离胞浆、胞核蛋白进行免疫沉淀实验进一步证明PHLPP与IRF3在细胞核内结合。
为进一步证明PHLPP调控TLR及RIG-I触发的Ⅰ型干扰素产生的靶分子是IRF3,我们采用了IRF3基因敲除小鼠(IRF3-/-)的腹腔巨噬细胞。实验发现过表达PHLPP对poly(LC)诱导的IRF3-/-巨噬细胞中Ⅰ型干扰素产生没有明显影响。在IRF3-/-巨噬细胞中瞬时转染IRF3质粒以恢复IRF3的表达后,发现过表达PHLPP也恢复了对poly(I:C)诱导的Ⅰ型干扰素产生的促进作用。由此确定PHLPP调控TLR及RIG-I触发的Ⅰ型干扰素产生的信号途径靶分子是IRF3。
IRF3作为TLR和RIG-I信号通路的共同下游分子之一,活化后发生磷酸化、二聚体化以及核转位,进而与CBP/P300结合,促进Ⅰ型干扰素的产生,随后IRF3蛋白通过蛋白酶体途径降解,从而保证Ⅰ型干扰素信号活化途径的平衡。我们研究发现PHLPP基因敲除小鼠的腹腔巨噬细胞在poly(I:C)刺激或者VSV、SeV感染后,其IRF3蛋白降解速度较对照小鼠的腹腔巨噬细胞增快；蛋白酶体抑制剂MG132可以完全抑制上述两种细胞中IRF3的降解。过表达PHLPP可以抑制SeV感染诱导的IRF3蛋白降解。PHLPP表达降低之后可以显著增强IRF3泛素化。以上实验结果表明,PHLPP能够与IRF3直接结合并维持IRF3蛋白的稳定,防止其泛素化而降解。
那么,PHLPP通过何种机制维持IRF3蛋白的稳定呢?考虑到PHLPP正向调控Ⅰ型干扰素的产生是依赖于其磷酸酶活性结构域PP2C,且有文献报道IRF3 339位丝氨酸(小鼠IRF3 332位丝氨酸(IRF3 S332))磷酸化可以促进IRF3的泛素化和降解,那么PHLPP是否通过使IRF3 S332位去磷酸化而稳定IRF3蛋白呢?我们构建了小鼠IRF3 S332位突变的表达载体(IRF3 S332A)。研究发现在HEK293细胞中瞬时转染PHLPP和野生型IRF3或IRF3 S332A,用SeV感染后,PHLPP对IRF3 S332A的降解和泛素化无明显影响,却可以抑制野生型IRF3的降解和泛素化。同时,报告基因实验显示PHLPP能促进野生型IRF3却不能促进IRF3 S332A突变体活化的IFN-β报告基因的活性。这些实验结果说明PHLPP的作用位点是IRF3 S332。
本部分研究结果表明巨噬细胞中PHLPP在细胞核内通过其LRR结构域与活化的IRF3 C端结合,通过其PP2C磷酸酶活性区使IRF3 S332去磷酸化,从而抑制S332磷酸化依赖的IRF3泛素化和降解,维持IRF3蛋白的稳定,促进Ⅰ型干扰素的产生。
三、PHLPP通过促进Ⅰ型干扰素产生而参与抵抗病毒感染的作用
为探讨PHLPP正向调控TLR及RIG-I触发的Ⅰ型干扰素的产生这一功能在感染性疾病,特别是病毒感染性疾病中的作用,我们首先观察了PHLPP对VSV复制的影响。实验发现PHLPP基因敲除小鼠的腹腔巨噬细胞在VSV感染后,培养上清中VSV病毒滴度(TCID50)显著增加,用培养上清处理的HEK293细胞迅速出现细胞变圆等病毒感染性病变及细胞死亡；而在巨噬细胞培养过程中中加入重组小鼠IFN-β,可以明显降低VSV病毒滴度,减少HEK293细胞死亡率。上述结果表明PHLPP通过促进Ⅰ型干扰素的产生而抑制了VSV的复制。
进一步我们取小鼠体内感染VSV 24-48小时之后的脏器检测病毒感染情况,发现PHLPP基因敲除小鼠的肝脏和脾脏组织中VSV的复制和病毒滴度(TCID50)较对照小鼠显著增加,表明PHLPP在体内参与了抵抗VSV感染的效应。
本部分研究表明PHLPP通过促进体内TLR及RIG-I触发的Ⅰ型干扰素的产生,抑制VSV的复制,提高小鼠对VSV体内感染的抵抗能力,提示其在抵御病毒感染性疾病中发挥着重要的作用。
综合以上三部分研究结果,我们发现磷酸酶PHLPP参与了巨噬细胞中TLR及RIG-I触发的Ⅰ型干扰素产生的正向调控；证明了PHLPP可以通过LRR结构域直接结合胞核内的IRF3,并依赖其磷酸酶活性使IRF3 332位丝氨酸去磷酸化而抑制IRF3的泛素化和降解,维持Ⅰ型干扰素产生的信号通路的持续活化,提高小鼠对VSV感染的抵抗力。本研究揭示了磷酸酶PHLPP在先天免疫应答调控中的作用,丰富了TLR信号及RIG-I信号转导调控的分子机制,为感染性疾病尤其是病毒性感染的免疫治疗提供了新的思路。
Innate immune cells recognize pathogen-associated molecular patterns (PAMPs) conserved in microbes by pattern recognition receptors (PRRs) to trigger innate immune responses. As one kind of key PRRs, TLRs comprise a large family consisting of at least 12 members and are mainly expressed in antigen-presenting cells (APCs) including macrophages and dendritic cells (DCs). Upon recognition of PAMPs, TLRs trigger myeloid differentiation factor 88 (MyD88)- and/or Toll/IL-1 receptor (TIR)-domain containing adaptor protein inducing IFN-β(TRIF)-dependent signaling pathway to activate mitogen-activated protein kinases (MAPKs), the transcription factors nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3) and/or IRF7, leading to the production of proinflammatory cytokines and typeⅠinterferon (IFN), and defense against invading pathogens.
Besides TLRs, retinoid acid-inducible gene I-like receptors (RLRs) are another important class of PRR families, which are composed of RNA helicase domain-containing proteins retinoic acid-inducible geneⅠ(RIG-Ⅰ), melanoma differentiation associated gene 5 (MDA5) and LGP2. They are localized in the cytoplasm and recognize the genomic RNA of dsRNA viruses and dsRNA generated as the replication intermediate of ssRNA viruses. Vesicular stomatitis virus (VSV) and Sendai virus (SeV) are the representative viruses which can activate RIG-Ⅰsignal pathway. RIG-Ⅰand MDA5 interact with adaptor protein IPS-1 (Interferon-beta promoter stimulator 1, also known as MAVS/Cardif/VISA), which mediates the gene expression of typeⅠinterferon and proinflammatory cytokines by activating IRF3/7 and NF-κB.
TLRs and RLRs play important roles in linking innate and adaptive immune responses. It is now well accepted that full activation of TLRs and RLRs is essential for initiating the innate immune response and enhancing adaptive immunity to eliminate invading pathogens. Less efficient activation of TLRs and RLRs or excessive activation of TLRs and RLRs may induce immune disorders and even immunopathological process. Up to now, various signal pathways are known to be involved in the tight regulation of TLR and RLR signaling to maintain the immunological balance. However, the underlying molecular mechanisms for the regulation of TLR and RLR signaling remain to be fully elucidated.
Phosphatase PHLPP (PH domain leucine-rich repeat protein phosphatase) is a serine/threonine phosphatase, which consist of a pleckstrin homology (PH) domain, a leucine-rich repeat (LRR) region, a PP2C (protein phosphatase 2C) domain, and a PDZ-binding motif. Previous studies showed that PHLPP can directly interact with K-Ras through its LRR domain to negatively regulate Ras-MAPK signal pathway in rat brain. PHLPP also functions as a negative regulator of MAPK and CREB-mediated transcription in hippocampal neurons to regulate rat memory formation. PHLPP is low expressed in many human tumor cells such as breast cancer, colon cancer and glioblastoma cell lines. PHLPP can specifically dephosphorylate the Ser473 of Akt depending on its PP2C and PDZ binding motif to suppress tumor growth and promote apoptosis of tumor cells. In addition, PHLPP controls the cellular levels of PKC by specifically dephosphorylating the hydrophobic motif, thus destabilizing the enzyme and promoting its degradation. All these reports suggest that PHLPP may show different functions depending on its different domain to involve in many cell preceess.
Nowadays, the important roles of protein phosphotase in regulating immune response draw more and more attention. In innate immunity, several protein phosphotases, such as SHP-1 (Src homology region 2 domain-containing phosphatase 1), SHP-2 (Src homology region 2 domain-containing phosphatase 2), SHIP-1 (Src homology 2 domain containing inositol-5'-phosphatase-1) and MKP-1 (MAPK phosphatase-1), have been reported to regulate TLR-and RIG-Ⅰ-triggered production of proinflammatory cytokines and/or typeⅠinterferon through different mechanism. However, whether phosphotase PHLPP can regulate TLR and RIG-Ⅰ-triggered immune response remains unknown.
The primary aim of this study is to investigate the regulation of TLR-and RIG-Ⅰ-triggered innate immune response by PHLPP in macrophages as well as the underlying mechanisms.
PartⅠ. PHLPP selectively promotes TLR-and RIG-Ⅰ-triggered typeⅠinterferon production in macrophages
We first examined the expression pattern of PHLPP. RT-PCR assay showed that the mRNA of PHLPP is ubiquitously expressed in various mouse tissues and immune cells, with highest levels in brain and spleen. LPS, poly(I:C) or VSV challenge upregulated PHLPP expression in mouse primary peritoneal macrophages.
Next, the effects of PHLPP silence or deficiency on the production of TLR- and RIG-Ⅰ-triggered cytokines were investigated. RNAi knockdown of PHLPP significantly suppressed TLR3,4- and RIG-Ⅰ-triggered production of IFN-α/βin macrophages, but had no effect on the production of IL-6 and TNF-α. Meanwhile, silencing of PHLPP expression had no influence on TLR9-triggered production of the above cytokines. Coincidently, overexpression of PHLPP significantly enhanced TLR3,4- and RIG-Ⅰ-triggered production of IFN-α/βin macrophages.
Overexpression of PHLPP significantly increased TRIF-, constitutively active RIG-Ⅰ-or MDA-5-activated expression of IFN-βreporter gene in a dose-dependent manner, but had no effect on TRIF-or MyD88-activated expression of TNF-αreporter gene, or MyD88-activated expression of IFN-βreporter gene.
In macrophages from PHLPP knockout mice generated by PB (PiggyBac) transposon system, PHLPP expression was downregulated over 80% as compared to that in macrophages from wild type mice. PHLPP deficiency significantly inhibited TLR3,4- and RIG-Ⅰ-triggered production of IFN-α/β, while had no effect on the production of IL-6 and TNF-αin macrophages. The production of above cytokines induced by CpG ODN remain unchanged in PHLPP-/- and PHLPP+/+ macrophages. PHLPP-/- mice and PHLPP+/+ mice were challenged by intraperitoneal injection with LPS, poly(I:C), or infection with VSV. After these challenges, the mRNA levels of IFN-α/βin primary peritoneal macrophages and IFN-p level in serum of PHLPP-/- mice were much lower than that in PHLPP+/+ mice, while there was no significant difference in the levels of proinflammatory cytokines.
Taken together, these results indicated that PHLPP selectively promotes TLR-triggered TRIF-dependent and RIG-Ⅰ-triggered production of typeⅠinterferon, but has no effect on the production of proinflammatory cytokines and MyD88-dependent production of cytokines.
PartⅡ. The underlying molecular mechanisms for the positive regulation of TLR and RIG-Ⅰ-triggered typeⅠinterferon production by PHLPP
To investigate the function of each domain of PHLPP in the enhancement of typeⅠinterferon production triggered by TLR3,4 and PIG-Ⅰ, various truncated or deleted mutants of PHLPP were constructed according to its known structural and functional domains, and then transfected into HEK293 cells to detect TRIF-activated expression of IFN-(3 reporter gene. We revealed that the mutants of PHLPP deleting LRR region or PP2C domain couldn't increase TRIF-activated expression of IFN-βreporter gene, while the mutants of PHLPP deleting PH domain or PDZ-binding motif significantly increased TRIF-dependent activation of IFN-βreporter gene as full-length PHLPP did. The mutant of PHLPP only expressing PP2C domain couldn't increase TRIF-activated expression of IFN-βreporter gene, while the mutant of PHLPP expressing LRR region and PP2C domain significantly could did so. Meanwhile, overexpression of the mutants of PHLPP deleting LRR region or PP2C domain failed to enhance poly(I:C)-induced production of IFN-(3 in macrophages. So, the results indicated that the LRR and PP2C domain were responsible for the positive regulation of TLR-and RIG-Ⅰ-triggered production of typeⅠinterferon by PHLPP.
Then, we tested whether PHLPP regulates TLR-and RIG-Ⅰ-triggered activation of MAPKs and NF-κB pathways. PHLPP deficiency had no significant effect on poly(I:C)-induced phosphorylation of ERK, JNK, p38 and IKKα/βin macrophages. Consistently, PHLPP overexpression did not affect MyD88-, TRIF- or constitutively active RIG-Ⅰ-activated expression of NF-κB reporter gene. So, PHLPP can't enhance TLR- and RIG-Ⅰ-triggered activation of MAPKs and NF-κB, which contributes to the unchanged production of inflammatory cytokines.
Next, we investigated which molecule in TLR and RIG-Ⅰsignal pathway PHLPP could interact with. Luciferase report gene assay showed that overexpression of PHLPP significantly increased TBK1- and IRF3-activated expression of IFN-βreporter gene, and TRIF-activated expression of IRF3 reporter gene in a dose-dependent manner. These results indicated IRF3 may be the potential target PHLPP can interact with. Immunoprecipitation experiment and GST pull-down assay confirmed PHLPP could directly interact with the C-terminal of IRF3 through its LRR region.
As we known, IRF3 resides in the cytoplasm in resting cells, and, upon stimulation, becomes activated leading to nuclear translocation. The confocal laser scanning microscope observed that PHLPP resided in both cytoplasm and nuclear of resting macrophages. Upon the stimulation of poly(I:C) or SeV, more PHLPP translocated into nuclear, and co-localized with IRF3 in the nuclei of macrophages. Immunoprecipitation experiment using separated extracts of cytoplasm and nuclei revealed that PHLPP could co-precipitate with IRF3 in the nuclei of macrophages.
To further confirm the target of PHLPP in regulation of TLR-and RIG-Ⅰ-triggered production of typeⅠinterferon is IRF3, we transduce PHLPP into IRF3 deficient (IRF3-/-) macrophages. Overexpression of PHLPP had no effect on poly(I:C)- or VSV-induced production of IFN-βin IRF3-/- macrophages, while PHLPP overexpression promoted poly(I:C) or VSV- triggered IFN-βproduction when rescuing the expression of IRF3 in IRF3-/- macrophages. So, the above results confirmed PHLPP can interact with IRF3 to promote TLR- and RIG-Ⅰ-triggered production of typeⅠinterferon.
As one of the key transcription factors in both TLR and RIG-I signal pathway, IRF3, upon stimulation, becomes activated via serine/threonine phosphorylation (between residues 385 and 405) leading to its dimerization, nuclear translocation and association with the coactivator CBP/p300 for inducing the production of typeⅠinterferon, then IRF3 degrades through proteasome-dependent pathway to maintain the balance of the production of typeⅠinterferon. We found that IRF3 of PHLPP-/- macrophages degraded faster and more significantly than that of wild-type macrophages challenged with poly(I:C), VSV or SeV. Considently, overexpression of PHLPP could delay and inhibit SeV-induced degradation of IRF3 in macrophages. MG132 could almost completely inhibit the degradation of IRF3 in PHLPP-/- and PHLPP+/+ macrophages. Meanwhile, PHLPP deficiency enhanced the poly(I:C)-, VSV- or SeV-induced polyubiquitination of IRF3. These results indicated PHLPP directly interacts with IRF3 to maintain the stability of IRF3, preventing it from ubiquitination and degradation.
Next, we wanted to know how PHLPP maintains the stability of IRF3. It has been reported that phosphorylation of Ser339 of human IRF3 (IRF3 Ser332 in mice IRF3) led to polyubiquitination of IRF3 and then proteasome-dependent degradation of IRF3. Combination with the above result that PHLPP promoted the production of typeⅠinterferon depending on its phosphatase activity of PP2C domain, we predicted that PHLPP may maintain the stability of IRF3 through dephosphorylating IRF3 Ser332. In orderto confirm the hypothesis, we generated the mutant IRF3 with alanine substituted for serine in residue 332 of IRF3 (IRF3 S332A). Overexpression of PHLPP had no effect on SeV-induced polyubiquitination and degradation of IRF3 S332A, while obviously inhibited polyubiquitination and degradation of wild-type IRF3. Reporter gene assay showed that overexpression of PHLPP couldn't enhance IRF3 S332A-activated expression of IFN-βreporter gene. These results demonstrated that PHLPP could dephosphorylate IRF3 Ser332 to inhibit its phosphorylation-dependent degradation of IRF3.
Taken together, these results demonstrated PHLPP directly interacted with the C-terminal of IRF3 through its LRR region in the nuclei of macrophages, and dephosphorylated IRF3 Ser332 through PP2C domain to maintain the stability of IRF3 by inhibiting IRF3 Ser332 phosphorylation-dependent degradation of IRF3, leading to the enhanced production of typeⅠinterferon triggered by TLR3,4 and RIG-Ⅰ.
PartⅢ. PHLPP enhances host defense against viral infection by promoting typeⅠinterferon production
Because PHLPP has been shown to be able to potentiate typeⅠinterferon production in macrophages, we wondered whether PHLPP-mediated enhancement of typeⅠinterferon production could protect the host from virus infection. We tested this with VSV infection model both in vitro and in vivo. Upon infection of VSV, VSV titers (TCID50) in the cultural supernatant of PHLPP-/- acrophages were significantly higher than that in wild type macrophages, and HEK293 cells cultured with the supernatant of PHLPP-/-macrophages quickly showed pathology of virus infection and almost all died. Addition of recombinant mouse IFN-(3 could decrease the titer of VSV in PHLPP-/- macrophages and rescue the death of HEK293 cells cultured with the supernatant of PHLPP-/- macrophages. These results indicated that PHLPP inhibits VSV replication through promoting the production of typeⅠinterferon during viral infection.
Next, we investigated the possible role of PHLPP in suppressing VSV infection in vivo. VSV RNA replicates and VSV titer in liver and spleen of PHLPP-/- mice were much higher than that of control mice after VSV challenge. These in vivo data revealed that PHLPP can inhibit VSV replication through promoting RIG-Ⅰ-triggered production of typeⅠinterferon, and protect the host from virus infection.
In conclusion, we have demonstrated that PHLPP can selectively promote the TLR3,4-and RIG-Ⅰ-triggered production of typeⅠinterferon in macrophages. PHLPP directly interacts with IRF3 through its LRR domain, dephosphorylates IRF3 Ser332 depending on the phosphatase activity of PP2C domain and subsequently inhibit polyubiquitination and Ser332 phosphorylation-dependent degradation of IRF3, thus maintaining the stability of IRF3. Our results indicate that phosphatase PHLPP may be an essential component of the host antiviral'machinery'.
另一类受到广泛关注的模式识别受体是维甲酸诱导的基因Ⅰ样受体(retinoid acid-inducible geneⅠ(RIG-I)-like receptors, RLRs),包括RIG-I和MDA5 (melanoma differentiation-associated gene 5),它们都可以识别胞浆中的病毒RNA。常用于活化RIG-I信号的RNA病毒有水泡口炎病毒(Vesicular stomatitis virus, VSV)、仙台病毒(Sendai virus, SeV)等。RIG-I/MDA5识别相应病毒RNA后,通过招募接头分子IPS-1 (Interferon-beta promoter stimulator 1)等激活下游信号,引起IRF-3以及NF-κB的核转位,最终促进Ⅰ型干扰素和炎性细胞因子产生,激发机体抗病毒反应。
TLR及RIG-I不仅启动天然免疫应答,控制炎症反应的性质、强度和持续时间,还可以调节获得性免疫应答的强度和类型,成为连接初始免疫应答和获得性免疫应答的桥梁。所以,TLR及RIG-I信号过度活化或活化不足会导致机体免疫功能异常和疾病的发生。许多其他信号通路参与对TLR及RIG-I信号的严密调控,然而到目前为止,对TLR和RIG-I信号通路调控的分子机制还没有完全研究清楚。因此,对TLR及RIG-I信号转导调控机制的深入研究具有重要的理论意义和应用价值。
PHLPP (PH domain leucine-rich repeat protein phosphatase)是一种具有PH(pleckstrin homology)结构域并且富含亮氨酸重复基序的丝/苏氨酸蛋白磷酸酶,它由N端的PH结构域、LRR (leucine-rich repeat)结构域、PP2C (protein phosphatase2C)样磷酸酶活性结构域以及C端的PDZ binding结构域组成,大鼠的同源物编码1687个氨基酸。以前的研究发现在大鼠脑组织细胞中,PHLPP能通过其LRR区域直接与K-Ras相结合,负向调控K-Ras和MAPK信号途径；PHLPP也能够抑制ERK通路活化,参与大鼠脑组织长时间记忆的形成。PHLPP在多种肿瘤细胞中低表达,能够通过PP2C样磷酸酶活性区特异性作用于Akt的473位丝氨酸,使其去磷酸化,从而抑制Akt依赖的肿瘤细胞增殖,促进肿瘤细胞凋亡。PHLPP还可以依赖其PH区域使PKC去磷酸化,从而调控细胞内PKC的水平。这些研究提示PHLPP可通过其不同的功能域参与多条信号转导通路的调控。
蛋白磷酸酶在免疫应答中的调控作用受到越来越多的重视。已经报道数个磷酸酶,包括SHP-1 (Src homology region 2 domain-containing phosphatase 1)、SHP-2、SHIP-1 (Src homology 2 domain containing inositol-5'-phosphatase-1)和MKP-1(MAPK phosphatase-1)等,分别通过不同的机制调控TLR或RIG-I触发的天然免疫应答中炎性细胞因子和/或Ⅰ型干扰素的产生。那么PHLPP作为另一重要的参与细胞信号转导调控的蛋白磷酸酶,是否参与天然免疫应答中TLR及RIG-I信号转导的调控尚不清楚。
本实验分三部分内容对PHLPP是否参与天然免疫的调控以及可能的相关机制等问题进行了研究。
一、PHLPP对TLR及RIG-I触发巨噬细胞产生Ⅰ型干扰素的选择性促进作用
我们研究发现PHLPP在小鼠脑组织、脾脏及淋巴结等组织和T细胞、B细胞、NK细胞、DC、腹腔巨噬细胞以及RAW264.7细胞株等免疫细胞中广泛表达。并且TLR配体LPS、poly(I:C)刺激以及RIG-I配体VSV感染可以诱导巨噬细胞表达PHLPP蛋白增加。
在小鼠巨噬细胞中,用靶向PHLPP的siRNA干扰PHLPP表达,发现可以显著抑制TLR4配体LPS、TLR3配体poly(I:C)以及RIG-I配体VSV诱导的Ⅰ型干扰素的产生,但对炎性细胞因子TNF-α、IL-6的产生无明显影响。另外,干扰PHLPP表达对CpG ODN诱导的Ⅰ型干扰素及炎性细胞因子产生均无显著影响。过表达PHLPP可以显著增强LPS、poly(I:C)以及VSV诱导的Ⅰ型干扰素的产生,而对炎性细胞因子TNF-α、IL-6的产生也无明显影响。
细胞因子报告基因结果显示,过表达PHLPP可以显著促进TRIF、组成性活化的RIG-I(RIG I-N)以及MDA5活化的IFN-β报告基因的活性,并且呈剂量依赖性；但对TRIF以及MyD88活化TNF-α报告基因的活性无明显影响,对MyD88活化IFN-β报告基因的活性也无明显影响。
通过PB转座子技术建立的PHLPP基因敲除小鼠,其腹腔巨噬细胞中PHLPP表达降低80%以上。实验发现PHLPP基因敲除小鼠的巨噬细胞在体外对LPS.poly(I:C)以及VSV诱导的IFN-α和IFN-β产生水平较野生型小鼠巨噬细胞明显下降,而炎性细胞因子TNF-α、IL-6的水平无明显差异。CpG ODN诱导的炎性细胞因子以及Ⅰ型干扰素的水平在两种细胞中均没有明显差异。在PHLPP基因敲除小鼠骨髓来源的DC中我们也得到了类似的结果。体内试验显示,PHLPP基因敲除小鼠及对照小鼠腹腔注射LPS.poly(I:C)或者VSV后,PHLPP基因敲除小鼠腹腔巨噬细胞中IFN-α和IFN-β的mRNA水平较对照小鼠均显著下降,而炎性细胞因子TNF-α和IL-6的mRNA水平无明显变化；PHLPP基因敲除小鼠血清中IFN-β水平较对照小鼠显著降低,而炎性细胞因子水平无明显变化。
以上研究结果显示PHLPP促进TLR配体诱导的TRIF依赖的以及RIG-I配体诱导的Ⅰ型干扰素的产生,而对其诱导的炎性细胞因子产生以及MyD88依赖的细胞因子产生无明显影响。
二、PHLPP选择性调控TLR及RIG-I触发Ⅰ型干扰素产生的分子机制研究
为明确PHLPP各个结构域的功能,我们根据PHLPP的分子结构构建了不同的PHLPP缺失突变载体。瞬时转染这些缺失突变载体后,检测对TRIF活化IFN-β报告基因的活性的影响。结果发现缺失LRR或者PP2C结构域的PHLPP突变体不能促进TRIF活化IFN-β报告基因的活性,而缺失PH或者PDZ结构域的PHLPP突变体仍然能够显著促进TRIF活化IFN-β报告基因的活性；单一表达PP2C结构域的PHLPP突变体不能促进TRIF活化IFN-β报告基因的活性,而表达LRR和PP2C结构域的PHLPP突变体能够显著促进TRIF活化IFN-β报告基因的活性。同时,我们发现在巨噬细胞中瞬时转染缺失LRR或者PP2C结构域的PHLPP突变体不能促进poly(I:C)诱导的Ⅰ型干扰素的产生。由此认为,PHLPP正向调控TLR及RIG-I触发的Ⅰ型干扰素的产生是依赖于其LRR结构域和磷酸酶活性结构域PP2C。
接着,我们观察了PHLPP对TLR及RIG-I信号活化的MAPKs、NF-κB的影响。发现PHLPP表达降低对poly(I:C)诱导的ERK、JNK、p38和IKKα/β的磷酸化没有明显影响。同时,报告基因结果显示过表达PHLPP对MyD88、TRIF或RIG-I活化的NF-κB报告基因活性也没有显著影响。因此,PHLPP不能促进TLR及RIG-I触发的MAPKs和NF-κB信号通路的活化,从而对炎性细胞因子产生没有显著影响。
那么PHLPP调控TLR及RIG-I信号触发的Ⅰ型干扰素产生的靶分子是什么呢?研究发现过表达PHLPP可以显著促进TBK1和IRF3活化的IFN-β报告基因的活性以及TRIF活化IRF3报告基因的活性,并且均具有剂量依赖性,提示PHLPP在Ⅰ型干扰素产生信号途径中作用在IRF3分子水平。
进一步免疫沉淀和GST-pull down实验表明PHLPP可以通过其LRR结构域直接与IRF3的C端相结合。共聚焦显微镜观察显示在巨噬细胞静息状态下PHLPP在胞浆、胞核中均有分布,而IRF3全部在胞浆中；poly(I:C)刺激或SeV感染后,PHLPP有往胞核中聚集的趋势,IRF3转位入核；并且PHLPP与IRF3在细胞核内产生共定位,且分离胞浆、胞核蛋白进行免疫沉淀实验进一步证明PHLPP与IRF3在细胞核内结合。
为进一步证明PHLPP调控TLR及RIG-I触发的Ⅰ型干扰素产生的靶分子是IRF3,我们采用了IRF3基因敲除小鼠(IRF3-/-)的腹腔巨噬细胞。实验发现过表达PHLPP对poly(LC)诱导的IRF3-/-巨噬细胞中Ⅰ型干扰素产生没有明显影响。在IRF3-/-巨噬细胞中瞬时转染IRF3质粒以恢复IRF3的表达后,发现过表达PHLPP也恢复了对poly(I:C)诱导的Ⅰ型干扰素产生的促进作用。由此确定PHLPP调控TLR及RIG-I触发的Ⅰ型干扰素产生的信号途径靶分子是IRF3。
IRF3作为TLR和RIG-I信号通路的共同下游分子之一,活化后发生磷酸化、二聚体化以及核转位,进而与CBP/P300结合,促进Ⅰ型干扰素的产生,随后IRF3蛋白通过蛋白酶体途径降解,从而保证Ⅰ型干扰素信号活化途径的平衡。我们研究发现PHLPP基因敲除小鼠的腹腔巨噬细胞在poly(I:C)刺激或者VSV、SeV感染后,其IRF3蛋白降解速度较对照小鼠的腹腔巨噬细胞增快；蛋白酶体抑制剂MG132可以完全抑制上述两种细胞中IRF3的降解。过表达PHLPP可以抑制SeV感染诱导的IRF3蛋白降解。PHLPP表达降低之后可以显著增强IRF3泛素化。以上实验结果表明,PHLPP能够与IRF3直接结合并维持IRF3蛋白的稳定,防止其泛素化而降解。
那么,PHLPP通过何种机制维持IRF3蛋白的稳定呢?考虑到PHLPP正向调控Ⅰ型干扰素的产生是依赖于其磷酸酶活性结构域PP2C,且有文献报道IRF3 339位丝氨酸(小鼠IRF3 332位丝氨酸(IRF3 S332))磷酸化可以促进IRF3的泛素化和降解,那么PHLPP是否通过使IRF3 S332位去磷酸化而稳定IRF3蛋白呢?我们构建了小鼠IRF3 S332位突变的表达载体(IRF3 S332A)。研究发现在HEK293细胞中瞬时转染PHLPP和野生型IRF3或IRF3 S332A,用SeV感染后,PHLPP对IRF3 S332A的降解和泛素化无明显影响,却可以抑制野生型IRF3的降解和泛素化。同时,报告基因实验显示PHLPP能促进野生型IRF3却不能促进IRF3 S332A突变体活化的IFN-β报告基因的活性。这些实验结果说明PHLPP的作用位点是IRF3 S332。
本部分研究结果表明巨噬细胞中PHLPP在细胞核内通过其LRR结构域与活化的IRF3 C端结合,通过其PP2C磷酸酶活性区使IRF3 S332去磷酸化,从而抑制S332磷酸化依赖的IRF3泛素化和降解,维持IRF3蛋白的稳定,促进Ⅰ型干扰素的产生。
三、PHLPP通过促进Ⅰ型干扰素产生而参与抵抗病毒感染的作用
为探讨PHLPP正向调控TLR及RIG-I触发的Ⅰ型干扰素的产生这一功能在感染性疾病,特别是病毒感染性疾病中的作用,我们首先观察了PHLPP对VSV复制的影响。实验发现PHLPP基因敲除小鼠的腹腔巨噬细胞在VSV感染后,培养上清中VSV病毒滴度(TCID50)显著增加,用培养上清处理的HEK293细胞迅速出现细胞变圆等病毒感染性病变及细胞死亡；而在巨噬细胞培养过程中中加入重组小鼠IFN-β,可以明显降低VSV病毒滴度,减少HEK293细胞死亡率。上述结果表明PHLPP通过促进Ⅰ型干扰素的产生而抑制了VSV的复制。
进一步我们取小鼠体内感染VSV 24-48小时之后的脏器检测病毒感染情况,发现PHLPP基因敲除小鼠的肝脏和脾脏组织中VSV的复制和病毒滴度(TCID50)较对照小鼠显著增加,表明PHLPP在体内参与了抵抗VSV感染的效应。
本部分研究表明PHLPP通过促进体内TLR及RIG-I触发的Ⅰ型干扰素的产生,抑制VSV的复制,提高小鼠对VSV体内感染的抵抗能力,提示其在抵御病毒感染性疾病中发挥着重要的作用。
综合以上三部分研究结果,我们发现磷酸酶PHLPP参与了巨噬细胞中TLR及RIG-I触发的Ⅰ型干扰素产生的正向调控；证明了PHLPP可以通过LRR结构域直接结合胞核内的IRF3,并依赖其磷酸酶活性使IRF3 332位丝氨酸去磷酸化而抑制IRF3的泛素化和降解,维持Ⅰ型干扰素产生的信号通路的持续活化,提高小鼠对VSV感染的抵抗力。本研究揭示了磷酸酶PHLPP在先天免疫应答调控中的作用,丰富了TLR信号及RIG-I信号转导调控的分子机制,为感染性疾病尤其是病毒性感染的免疫治疗提供了新的思路。
Innate immune cells recognize pathogen-associated molecular patterns (PAMPs) conserved in microbes by pattern recognition receptors (PRRs) to trigger innate immune responses. As one kind of key PRRs, TLRs comprise a large family consisting of at least 12 members and are mainly expressed in antigen-presenting cells (APCs) including macrophages and dendritic cells (DCs). Upon recognition of PAMPs, TLRs trigger myeloid differentiation factor 88 (MyD88)- and/or Toll/IL-1 receptor (TIR)-domain containing adaptor protein inducing IFN-β(TRIF)-dependent signaling pathway to activate mitogen-activated protein kinases (MAPKs), the transcription factors nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3) and/or IRF7, leading to the production of proinflammatory cytokines and typeⅠinterferon (IFN), and defense against invading pathogens.
Besides TLRs, retinoid acid-inducible gene I-like receptors (RLRs) are another important class of PRR families, which are composed of RNA helicase domain-containing proteins retinoic acid-inducible geneⅠ(RIG-Ⅰ), melanoma differentiation associated gene 5 (MDA5) and LGP2. They are localized in the cytoplasm and recognize the genomic RNA of dsRNA viruses and dsRNA generated as the replication intermediate of ssRNA viruses. Vesicular stomatitis virus (VSV) and Sendai virus (SeV) are the representative viruses which can activate RIG-Ⅰsignal pathway. RIG-Ⅰand MDA5 interact with adaptor protein IPS-1 (Interferon-beta promoter stimulator 1, also known as MAVS/Cardif/VISA), which mediates the gene expression of typeⅠinterferon and proinflammatory cytokines by activating IRF3/7 and NF-κB.
TLRs and RLRs play important roles in linking innate and adaptive immune responses. It is now well accepted that full activation of TLRs and RLRs is essential for initiating the innate immune response and enhancing adaptive immunity to eliminate invading pathogens. Less efficient activation of TLRs and RLRs or excessive activation of TLRs and RLRs may induce immune disorders and even immunopathological process. Up to now, various signal pathways are known to be involved in the tight regulation of TLR and RLR signaling to maintain the immunological balance. However, the underlying molecular mechanisms for the regulation of TLR and RLR signaling remain to be fully elucidated.
Phosphatase PHLPP (PH domain leucine-rich repeat protein phosphatase) is a serine/threonine phosphatase, which consist of a pleckstrin homology (PH) domain, a leucine-rich repeat (LRR) region, a PP2C (protein phosphatase 2C) domain, and a PDZ-binding motif. Previous studies showed that PHLPP can directly interact with K-Ras through its LRR domain to negatively regulate Ras-MAPK signal pathway in rat brain. PHLPP also functions as a negative regulator of MAPK and CREB-mediated transcription in hippocampal neurons to regulate rat memory formation. PHLPP is low expressed in many human tumor cells such as breast cancer, colon cancer and glioblastoma cell lines. PHLPP can specifically dephosphorylate the Ser473 of Akt depending on its PP2C and PDZ binding motif to suppress tumor growth and promote apoptosis of tumor cells. In addition, PHLPP controls the cellular levels of PKC by specifically dephosphorylating the hydrophobic motif, thus destabilizing the enzyme and promoting its degradation. All these reports suggest that PHLPP may show different functions depending on its different domain to involve in many cell preceess.
Nowadays, the important roles of protein phosphotase in regulating immune response draw more and more attention. In innate immunity, several protein phosphotases, such as SHP-1 (Src homology region 2 domain-containing phosphatase 1), SHP-2 (Src homology region 2 domain-containing phosphatase 2), SHIP-1 (Src homology 2 domain containing inositol-5'-phosphatase-1) and MKP-1 (MAPK phosphatase-1), have been reported to regulate TLR-and RIG-Ⅰ-triggered production of proinflammatory cytokines and/or typeⅠinterferon through different mechanism. However, whether phosphotase PHLPP can regulate TLR and RIG-Ⅰ-triggered immune response remains unknown.
The primary aim of this study is to investigate the regulation of TLR-and RIG-Ⅰ-triggered innate immune response by PHLPP in macrophages as well as the underlying mechanisms.
PartⅠ. PHLPP selectively promotes TLR-and RIG-Ⅰ-triggered typeⅠinterferon production in macrophages
We first examined the expression pattern of PHLPP. RT-PCR assay showed that the mRNA of PHLPP is ubiquitously expressed in various mouse tissues and immune cells, with highest levels in brain and spleen. LPS, poly(I:C) or VSV challenge upregulated PHLPP expression in mouse primary peritoneal macrophages.
Next, the effects of PHLPP silence or deficiency on the production of TLR- and RIG-Ⅰ-triggered cytokines were investigated. RNAi knockdown of PHLPP significantly suppressed TLR3,4- and RIG-Ⅰ-triggered production of IFN-α/βin macrophages, but had no effect on the production of IL-6 and TNF-α. Meanwhile, silencing of PHLPP expression had no influence on TLR9-triggered production of the above cytokines. Coincidently, overexpression of PHLPP significantly enhanced TLR3,4- and RIG-Ⅰ-triggered production of IFN-α/βin macrophages.
Overexpression of PHLPP significantly increased TRIF-, constitutively active RIG-Ⅰ-or MDA-5-activated expression of IFN-βreporter gene in a dose-dependent manner, but had no effect on TRIF-or MyD88-activated expression of TNF-αreporter gene, or MyD88-activated expression of IFN-βreporter gene.
In macrophages from PHLPP knockout mice generated by PB (PiggyBac) transposon system, PHLPP expression was downregulated over 80% as compared to that in macrophages from wild type mice. PHLPP deficiency significantly inhibited TLR3,4- and RIG-Ⅰ-triggered production of IFN-α/β, while had no effect on the production of IL-6 and TNF-αin macrophages. The production of above cytokines induced by CpG ODN remain unchanged in PHLPP-/- and PHLPP+/+ macrophages. PHLPP-/- mice and PHLPP+/+ mice were challenged by intraperitoneal injection with LPS, poly(I:C), or infection with VSV. After these challenges, the mRNA levels of IFN-α/βin primary peritoneal macrophages and IFN-p level in serum of PHLPP-/- mice were much lower than that in PHLPP+/+ mice, while there was no significant difference in the levels of proinflammatory cytokines.
Taken together, these results indicated that PHLPP selectively promotes TLR-triggered TRIF-dependent and RIG-Ⅰ-triggered production of typeⅠinterferon, but has no effect on the production of proinflammatory cytokines and MyD88-dependent production of cytokines.
PartⅡ. The underlying molecular mechanisms for the positive regulation of TLR and RIG-Ⅰ-triggered typeⅠinterferon production by PHLPP
To investigate the function of each domain of PHLPP in the enhancement of typeⅠinterferon production triggered by TLR3,4 and PIG-Ⅰ, various truncated or deleted mutants of PHLPP were constructed according to its known structural and functional domains, and then transfected into HEK293 cells to detect TRIF-activated expression of IFN-(3 reporter gene. We revealed that the mutants of PHLPP deleting LRR region or PP2C domain couldn't increase TRIF-activated expression of IFN-βreporter gene, while the mutants of PHLPP deleting PH domain or PDZ-binding motif significantly increased TRIF-dependent activation of IFN-βreporter gene as full-length PHLPP did. The mutant of PHLPP only expressing PP2C domain couldn't increase TRIF-activated expression of IFN-βreporter gene, while the mutant of PHLPP expressing LRR region and PP2C domain significantly could did so. Meanwhile, overexpression of the mutants of PHLPP deleting LRR region or PP2C domain failed to enhance poly(I:C)-induced production of IFN-(3 in macrophages. So, the results indicated that the LRR and PP2C domain were responsible for the positive regulation of TLR-and RIG-Ⅰ-triggered production of typeⅠinterferon by PHLPP.
Then, we tested whether PHLPP regulates TLR-and RIG-Ⅰ-triggered activation of MAPKs and NF-κB pathways. PHLPP deficiency had no significant effect on poly(I:C)-induced phosphorylation of ERK, JNK, p38 and IKKα/βin macrophages. Consistently, PHLPP overexpression did not affect MyD88-, TRIF- or constitutively active RIG-Ⅰ-activated expression of NF-κB reporter gene. So, PHLPP can't enhance TLR- and RIG-Ⅰ-triggered activation of MAPKs and NF-κB, which contributes to the unchanged production of inflammatory cytokines.
Next, we investigated which molecule in TLR and RIG-Ⅰsignal pathway PHLPP could interact with. Luciferase report gene assay showed that overexpression of PHLPP significantly increased TBK1- and IRF3-activated expression of IFN-βreporter gene, and TRIF-activated expression of IRF3 reporter gene in a dose-dependent manner. These results indicated IRF3 may be the potential target PHLPP can interact with. Immunoprecipitation experiment and GST pull-down assay confirmed PHLPP could directly interact with the C-terminal of IRF3 through its LRR region.
As we known, IRF3 resides in the cytoplasm in resting cells, and, upon stimulation, becomes activated leading to nuclear translocation. The confocal laser scanning microscope observed that PHLPP resided in both cytoplasm and nuclear of resting macrophages. Upon the stimulation of poly(I:C) or SeV, more PHLPP translocated into nuclear, and co-localized with IRF3 in the nuclei of macrophages. Immunoprecipitation experiment using separated extracts of cytoplasm and nuclei revealed that PHLPP could co-precipitate with IRF3 in the nuclei of macrophages.
To further confirm the target of PHLPP in regulation of TLR-and RIG-Ⅰ-triggered production of typeⅠinterferon is IRF3, we transduce PHLPP into IRF3 deficient (IRF3-/-) macrophages. Overexpression of PHLPP had no effect on poly(I:C)- or VSV-induced production of IFN-βin IRF3-/- macrophages, while PHLPP overexpression promoted poly(I:C) or VSV- triggered IFN-βproduction when rescuing the expression of IRF3 in IRF3-/- macrophages. So, the above results confirmed PHLPP can interact with IRF3 to promote TLR- and RIG-Ⅰ-triggered production of typeⅠinterferon.
As one of the key transcription factors in both TLR and RIG-I signal pathway, IRF3, upon stimulation, becomes activated via serine/threonine phosphorylation (between residues 385 and 405) leading to its dimerization, nuclear translocation and association with the coactivator CBP/p300 for inducing the production of typeⅠinterferon, then IRF3 degrades through proteasome-dependent pathway to maintain the balance of the production of typeⅠinterferon. We found that IRF3 of PHLPP-/- macrophages degraded faster and more significantly than that of wild-type macrophages challenged with poly(I:C), VSV or SeV. Considently, overexpression of PHLPP could delay and inhibit SeV-induced degradation of IRF3 in macrophages. MG132 could almost completely inhibit the degradation of IRF3 in PHLPP-/- and PHLPP+/+ macrophages. Meanwhile, PHLPP deficiency enhanced the poly(I:C)-, VSV- or SeV-induced polyubiquitination of IRF3. These results indicated PHLPP directly interacts with IRF3 to maintain the stability of IRF3, preventing it from ubiquitination and degradation.
Next, we wanted to know how PHLPP maintains the stability of IRF3. It has been reported that phosphorylation of Ser339 of human IRF3 (IRF3 Ser332 in mice IRF3) led to polyubiquitination of IRF3 and then proteasome-dependent degradation of IRF3. Combination with the above result that PHLPP promoted the production of typeⅠinterferon depending on its phosphatase activity of PP2C domain, we predicted that PHLPP may maintain the stability of IRF3 through dephosphorylating IRF3 Ser332. In orderto confirm the hypothesis, we generated the mutant IRF3 with alanine substituted for serine in residue 332 of IRF3 (IRF3 S332A). Overexpression of PHLPP had no effect on SeV-induced polyubiquitination and degradation of IRF3 S332A, while obviously inhibited polyubiquitination and degradation of wild-type IRF3. Reporter gene assay showed that overexpression of PHLPP couldn't enhance IRF3 S332A-activated expression of IFN-βreporter gene. These results demonstrated that PHLPP could dephosphorylate IRF3 Ser332 to inhibit its phosphorylation-dependent degradation of IRF3.
Taken together, these results demonstrated PHLPP directly interacted with the C-terminal of IRF3 through its LRR region in the nuclei of macrophages, and dephosphorylated IRF3 Ser332 through PP2C domain to maintain the stability of IRF3 by inhibiting IRF3 Ser332 phosphorylation-dependent degradation of IRF3, leading to the enhanced production of typeⅠinterferon triggered by TLR3,4 and RIG-Ⅰ.
PartⅢ. PHLPP enhances host defense against viral infection by promoting typeⅠinterferon production
Because PHLPP has been shown to be able to potentiate typeⅠinterferon production in macrophages, we wondered whether PHLPP-mediated enhancement of typeⅠinterferon production could protect the host from virus infection. We tested this with VSV infection model both in vitro and in vivo. Upon infection of VSV, VSV titers (TCID50) in the cultural supernatant of PHLPP-/- acrophages were significantly higher than that in wild type macrophages, and HEK293 cells cultured with the supernatant of PHLPP-/-macrophages quickly showed pathology of virus infection and almost all died. Addition of recombinant mouse IFN-(3 could decrease the titer of VSV in PHLPP-/- macrophages and rescue the death of HEK293 cells cultured with the supernatant of PHLPP-/- macrophages. These results indicated that PHLPP inhibits VSV replication through promoting the production of typeⅠinterferon during viral infection.
Next, we investigated the possible role of PHLPP in suppressing VSV infection in vivo. VSV RNA replicates and VSV titer in liver and spleen of PHLPP-/- mice were much higher than that of control mice after VSV challenge. These in vivo data revealed that PHLPP can inhibit VSV replication through promoting RIG-Ⅰ-triggered production of typeⅠinterferon, and protect the host from virus infection.
In conclusion, we have demonstrated that PHLPP can selectively promote the TLR3,4-and RIG-Ⅰ-triggered production of typeⅠinterferon in macrophages. PHLPP directly interacts with IRF3 through its LRR domain, dephosphorylates IRF3 Ser332 depending on the phosphatase activity of PP2C domain and subsequently inhibit polyubiquitination and Ser332 phosphorylation-dependent degradation of IRF3, thus maintaining the stability of IRF3. Our results indicate that phosphatase PHLPP may be an essential component of the host antiviral'machinery'.
引文
1. Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol.2003;21:335-376.
2. Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity:update on Toll-like receptors. Nat Immunol.2010;11:373-384.
3. Takeuchi O, Akira S. Pattern recognition receptors and inflammation. Cell.2010;140:805-820.
4. Akira S, Takeda K. Toll-like receptor signaling. Nat Rev Immunol.2004;4:499-511.
5. Yamamoto M, Sato S, Hemmi H, Hoshino K, Kaisho T, Sanjo H, Takeuchi O, Sugiyama M, Okabe M, Takeda K, Akira S. Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. Science.2003;301:640-643.
6. Bowie AG, Unterholzner L. Viral evasion and subversion of pattern-recognition receptor signalling. Nat Rev Immunol.2008;8:911-922.
7. Takeuchi O, Akira S. Innate immunity to virus infection. Immunol Rev.2009;227:75-86.
8. Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, Matsui K, Uematsu S, Jung A, Kawai T, Ishii KJ, Yamaguchi O, Otsu K, Tsujimura T, Koh CS, Reis e Sousa C, Matsuura Y, Fujita T, Akira S. Differential roles of MDA5 and RIG-Ⅰ helicases in the recognition of RNA viruses. Nature. 2006;441:101-105.
9. Kawai T, Takahashi K, Sato S, Coban C, Kumar H, Kato H, Ishii KJ, Takeuchi O, Akira S. IPS-1, an adaptor triggering RIG-Ⅰ- and Mda5-mediated type Ⅰ interferon induction. Nat Immunol. 2005;6:981-988.
10. Seth RB, Sun L, Ea CK, Chen ZJ. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3.Cell.2005;122:669-682.
11. Xu LG, Wang YY, Han KJ, Li LY, Zhai Z, Shu HB. VISA is an adapter protein required for virus-triggered IFN-beta signaling. Mol Cell.2005;19:727-740.
12. Meylan E, Curran J, Hofmann K, Moradpour D, Binder M, Bartenschlager R, Tschopp J. Cardif is an adaptor protein in the RIG-Ⅰ antiviral pathway and is targeted by hepatitis C virus. Nature. 2005;437:1167-1172.
13. Akira S, Takeda K, Kaisho T. Toll-like receptors:critical proteins linking innate and acquired immunity. Nat Immunol.2001;2:675-680.
14. Liew FY, Xu D, Brint EK, O'Neill LA. Negative regulation of toll-like receptor-mediated immune responses. Nat Rev Immunol.2005;5:446-458.
15. Luke A.J. O'Neill. When Signaling Pathways Collide:Positive and Negative Regulation of Toll-like Receptor Signal Transduction. Immunity.2008;29:12-20.
16. Gao T, Furnari F, Newton AC. PHLPP:a phosphatase that directly dephosphorylates Akt, promotes apoptosis, and suppresses tumor growth. Mol Cell.2005;18:13-24.
17. Shimizu K, Okada M, Takano A, Nagai K. SCOP, a novel gene product expressed in a circadian manner in rat suprachiasmatic nucleus. FEBS Lett.1999;458:363-369.
18. Shimizu K, Okada M, Nagai K, Fukada Y. Suprachiasmatic nucleus circadian oscillatory protein, a Novel binding Partner of K-Ras in the Membrane Rafts, Negatively Regulates MAPK Pathway. J Biol Chem.2003;278:14920-14925.
19. Shimizu K, Mackenziebc SM, Storm DR. SCOP/PHLPP and its functional role in the brain. Mol BioSyst.2010;6:38-43.
20. Shimizu K, Phan T, Mansuy IM, Storm DR. Proteolytic degradation of SCOP in the hippocampus contributes to activation of MAP kinase and memory. Cell.2007;128:1219-1229.
21. Gao T, Brognard J, and Newton AC. The phosphatase PHLPP controls the cellular levels of protein kinase C. J Biol Chem.2008;283:6300-6311.
22. Karen M. Doody, Annie Bourdeau, Michel L. Tremblay. T-cell protein tyrosine phosphatase is a key regulator in immune cell signaling:lessons from the knockout mouse model and implications in human disease. Immunol Rev.2009;228:325-341.
23. An H, Hou J, Zhou J, Zhao W, Xu H, Zheng Y, Yu Y, Liu S, Cao X. Phosphatase SHP-1 promotes TLR- and RIG-Ⅰ-activated production of type Ⅰ interferon by inhibiting the kinase IRAK1. Nat Immunol.2008;9:542-550.
24. An H, Zhao W, Hou J, Zhang Y, Xie Y, Zheng Y, Xu H, Qian C, Zhou J, Yu Y, Liu S, Feng G, Cao X. SHP-2 Phosphatase Negatively Regulates the TRIF Adaptor Protein-Dependent Type I Interferon and Proinflammatory Cytokine Production. Immunity.2006;25:919-928.
25. An H, Xu H, Zhang M, Zhou J, Feng T, Qian C, Qi R, Cao X. Src homology 2 domain-containing inositol-5-phosphatase 1 (SHIP1) negatively regulates TLR4-mediated LPS response primarily through a phosphatase activity-and PI-3K-independent mechanism. Blood.2005;105:4685-4692.
26. Leavy O. MKP1:A dynamic immune regulator. Nat Rev Immunol.2006;6:170-171.
27. Dong C, Davis RJ, Flavell RA. MAP kinases in immune response. Annu Rev Immunol. 2002;20:55-72.
28. Smith H, Peggie M, Campbell DG, Vandermoere F, Carrick E, Cohen P. Identification of the phosphorylation sites on the E3 ubiquitin ligase Pellino that are critical for activation by IRAKI and IRAK4. Proc Natl Acad Sci USA.2009;106:4584-4590.
29. Huang G, Shi LZ, Chi H. Regulation of JNK and p38 MAPK in the immune system:signal integration, propagation and termination. Cytokine.2009;48:161-169.
30. Noyce RS, Collins SE, Mossman KL. Differential modification of interferon regulatory factor 3 following virus particle entry. J Virol.2009;83:4013-4022.
31. Ding S, Wu X, Li G, Han M, Zhuang Y, Xu T. Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice. Cell.2005;122:473-483.
32. Salojin KV, Owusu IB, Millerchip KA, Potter M, Platt KA, Oravecz T. Essential role of MAPK phosphatase-1 in the negative control of innate immune responses. J Immunol. 2006;176:1899-1907.
33. Gabhann JN, Higgs R, Brennan K, Thomas W, Damen JE, Ben Larbi N, Krystal G, Jefferies CA. Absence of SHIP-1 results in constitutive phosphorylation of Tank-binding kinase 1 and enhanced TLR3-dependent IFN-b production. J Immunol.2010;184:2314-2320.
34. Abu-Dayyeh I, Shio MT, Sato S, Akira S, Cousineau B, Olivier M. Leishmania-induced IRAK-1 inactivation is mediated by SHP-1 interacting with an evolutionarily conserved KTIM motif. PLoS Negl Trop Dis.2008;2:e305.
35. Zhou D, Collins CA, Wu P, Brown EJ. Protein tyrosine phosphatase SHP-1 positively regulates TLR-induced IL-12p40 production in macrophages through inhibition of phosphatidylinositol 3-kinase. J Leukoc Biol.2010;87:845-855.
36. Yoneyama M, Suhara W, Fujita T. Control of IRF-3 activation by phosphorylation. J Interferon Cytokine Res.2002;22:73-76.
37. McWhirter SM, Fitzgerald KA, Rosains J, Rowe DC, Golenbock DT, Maniatis T. IFN-regulatory factor 3-dependent gene expression is defective in Tbkl-deficient mouse embryonic fibroblasts. Proc Natl Acad Sci USA.2004;101:233-238.
38. Saitoh T, Tun-Kyi A, Ryo A, Yamamoto M, Finn G, Fujita T, Akira S, Yamamoto N, Lu KP, Yamaoka S. Negative regulation of interferon-regulatory factor 3-dependent innate antiviral response by the prolyl isomerase Pin1. Nat Immunol.2006;7:598-605.
39. Honda K, Taniguchi T. IRFs:master regulators of signalling by Toll-like receptors and cytosolic pattern-recognition receptors. Nat Rev Immunol.2006;6:644-658.
40. Ojaniemi M, Glumoff V, Harju K, Liljeroos M, Vuori K, Hallman M. Phosphatidylinositol 3-kinase is involved in Toll-like receptor 4-mediated cytokine expression in mouse macrophages. Eur J Immunol.2003;33:597-605.
41. Sarkar SN, Peters KL, Elco CP, Sakamoto S, Pal S, Sen GC. Novel roles of TLR3 tyrosine phosphorylation and PI3 kinase in double-stranded RNA signaling. Nat Struct Mol Biol. 2004;11:1060-1067.
42. Eskan MA, Rose BG, Benakanakere MR, Lee MJ, Kinane DF. Sphingosine 1-phosphate 1 and TLR4 mediate IFN-beta expression in human gingival epithelial cells. J Immunol. 2008;180:1818-1825.
43. Nistal-Villan E, Gack MU, Martinez-Delgado G, Maharaj NP, Inn K, Yang H, Wang R, Aggarwal AK, Jung JU, Garcia-Sastre A. Negative role of RIG-Ⅰ serine 8 phosphorylation in the regulation of IFN-{beta} production. J Biol Chem.2010 Apr 20. [Epub ahead of print]
44. Sharma S, BR tenOever, N Grandvaux, GP Zhou, R Lin, J Hiscott. Triggering the interferon antiviral response through an IKK-related pathway. Science.2003;300:1148-1151.
45. Fitzgerald KA, SM McWhirter, KL Faia, DC Rowe, E Latz, DT Golenbock, AJ Coyle, SM Liao, T Maniatis. IKK- and TBK1 are essential components of the IRF3 signaling pathway. Nat Immunol. 2003;4:491-496.
46. Foy E, Li K, Wang C, Sumpter R Jr, Ikeda M, Lemon SM, Gale M Jr. Regulation of interferon regulatory factor-3 by the hepatitis C virus serine protease. Science 2003;300:1145-1148.
47. Li K, Foy E, Ferreon JC, Nakamura M, Ferreon AC, Ikeda M, Ray SC, Gale M Jr, Lemon SM. Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF. Proc Natl Acad Sci USA.2005;102:2992-2997.
48. Li XD, Sun L, Seth RB, Pineda G, Chen ZJ. Hepatitis C virus protease NS3/4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity. Proc Natl Acad Sci USA.2005;102(49):17717-17722.
49. Barro M, Patton JT. Rotavirus nonstructural protein 1 subverts innate immune response by inducing degradation of IFN regulatory factor 3. Proc Natl Acad Sci USA.2005;102:4114-4119.
50. Chen Z, Rijnbrand R, Jangra RK, Devaraj SG, Qu L, Ma Y, Lemon SM, Li K.Ubiquitination and proteasomal degradation of interferon regulatory factor-3 induced by Npro from a cytopathic bovine viral diarrhea virus. Virology.2007;366:277-292.
51. Bibeau-Poirier A, Gravel SP, Clement JF, Rolland S, Rodier G, Coulombe P, Hiscott J, Grandvaux N, Meloche S, Servant MJ. Involvement of the IkappaB kinase (IKK)-related kinases tank-binding kinase 1/IKKi and cullin-based ubiquitin ligases in IFN regulatory factor-3 degradation. J Immunol. 2006;177:5059-5067.
52. Zhang J, Xu LG, Han KJ, Wei X, Shu HB. PIASy represses TRIF-induced ISRE and NF-kappaB activation but not apoptosis. FEBS Lett. 2004;570:97-101.
53. Kubota T, Matsuoka M, Chang TH, Tailor P, Sasaki T, Tashiro M, Kato A, Ozato K. Virus infection triggers SUMOylation of IRF3 and IRF7, leading to the negative regulation of type Ⅰ interferon gene expression. J Biol Chem.2008;283:25660-25670.
54. Stetson DB, Medzhitov R. Type Ⅰ interferons in host defense. Immunity.2006;25:373-381.
55. Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, Miyagishi M, Taira K, Akira S, Fujita T. The RNA helicase RIG-Ⅰ has an essential function in double-stranded RNA-induced innate antiviral responses. Nat Immunol.2004;5:730-737.
1. Shi Y. Serine/Threonine Phosphatases:Mechanism through Structure. Cell.2009;139:468-484.
2. Mumby MC, Walter G. Protein Serine/Threonine Phosphatases:Structure, Regulation, and Functions in Cell Growth. Physiol Rev.1993;73:673-699.
3. Zhang Z. Protein Tyrosine Phosphatase:Structure and Function, Substrate Specificity, and Inhibitor Development. Annu Rev Pharmacol Toxicol.2002;42:209-234.
4. Camps M, Nichols A, Arkinstall S. Dual specificity phosphatases:a gene family for control of MAP kinase function. FASEB J.2000;14:6-16.
5. Maehama T, Okahara F, Kanaho Y. The tumour suppressor PTEN:involvement of a tumour suppressor candidate protein in PTEN turnover. Biochem Soc Trans.2004;32:343-347.
6. Baumer N, Maurer A, Krieglstein J, Klumpp S. Expression of Protein Histidine Phosphatase in Escherichia coli, Purification, and Determination of Enzyme Activity. Methods Mol Biol. 2006;365:247-260.
7. Seger R, Krebs EG. The MAPK Signalling cascade. FASEB J.1995;9:726-735.
8. Ladbury JE. Measurement of the formation of complexes in tyrosine kinase-mediated signal transduction.Acta Crystallogr D Biol Crystallogr.2007;63:26-31.
9. Doody KM, Bourdeau A, Tremblay ML. T-cell protein tyrosine phosphatase is a key regulator in immune cell signaling:lessons from the knockout mouse model and implications in human disease. Immunol Rev.2009;28:325-341.
10. O'Neill LA. When Signaling Pathways Collide:Positive and Negative Regulation of Toll-like Receptor Signal Transduction. Immunity.2008;29:12-20.
11. Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell. 2006;124:783-801.
12. Akira S, Takeda K. Toll-like receptor signaling. Nat Rev Immunol.2004;4:499-511.
13. Takeuchi O, Akira S. Innate immunity to virus infection. Immunol Rev.2009;227:75-86.
14. Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, Matsui K, Uematsu S, Jung A, Kawai T, Ishii KJ, Yamaguchi O, Otsu K, Tsujimura T, Koh CS, Reis e Sousa C, Matsuura Y, Fujita T, Akira S. Differential roles of MDA5 and RIG-Ⅰ helicases in the recognition of RNA viruses. Nature. 2006;441:101-105.
15. Akira S, Takeda K, Kaisho T. Toll-like receptors:critical proteins linking innate and acquired immunity. Nat Immunol.2001;2:675-680.
16. Liew FY, Xu D, Brint EK, O'Neill LA. Negative regulation of toll-like receptor-mediated immune responses. Nat Rev Immunol.2005;5:446-458.
17. Kyriakis JM, Avruch J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by. stress and inflammation. Physiol Rev.2001;81:807-869.
18. Dong C, Davis RJ, Flavell RA. MAP kinases in immune response. Annu Rev Immunol. 2002;20:55-72.
19. Dickinson RJ, Keyse SM. Diverse physiological functions for dual-specificity MAP kinase phosphatases. J Cell Sci.2006;119:4607-4615.
20. Owens DM, Keyse SM. Differential regulation of MAP kinase signalling by dual-specificity protein phosphatases. Oncogene.2007;26:3203-3213.
21. Chi H, Barry SP, Roth RJ, Wu JJ, Jones EA, Bennett AM, Flavell RA. Dynamic regulation of pro-and anti-inflammatory cytokines by MAPK phosphatase 1 (MKP-1) in innate immune responses. Proc Natl Acad Sci USA.2006;103:2274-2279.
22. Salojin KV, Owusu IB, Millerchip KA, Potter M, Platt KA, Oravecz T. Essential role of MAPK phosphatase-1 in the negative control of innate immune responses. J Immunol. 2006;176:1899-1907.
23. Zhao Q, Wang X, Nelin LD, Yao Y, Matta R, Manson ME, Baliga RS, Meng X, Smith CV, Bauer JA, Chang CH, Liu Y. MAP kinase phosphatase 1 controls innate immune responses and suppresses endotoxic shock. J Exp Med.2006;203:131-140.
24. Hammer M, Mages J, Dietrich H, Servatius A, Howells N, Cato AC, Lang R. Dual specificity phosphatase 1 (DUSP1) regulates a subset of LPS-induced genes and protects mice from lethal endotoxin shock. J Exp Med.2006;203:15-20.
25. Leavy O. MKP1:A dynamic immune regulator. Nat Rev Immunol.2006;6:170-171.
26. March ME, Ravichandran K. Regulation of the immune response by SHIP. Semin Immunol. 2002;14:37-47.
27. Leung W, Tarasenko T, Bolland S. Differential roles for the inositol phosphatase SHIP in the regulation of macrophages and lymphocytes. Immunol Res.2009;43:243-251.
28. Helgason CD, Damen JE, Rosten P, Grewal R, Sorensen P, Chappel SM, Borowski A, Jirik F, Krystal G, Humphries RK. Targeted disruption of SHIP leads to hemopoietic perturbations, lung pathology, and a shortened life span. Genes Dev.1998;12:1610-1620.
29. Liu Q, Sasaki T, Kozieradzki I, Wakeham A, Itie A, Dumont DJ, Penninger JM. SHIP is a negative regulator of growth factor receptor-mediated PKB/Akt activation andmyeloid cell survival. Genes Dev.1999;13:786-791.
30. Sly LM, Rauh MJ, Kalesnikoff J, Song CH, Krystal G. LPS-induced upregulation of sHIP is essential for endotoxin tolerance. Immunity.2004;21:227-239.
31. Hayashia T, Graya CS, Chana M, Tawataoa RI, Ronacherb L, McGargillc MA, Dattad SK, Carsona DA, Corrb M. Prevention of autoimmune disease by induction of tolerance to Toll-like receptor 7. Proc Natl Acad Sci USA.2009;106:2764-2769.
32. An H, Xu H, Zhang M, Zhou J, Feng T, Qian C, Qi R, Cao X. Src homology 2 domain-containing inositol-5-phosphatase 1 (SHIP1) negatively regulates TLR4-mediated LPS response primarily through a phosphatase activity-and PI-3K-independent mechanism. Blood.2005;105:4685-4692.
33. Gabhann JN, Higgs R, Brennan K, Thomas W, Damen JE, Ben Larbi N, Krystal G, Jefferies CA. Absence of SHIP-1 Results in Constitutive Phosphorylation of Tank-Binding Kinase 1 and Enhanced TLR3-Dependent IFN-b Production. J Immunol.2010;184:2314-2320.
34. Khaled AR, Butfiloski EJ, Sobel ES, Schiffenbauer J. Functional consequences of the SHP-1 defect in motheaten viable mice:role of NF-kB. Cell Immunol.1998;185:49-58.
35. Nandan D, Lo R, Reiner NE. Activation of phosphotyrosine phosphatase activity attenuates mitogen-activated protein kinase signaling and inhibits c-FOS and nitric oxide synthase expression in macrophages infected with Leishmania donovani. Infect Immun.1999;67:4055-4063.
36. Zhang Z, Jimi E, Bothwell AL. Receptor activator of NF-kB ligand stimulates recruitment of SHP-1 to the complex containing TNFR-associated factor 6 that regulates osteoclastogenesis. J Immunol.2003;171:3620-3626.
37. An H, Hou J, Zhou J, Zhao W, Xu H, Zheng Y, Yu Y, Liu S, Cao X. Phosphatase SHP-1 promotes TLR- and RIG-Ⅰ-activated production of type Ⅰ interferon by inhibiting the kinase IRAK1. Nat Immunol.2008;9:542-550.
38. Abu-Dayyeh I, Shio MT, Sato S, Akira S, Cousineau B, Olivier M. Leishmania-induced IRAK-1 inactivation is mediated by SHP-1 interacting with an evolutionarily conserved KTIM motif. PLoS Negl Trop Dis.2008;2:e305.
39. Zhou D, Collins CA, Wu P, Brown EJ. Protein tyrosine phosphatase SHP-1 positively regulates TLR-induced IL-12p40 production in macrophages through inhibition of phosphatidylinositol 3-kinase. J Leukoc Biol.2010;87:845-855.
40. Salmond RJ, Alexander DR. SHP2 forecast for the immune system:fog gradually clearing. Trends Immunol.2006;27:154-1560.
41. Wang N, Li Z, Ding R, Frank GD, Senbonmatsu T, Landon EJ, Inagami T, Zhao ZJ. Antagonism or synergism. Role of tyrosine phosphatases SHP-1 and SHP-2 in growth factor signaling. J Biol Chem.2006;281:21878-21883.
42. Neel BG, Gu H, Pao L. The'Shp'ing news:SH2 domain-containing tyrosine phosphatases in cell signaling. Trends Biochem Sci.2003;28:284-293.
43. Frearson JA, Alexander DR. The phosphotyrosine phosphatase SHP-2 participates in a multimeric signaling complex and regulates T cell receptor (TCR) coupling to the Ras/mitogen-activated protein kinase (MAPK) pathway in Jurkat T cells. J Exp Med.1998;187:1417-1426.
44. Qu CK, Nguyen S, Chen J, Feng GS. Requirement of Shp-2 tyrosine phosphatase in lymphoid and hematopoietic cell development. Blood.2001;97:911-914.
45. Qu CK. Role of the SHP-2 tyrosine phosphatase in cytokineinduced signaling and cellular response. Biochim Biophys Acta.2002;1592:297-301.
46. Wang Q, Downey GP, Herrera-Abreu MT, Kapus A, McCulloch CA. SHP-2 modulates interleukin-1-induced Ca2+ flux and ERK activation via phosphorylation of phospholipase Cgamma1. J Biol Chem.2005;280:8397-8406.
47. Gadina M, Stancato LM, Bacon CM, Larner AC, O'Shea JJ. Involvement of SHP-2 in multiple aspects of IL-2 signaling:evidence for a positive regulatory role. J Immunol.1998;160:4657-4661.
48. Lundin Brockdorff J, Woetmann A, Mustelin T, Kaltoft K, Zhang Q, Wasik MA, Ropke C,Φdum N. SHP2 regulates IL-2 induced MAPK activation, but not Stat3 or Stat5 tyrosine phosphorylation, in cutaneous T cell lymphoma cells. Cytokine.2002;20:141-147.
49. Salmond RJ, Huyer G, Kotsoni A, Clements L, Alexander DR. The src homology 2 domain-containing tyrosine phosphatase 2 regulates primary T-dependent immune responses and Th cell differentiation. J Immunol.2005;175:6498-6508.
50. Wheadon H, Edmead C, Welham MJ. Regulation of interleukin-3-induced substrate phosphorylation and cell survival by SHP-2 (Src-homology protein tyrosine phosphatase 2). Biochem J.2003;376:147-157.
51. Yu WM, Hawley TS, Hawley RG, Qu CK. Catalytic-dependent and-independent roles of SHP-2 tyrosine phosphatase in interleukin-3 signaling. Oncogene.2003;22:5995-6004.
52. Ohtani T, Ishihara K, Atsumi T, Nishida K, Kaneko Y, Miyata T, Itoh S, Narimatsu M, Maeda H, Fukada T, Itoh M, Okano H, Hibi M, Hirano T. Dissection of signaling cascades through gp130 in vivo:reciprocal roles for STAT3- and SHP2-mediated signals in immune responses. Immunity. 2000;12:95-105.
53. Gu H, Neel BG. The 'Gab' in signal transduction. Trends Cell Biol.2003;13:122-130.
54. Zhang SQ, Yang W, Kontaridis MI, Bivona TG, Wen G, Araki T, Luo J, Thompson JA, Schraven BL, Philips MR, Neel BG. Shp2 regulates Src family kinase activity and Ras/Erk activation by controlling Csk recruitment. Mol Cell.2004;13:341-355.
55. Yu CL, Jin YJ, Burakoff SJ. Cytosolic tyrosine dephosphorylation of STAT5:potential role of SHP-2 in STAT5 regulation. J Biol Chem.2000;275:599-604.
56. Chen Y, Wen R, Yang S, Schuman J, Zhang EE, Yi T, Feng GS, Wang D. Identification of Shp-2 as a Stat5A phosphatase. J Biol Chem.2003;278:16520-16527.
57. Lehmann U, Schmitz J, Weissenbach M, Sobota RM, Hortner M, Friederichs K, Behrmann I, Tsiaris W, Sasaki A, Schneider-Mergener J, Yoshimura A, Neel BG, Heinrich PC, Schaper F. SHP2 and SOCS3 contribute to Tyr-759-dependent attenuation of interleukin-6 signaling through gp130. J Biol Chem.2003;278:661-671.
58. Du Z, Shen Y, Yang W, Mecklenbrauker I, Neel BG, Ivashkiv LB. Inhibition of IFN-a signaling by a PKC- and protein tyrosine phosphatase SHP-2-dependent pathway. Proc Natl Acad Sci USA. 2005;102:10267-10272.
59. You M, Yu DH, Feng GS. Shp-2 tyrosine phosphatase functions as a negative regulator of the interferon-stimulated JAK/STAT pathway. Mol Cell Biol.1999; 19:2416-2424.
60. An H, Zhao W, Hou J, Zhang Y, Xie Y, Zheng Y, Xu H, Qian C, Zhou J, Yu Y, Liu S, Feng G, Cao X. SHP-2 phosphatase negatively regulates the TRIF adaptor protein-dependent type Ⅰ interferon and proinflammatory cytokine production. Immunity.2006;25:919-928.
61. Alberola-Ila J, Takaki S, Kerner JD, Perlmutter RM. Differential signaling by lymphocyte antigen receptors. Annu Rev Immunol.1997;15:125-154.
62. Healy JI, Goodnow CC. Positive versus negative signaling by lymphocyte antigen receptors. Annu Rev Immunol.1998;16:645-670.
63. van Oers NS. T cell receptor-mediated signs and signals governing T cell development. Semin Immunol.1999;11:227-237.
64. Campbell KS. Signal transduction from the B cell antigen-receptor. Curr Opin Immunol. 1999;11:256-264.
65. Green MC, Shultz LD. Motheaten, an immunodeficient mutant of the mouse. I. Genetics and pathology. J Hered.1975;66:250-258.
66. Bignon JS, Siminovitch KA. Identification of PTPIC mutation as the genetic defect in motheaten and viable motheaten mice:a step toward defining the roles of protein tyrosine phosphatases in the regulation of hemopoietic cell differentiation and function. Clin Immunol Immunopathol. 1994;73:168-179.
67. Koo GC, Rosen H, Sirotina A, Ma XD, Shultz LD. Anti-CD11b antibody prevents immunopathologic changes in viable moth-eaten bone marrow chimeric mice. J Immunol. 1993;151:6733-6741.
68. Pani G, Kozlowski M, Cambier JC, Mills GB, Siminovitch KA. Identification of the tyrosine phosphatase PTP1C as a B cell antigen receptor-associated protein involved in the regulation of B cell signaling. J Exp Med.1995;181:2077-2084.
69. Ono M, Okada H, Bolland S, Yanagi S, Kurosaki T, Ravetch JV. Deletion of SHIP or SHP-1 reveals two distinct pathways for inhibitory signaling. Cell.1997;90:293-301.
70. Nitschke L, Carsetti R, Ocker B, Kohler G, Lamers MC. CD22 is a negative regulator of B-cell receptor signalling. Curr Biol.1997;7:133-143.
71. Fujimoto M, Bradney AP, Poe JC, Steeber DA, Tedder TF. Modulation of B lymphocyte antigen receptor signal transduction by a CD19/CD22 regulatory loop. Immunity.1999;11:191-200.
72. Tsubata T. Co-receptors on B lymphocytes. Curr Opin Immunol.1999;11:249-255.
73. Maeda A, Kurosaki M, Ono M, Takai T, Kurosaki T. Requirement of SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 for paired immunoglobulin-like receptor B (PIR-B)-mediated inhibitory signal. J Exp Med.1998;187:1355-1360.
74. Dustin LB, Plas DR, Wong J, Hu YT, Soto C, Chan AC, Thomas ML. Expression of dominant-negative src-homology domain 2-containing protein tyrosine phosphatase-1 results in increased Syk tyrosine kinase activity and B cell activation. J Immunol.1999; 162:2717-2724.
75. Lorenz U, Ravichandran KS, Burakoff SJ, Neel BG. Lack of SHPTP1 results in src-family kinase hyperactivation and thymocyte hyperresponsiveness. Proc Natl Acad Sci USA.1996;93:9624-9629.
76. Tarakhovsky A, Kanner SB, Hombach J, Ledbetter JA, Muller W, Killeen N, Rajewsky K. A role for CD5 in TCR-mediated signal transduction and thymocyte selection. Science.1995;269:535-537.
77. Johnson KG, LeRoy FG, Borysiewicz LK, Matthews RJ. TCR signaling thresholds regulating T cell development and activation are dependent upon SHP-1. J Immunol.1999;162:3802-3813.
78. Zhang J, Somani AK, Watt S, Mills GB, Siminovitch KA. The Src-homology domain 2-bearing protein tyrosine phosphatase-1 inhibits antigen receptor-induced apoptosis of activated peripheral T cells. J Immunol.1999;162:6359-6367.
79. Cuevas B, Lu Y, Watt S, Kumar R, Zhang J, Siminovitch KA, Mills GB. SHP-1 regulates Lck-induced phosphatidylinositol 3-kinase phosphorylation and activity. J Biol Chem. 1999;274:27583-27589.
80. Bertone S, Schiavetti F, Bellomo R, Vitale C, Ponte M, Moretta L, Mingari MC. Transforming growth factor-beta-induced expression of CD94/NKG2A inhibitory receptors in human T lymphocytes. Eur J Immunol.1999;29:23-29.
81. Ashwell JD, D'Oro U. CD45 and Src-family kinases:and now for something completely different. Immunol Today.1999;20:412-416.
82. Thomas ML. The regulation of antigen-receptor signaling by protein tyrosine phosphatases:a hole in the story. Curr Opin Immunol.1999;11:270-276.
83. Leitenberg D, Boutin Y, Lu DD, Bottomly K. Biochemical association of CD45 with the T cell receptor complex:regulation by CD45 isoform and during T cell activation. Immunity. 1999;10:701-711.
84. D'Oro U, Ashwell JD. Cutting edge:the CD45 tyrosine phosphatase is an inhibitor of Lck activity in thymocytes. J Immunol.1999; 162:1879-1883.
85. Katagiri T, Ogimoto M, Hasegawa K, Arimura Y, Mitomo K, Okada M, Clark MR, Mizuno K, Yakura H. CD45 negatively regulates lyn activity by dephosphorylating both positive and negative regulatory tyrosine residues in immature B cells. J Immunol.1999;163:1321-1326.
86. Shibasaki F, Hallin U, Uchino H. Calcineurin as a multifunctional regulator. J Biochem. 2002;131:1-15.
87. Rao A, Luo C, Hogan PG. Transcription factors of the NFAT family:regulation and function. Annu Rev Immunol.1997;15:707-747.
88. Im SH, Rao A. Activation and deactivation of gene expression by Ca2+/calcineurin-NFAT-mediated signaling. Mol Cells.2004;18:1-9.
89. Kissinger CR, Parge HE, Knighton DR, Lewis CT, Pelletier LA, Tempczyk A, Kalish VJ, Tucker KD, Showalter RE, Moomaw EW, Gastinel LN, Habuka N, Chen X, Maldonado F, Barker JE, Bacquet R, Ernest Villafranca J. Crystal structures of human calcineurin and the human FKBP12- FK506-calcineurin complex. Nature.1995;378:641-644.
90. Huai Q, Kim HY, Liu Y, Zhao Y, Mondragon A, Liu JO, Ke H. Crystal structure of calcineurin-cyclophilin-cyclosporin shows common but distinct recognition of immunophilin-drug complexes. Proc Natl Acad Sci USA.2002;99:12037-12042.
91. Greenblatt MB, Aliprantis A, Hu B, Glimcher LH. Calcineurin regulates innate antifungal immunity in neutrophils. J Exp Med.2010;207:923-931.
2. Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity:update on Toll-like receptors. Nat Immunol.2010;11:373-384.
3. Takeuchi O, Akira S. Pattern recognition receptors and inflammation. Cell.2010;140:805-820.
4. Akira S, Takeda K. Toll-like receptor signaling. Nat Rev Immunol.2004;4:499-511.
5. Yamamoto M, Sato S, Hemmi H, Hoshino K, Kaisho T, Sanjo H, Takeuchi O, Sugiyama M, Okabe M, Takeda K, Akira S. Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. Science.2003;301:640-643.
6. Bowie AG, Unterholzner L. Viral evasion and subversion of pattern-recognition receptor signalling. Nat Rev Immunol.2008;8:911-922.
7. Takeuchi O, Akira S. Innate immunity to virus infection. Immunol Rev.2009;227:75-86.
8. Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, Matsui K, Uematsu S, Jung A, Kawai T, Ishii KJ, Yamaguchi O, Otsu K, Tsujimura T, Koh CS, Reis e Sousa C, Matsuura Y, Fujita T, Akira S. Differential roles of MDA5 and RIG-Ⅰ helicases in the recognition of RNA viruses. Nature. 2006;441:101-105.
9. Kawai T, Takahashi K, Sato S, Coban C, Kumar H, Kato H, Ishii KJ, Takeuchi O, Akira S. IPS-1, an adaptor triggering RIG-Ⅰ- and Mda5-mediated type Ⅰ interferon induction. Nat Immunol. 2005;6:981-988.
10. Seth RB, Sun L, Ea CK, Chen ZJ. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3.Cell.2005;122:669-682.
11. Xu LG, Wang YY, Han KJ, Li LY, Zhai Z, Shu HB. VISA is an adapter protein required for virus-triggered IFN-beta signaling. Mol Cell.2005;19:727-740.
12. Meylan E, Curran J, Hofmann K, Moradpour D, Binder M, Bartenschlager R, Tschopp J. Cardif is an adaptor protein in the RIG-Ⅰ antiviral pathway and is targeted by hepatitis C virus. Nature. 2005;437:1167-1172.
13. Akira S, Takeda K, Kaisho T. Toll-like receptors:critical proteins linking innate and acquired immunity. Nat Immunol.2001;2:675-680.
14. Liew FY, Xu D, Brint EK, O'Neill LA. Negative regulation of toll-like receptor-mediated immune responses. Nat Rev Immunol.2005;5:446-458.
15. Luke A.J. O'Neill. When Signaling Pathways Collide:Positive and Negative Regulation of Toll-like Receptor Signal Transduction. Immunity.2008;29:12-20.
16. Gao T, Furnari F, Newton AC. PHLPP:a phosphatase that directly dephosphorylates Akt, promotes apoptosis, and suppresses tumor growth. Mol Cell.2005;18:13-24.
17. Shimizu K, Okada M, Takano A, Nagai K. SCOP, a novel gene product expressed in a circadian manner in rat suprachiasmatic nucleus. FEBS Lett.1999;458:363-369.
18. Shimizu K, Okada M, Nagai K, Fukada Y. Suprachiasmatic nucleus circadian oscillatory protein, a Novel binding Partner of K-Ras in the Membrane Rafts, Negatively Regulates MAPK Pathway. J Biol Chem.2003;278:14920-14925.
19. Shimizu K, Mackenziebc SM, Storm DR. SCOP/PHLPP and its functional role in the brain. Mol BioSyst.2010;6:38-43.
20. Shimizu K, Phan T, Mansuy IM, Storm DR. Proteolytic degradation of SCOP in the hippocampus contributes to activation of MAP kinase and memory. Cell.2007;128:1219-1229.
21. Gao T, Brognard J, and Newton AC. The phosphatase PHLPP controls the cellular levels of protein kinase C. J Biol Chem.2008;283:6300-6311.
22. Karen M. Doody, Annie Bourdeau, Michel L. Tremblay. T-cell protein tyrosine phosphatase is a key regulator in immune cell signaling:lessons from the knockout mouse model and implications in human disease. Immunol Rev.2009;228:325-341.
23. An H, Hou J, Zhou J, Zhao W, Xu H, Zheng Y, Yu Y, Liu S, Cao X. Phosphatase SHP-1 promotes TLR- and RIG-Ⅰ-activated production of type Ⅰ interferon by inhibiting the kinase IRAK1. Nat Immunol.2008;9:542-550.
24. An H, Zhao W, Hou J, Zhang Y, Xie Y, Zheng Y, Xu H, Qian C, Zhou J, Yu Y, Liu S, Feng G, Cao X. SHP-2 Phosphatase Negatively Regulates the TRIF Adaptor Protein-Dependent Type I Interferon and Proinflammatory Cytokine Production. Immunity.2006;25:919-928.
25. An H, Xu H, Zhang M, Zhou J, Feng T, Qian C, Qi R, Cao X. Src homology 2 domain-containing inositol-5-phosphatase 1 (SHIP1) negatively regulates TLR4-mediated LPS response primarily through a phosphatase activity-and PI-3K-independent mechanism. Blood.2005;105:4685-4692.
26. Leavy O. MKP1:A dynamic immune regulator. Nat Rev Immunol.2006;6:170-171.
27. Dong C, Davis RJ, Flavell RA. MAP kinases in immune response. Annu Rev Immunol. 2002;20:55-72.
28. Smith H, Peggie M, Campbell DG, Vandermoere F, Carrick E, Cohen P. Identification of the phosphorylation sites on the E3 ubiquitin ligase Pellino that are critical for activation by IRAKI and IRAK4. Proc Natl Acad Sci USA.2009;106:4584-4590.
29. Huang G, Shi LZ, Chi H. Regulation of JNK and p38 MAPK in the immune system:signal integration, propagation and termination. Cytokine.2009;48:161-169.
30. Noyce RS, Collins SE, Mossman KL. Differential modification of interferon regulatory factor 3 following virus particle entry. J Virol.2009;83:4013-4022.
31. Ding S, Wu X, Li G, Han M, Zhuang Y, Xu T. Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice. Cell.2005;122:473-483.
32. Salojin KV, Owusu IB, Millerchip KA, Potter M, Platt KA, Oravecz T. Essential role of MAPK phosphatase-1 in the negative control of innate immune responses. J Immunol. 2006;176:1899-1907.
33. Gabhann JN, Higgs R, Brennan K, Thomas W, Damen JE, Ben Larbi N, Krystal G, Jefferies CA. Absence of SHIP-1 results in constitutive phosphorylation of Tank-binding kinase 1 and enhanced TLR3-dependent IFN-b production. J Immunol.2010;184:2314-2320.
34. Abu-Dayyeh I, Shio MT, Sato S, Akira S, Cousineau B, Olivier M. Leishmania-induced IRAK-1 inactivation is mediated by SHP-1 interacting with an evolutionarily conserved KTIM motif. PLoS Negl Trop Dis.2008;2:e305.
35. Zhou D, Collins CA, Wu P, Brown EJ. Protein tyrosine phosphatase SHP-1 positively regulates TLR-induced IL-12p40 production in macrophages through inhibition of phosphatidylinositol 3-kinase. J Leukoc Biol.2010;87:845-855.
36. Yoneyama M, Suhara W, Fujita T. Control of IRF-3 activation by phosphorylation. J Interferon Cytokine Res.2002;22:73-76.
37. McWhirter SM, Fitzgerald KA, Rosains J, Rowe DC, Golenbock DT, Maniatis T. IFN-regulatory factor 3-dependent gene expression is defective in Tbkl-deficient mouse embryonic fibroblasts. Proc Natl Acad Sci USA.2004;101:233-238.
38. Saitoh T, Tun-Kyi A, Ryo A, Yamamoto M, Finn G, Fujita T, Akira S, Yamamoto N, Lu KP, Yamaoka S. Negative regulation of interferon-regulatory factor 3-dependent innate antiviral response by the prolyl isomerase Pin1. Nat Immunol.2006;7:598-605.
39. Honda K, Taniguchi T. IRFs:master regulators of signalling by Toll-like receptors and cytosolic pattern-recognition receptors. Nat Rev Immunol.2006;6:644-658.
40. Ojaniemi M, Glumoff V, Harju K, Liljeroos M, Vuori K, Hallman M. Phosphatidylinositol 3-kinase is involved in Toll-like receptor 4-mediated cytokine expression in mouse macrophages. Eur J Immunol.2003;33:597-605.
41. Sarkar SN, Peters KL, Elco CP, Sakamoto S, Pal S, Sen GC. Novel roles of TLR3 tyrosine phosphorylation and PI3 kinase in double-stranded RNA signaling. Nat Struct Mol Biol. 2004;11:1060-1067.
42. Eskan MA, Rose BG, Benakanakere MR, Lee MJ, Kinane DF. Sphingosine 1-phosphate 1 and TLR4 mediate IFN-beta expression in human gingival epithelial cells. J Immunol. 2008;180:1818-1825.
43. Nistal-Villan E, Gack MU, Martinez-Delgado G, Maharaj NP, Inn K, Yang H, Wang R, Aggarwal AK, Jung JU, Garcia-Sastre A. Negative role of RIG-Ⅰ serine 8 phosphorylation in the regulation of IFN-{beta} production. J Biol Chem.2010 Apr 20. [Epub ahead of print]
44. Sharma S, BR tenOever, N Grandvaux, GP Zhou, R Lin, J Hiscott. Triggering the interferon antiviral response through an IKK-related pathway. Science.2003;300:1148-1151.
45. Fitzgerald KA, SM McWhirter, KL Faia, DC Rowe, E Latz, DT Golenbock, AJ Coyle, SM Liao, T Maniatis. IKK- and TBK1 are essential components of the IRF3 signaling pathway. Nat Immunol. 2003;4:491-496.
46. Foy E, Li K, Wang C, Sumpter R Jr, Ikeda M, Lemon SM, Gale M Jr. Regulation of interferon regulatory factor-3 by the hepatitis C virus serine protease. Science 2003;300:1145-1148.
47. Li K, Foy E, Ferreon JC, Nakamura M, Ferreon AC, Ikeda M, Ray SC, Gale M Jr, Lemon SM. Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF. Proc Natl Acad Sci USA.2005;102:2992-2997.
48. Li XD, Sun L, Seth RB, Pineda G, Chen ZJ. Hepatitis C virus protease NS3/4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity. Proc Natl Acad Sci USA.2005;102(49):17717-17722.
49. Barro M, Patton JT. Rotavirus nonstructural protein 1 subverts innate immune response by inducing degradation of IFN regulatory factor 3. Proc Natl Acad Sci USA.2005;102:4114-4119.
50. Chen Z, Rijnbrand R, Jangra RK, Devaraj SG, Qu L, Ma Y, Lemon SM, Li K.Ubiquitination and proteasomal degradation of interferon regulatory factor-3 induced by Npro from a cytopathic bovine viral diarrhea virus. Virology.2007;366:277-292.
51. Bibeau-Poirier A, Gravel SP, Clement JF, Rolland S, Rodier G, Coulombe P, Hiscott J, Grandvaux N, Meloche S, Servant MJ. Involvement of the IkappaB kinase (IKK)-related kinases tank-binding kinase 1/IKKi and cullin-based ubiquitin ligases in IFN regulatory factor-3 degradation. J Immunol. 2006;177:5059-5067.
52. Zhang J, Xu LG, Han KJ, Wei X, Shu HB. PIASy represses TRIF-induced ISRE and NF-kappaB activation but not apoptosis. FEBS Lett. 2004;570:97-101.
53. Kubota T, Matsuoka M, Chang TH, Tailor P, Sasaki T, Tashiro M, Kato A, Ozato K. Virus infection triggers SUMOylation of IRF3 and IRF7, leading to the negative regulation of type Ⅰ interferon gene expression. J Biol Chem.2008;283:25660-25670.
54. Stetson DB, Medzhitov R. Type Ⅰ interferons in host defense. Immunity.2006;25:373-381.
55. Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, Miyagishi M, Taira K, Akira S, Fujita T. The RNA helicase RIG-Ⅰ has an essential function in double-stranded RNA-induced innate antiviral responses. Nat Immunol.2004;5:730-737.
1. Shi Y. Serine/Threonine Phosphatases:Mechanism through Structure. Cell.2009;139:468-484.
2. Mumby MC, Walter G. Protein Serine/Threonine Phosphatases:Structure, Regulation, and Functions in Cell Growth. Physiol Rev.1993;73:673-699.
3. Zhang Z. Protein Tyrosine Phosphatase:Structure and Function, Substrate Specificity, and Inhibitor Development. Annu Rev Pharmacol Toxicol.2002;42:209-234.
4. Camps M, Nichols A, Arkinstall S. Dual specificity phosphatases:a gene family for control of MAP kinase function. FASEB J.2000;14:6-16.
5. Maehama T, Okahara F, Kanaho Y. The tumour suppressor PTEN:involvement of a tumour suppressor candidate protein in PTEN turnover. Biochem Soc Trans.2004;32:343-347.
6. Baumer N, Maurer A, Krieglstein J, Klumpp S. Expression of Protein Histidine Phosphatase in Escherichia coli, Purification, and Determination of Enzyme Activity. Methods Mol Biol. 2006;365:247-260.
7. Seger R, Krebs EG. The MAPK Signalling cascade. FASEB J.1995;9:726-735.
8. Ladbury JE. Measurement of the formation of complexes in tyrosine kinase-mediated signal transduction.Acta Crystallogr D Biol Crystallogr.2007;63:26-31.
9. Doody KM, Bourdeau A, Tremblay ML. T-cell protein tyrosine phosphatase is a key regulator in immune cell signaling:lessons from the knockout mouse model and implications in human disease. Immunol Rev.2009;28:325-341.
10. O'Neill LA. When Signaling Pathways Collide:Positive and Negative Regulation of Toll-like Receptor Signal Transduction. Immunity.2008;29:12-20.
11. Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell. 2006;124:783-801.
12. Akira S, Takeda K. Toll-like receptor signaling. Nat Rev Immunol.2004;4:499-511.
13. Takeuchi O, Akira S. Innate immunity to virus infection. Immunol Rev.2009;227:75-86.
14. Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, Matsui K, Uematsu S, Jung A, Kawai T, Ishii KJ, Yamaguchi O, Otsu K, Tsujimura T, Koh CS, Reis e Sousa C, Matsuura Y, Fujita T, Akira S. Differential roles of MDA5 and RIG-Ⅰ helicases in the recognition of RNA viruses. Nature. 2006;441:101-105.
15. Akira S, Takeda K, Kaisho T. Toll-like receptors:critical proteins linking innate and acquired immunity. Nat Immunol.2001;2:675-680.
16. Liew FY, Xu D, Brint EK, O'Neill LA. Negative regulation of toll-like receptor-mediated immune responses. Nat Rev Immunol.2005;5:446-458.
17. Kyriakis JM, Avruch J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by. stress and inflammation. Physiol Rev.2001;81:807-869.
18. Dong C, Davis RJ, Flavell RA. MAP kinases in immune response. Annu Rev Immunol. 2002;20:55-72.
19. Dickinson RJ, Keyse SM. Diverse physiological functions for dual-specificity MAP kinase phosphatases. J Cell Sci.2006;119:4607-4615.
20. Owens DM, Keyse SM. Differential regulation of MAP kinase signalling by dual-specificity protein phosphatases. Oncogene.2007;26:3203-3213.
21. Chi H, Barry SP, Roth RJ, Wu JJ, Jones EA, Bennett AM, Flavell RA. Dynamic regulation of pro-and anti-inflammatory cytokines by MAPK phosphatase 1 (MKP-1) in innate immune responses. Proc Natl Acad Sci USA.2006;103:2274-2279.
22. Salojin KV, Owusu IB, Millerchip KA, Potter M, Platt KA, Oravecz T. Essential role of MAPK phosphatase-1 in the negative control of innate immune responses. J Immunol. 2006;176:1899-1907.
23. Zhao Q, Wang X, Nelin LD, Yao Y, Matta R, Manson ME, Baliga RS, Meng X, Smith CV, Bauer JA, Chang CH, Liu Y. MAP kinase phosphatase 1 controls innate immune responses and suppresses endotoxic shock. J Exp Med.2006;203:131-140.
24. Hammer M, Mages J, Dietrich H, Servatius A, Howells N, Cato AC, Lang R. Dual specificity phosphatase 1 (DUSP1) regulates a subset of LPS-induced genes and protects mice from lethal endotoxin shock. J Exp Med.2006;203:15-20.
25. Leavy O. MKP1:A dynamic immune regulator. Nat Rev Immunol.2006;6:170-171.
26. March ME, Ravichandran K. Regulation of the immune response by SHIP. Semin Immunol. 2002;14:37-47.
27. Leung W, Tarasenko T, Bolland S. Differential roles for the inositol phosphatase SHIP in the regulation of macrophages and lymphocytes. Immunol Res.2009;43:243-251.
28. Helgason CD, Damen JE, Rosten P, Grewal R, Sorensen P, Chappel SM, Borowski A, Jirik F, Krystal G, Humphries RK. Targeted disruption of SHIP leads to hemopoietic perturbations, lung pathology, and a shortened life span. Genes Dev.1998;12:1610-1620.
29. Liu Q, Sasaki T, Kozieradzki I, Wakeham A, Itie A, Dumont DJ, Penninger JM. SHIP is a negative regulator of growth factor receptor-mediated PKB/Akt activation andmyeloid cell survival. Genes Dev.1999;13:786-791.
30. Sly LM, Rauh MJ, Kalesnikoff J, Song CH, Krystal G. LPS-induced upregulation of sHIP is essential for endotoxin tolerance. Immunity.2004;21:227-239.
31. Hayashia T, Graya CS, Chana M, Tawataoa RI, Ronacherb L, McGargillc MA, Dattad SK, Carsona DA, Corrb M. Prevention of autoimmune disease by induction of tolerance to Toll-like receptor 7. Proc Natl Acad Sci USA.2009;106:2764-2769.
32. An H, Xu H, Zhang M, Zhou J, Feng T, Qian C, Qi R, Cao X. Src homology 2 domain-containing inositol-5-phosphatase 1 (SHIP1) negatively regulates TLR4-mediated LPS response primarily through a phosphatase activity-and PI-3K-independent mechanism. Blood.2005;105:4685-4692.
33. Gabhann JN, Higgs R, Brennan K, Thomas W, Damen JE, Ben Larbi N, Krystal G, Jefferies CA. Absence of SHIP-1 Results in Constitutive Phosphorylation of Tank-Binding Kinase 1 and Enhanced TLR3-Dependent IFN-b Production. J Immunol.2010;184:2314-2320.
34. Khaled AR, Butfiloski EJ, Sobel ES, Schiffenbauer J. Functional consequences of the SHP-1 defect in motheaten viable mice:role of NF-kB. Cell Immunol.1998;185:49-58.
35. Nandan D, Lo R, Reiner NE. Activation of phosphotyrosine phosphatase activity attenuates mitogen-activated protein kinase signaling and inhibits c-FOS and nitric oxide synthase expression in macrophages infected with Leishmania donovani. Infect Immun.1999;67:4055-4063.
36. Zhang Z, Jimi E, Bothwell AL. Receptor activator of NF-kB ligand stimulates recruitment of SHP-1 to the complex containing TNFR-associated factor 6 that regulates osteoclastogenesis. J Immunol.2003;171:3620-3626.
37. An H, Hou J, Zhou J, Zhao W, Xu H, Zheng Y, Yu Y, Liu S, Cao X. Phosphatase SHP-1 promotes TLR- and RIG-Ⅰ-activated production of type Ⅰ interferon by inhibiting the kinase IRAK1. Nat Immunol.2008;9:542-550.
38. Abu-Dayyeh I, Shio MT, Sato S, Akira S, Cousineau B, Olivier M. Leishmania-induced IRAK-1 inactivation is mediated by SHP-1 interacting with an evolutionarily conserved KTIM motif. PLoS Negl Trop Dis.2008;2:e305.
39. Zhou D, Collins CA, Wu P, Brown EJ. Protein tyrosine phosphatase SHP-1 positively regulates TLR-induced IL-12p40 production in macrophages through inhibition of phosphatidylinositol 3-kinase. J Leukoc Biol.2010;87:845-855.
40. Salmond RJ, Alexander DR. SHP2 forecast for the immune system:fog gradually clearing. Trends Immunol.2006;27:154-1560.
41. Wang N, Li Z, Ding R, Frank GD, Senbonmatsu T, Landon EJ, Inagami T, Zhao ZJ. Antagonism or synergism. Role of tyrosine phosphatases SHP-1 and SHP-2 in growth factor signaling. J Biol Chem.2006;281:21878-21883.
42. Neel BG, Gu H, Pao L. The'Shp'ing news:SH2 domain-containing tyrosine phosphatases in cell signaling. Trends Biochem Sci.2003;28:284-293.
43. Frearson JA, Alexander DR. The phosphotyrosine phosphatase SHP-2 participates in a multimeric signaling complex and regulates T cell receptor (TCR) coupling to the Ras/mitogen-activated protein kinase (MAPK) pathway in Jurkat T cells. J Exp Med.1998;187:1417-1426.
44. Qu CK, Nguyen S, Chen J, Feng GS. Requirement of Shp-2 tyrosine phosphatase in lymphoid and hematopoietic cell development. Blood.2001;97:911-914.
45. Qu CK. Role of the SHP-2 tyrosine phosphatase in cytokineinduced signaling and cellular response. Biochim Biophys Acta.2002;1592:297-301.
46. Wang Q, Downey GP, Herrera-Abreu MT, Kapus A, McCulloch CA. SHP-2 modulates interleukin-1-induced Ca2+ flux and ERK activation via phosphorylation of phospholipase Cgamma1. J Biol Chem.2005;280:8397-8406.
47. Gadina M, Stancato LM, Bacon CM, Larner AC, O'Shea JJ. Involvement of SHP-2 in multiple aspects of IL-2 signaling:evidence for a positive regulatory role. J Immunol.1998;160:4657-4661.
48. Lundin Brockdorff J, Woetmann A, Mustelin T, Kaltoft K, Zhang Q, Wasik MA, Ropke C,Φdum N. SHP2 regulates IL-2 induced MAPK activation, but not Stat3 or Stat5 tyrosine phosphorylation, in cutaneous T cell lymphoma cells. Cytokine.2002;20:141-147.
49. Salmond RJ, Huyer G, Kotsoni A, Clements L, Alexander DR. The src homology 2 domain-containing tyrosine phosphatase 2 regulates primary T-dependent immune responses and Th cell differentiation. J Immunol.2005;175:6498-6508.
50. Wheadon H, Edmead C, Welham MJ. Regulation of interleukin-3-induced substrate phosphorylation and cell survival by SHP-2 (Src-homology protein tyrosine phosphatase 2). Biochem J.2003;376:147-157.
51. Yu WM, Hawley TS, Hawley RG, Qu CK. Catalytic-dependent and-independent roles of SHP-2 tyrosine phosphatase in interleukin-3 signaling. Oncogene.2003;22:5995-6004.
52. Ohtani T, Ishihara K, Atsumi T, Nishida K, Kaneko Y, Miyata T, Itoh S, Narimatsu M, Maeda H, Fukada T, Itoh M, Okano H, Hibi M, Hirano T. Dissection of signaling cascades through gp130 in vivo:reciprocal roles for STAT3- and SHP2-mediated signals in immune responses. Immunity. 2000;12:95-105.
53. Gu H, Neel BG. The 'Gab' in signal transduction. Trends Cell Biol.2003;13:122-130.
54. Zhang SQ, Yang W, Kontaridis MI, Bivona TG, Wen G, Araki T, Luo J, Thompson JA, Schraven BL, Philips MR, Neel BG. Shp2 regulates Src family kinase activity and Ras/Erk activation by controlling Csk recruitment. Mol Cell.2004;13:341-355.
55. Yu CL, Jin YJ, Burakoff SJ. Cytosolic tyrosine dephosphorylation of STAT5:potential role of SHP-2 in STAT5 regulation. J Biol Chem.2000;275:599-604.
56. Chen Y, Wen R, Yang S, Schuman J, Zhang EE, Yi T, Feng GS, Wang D. Identification of Shp-2 as a Stat5A phosphatase. J Biol Chem.2003;278:16520-16527.
57. Lehmann U, Schmitz J, Weissenbach M, Sobota RM, Hortner M, Friederichs K, Behrmann I, Tsiaris W, Sasaki A, Schneider-Mergener J, Yoshimura A, Neel BG, Heinrich PC, Schaper F. SHP2 and SOCS3 contribute to Tyr-759-dependent attenuation of interleukin-6 signaling through gp130. J Biol Chem.2003;278:661-671.
58. Du Z, Shen Y, Yang W, Mecklenbrauker I, Neel BG, Ivashkiv LB. Inhibition of IFN-a signaling by a PKC- and protein tyrosine phosphatase SHP-2-dependent pathway. Proc Natl Acad Sci USA. 2005;102:10267-10272.
59. You M, Yu DH, Feng GS. Shp-2 tyrosine phosphatase functions as a negative regulator of the interferon-stimulated JAK/STAT pathway. Mol Cell Biol.1999; 19:2416-2424.
60. An H, Zhao W, Hou J, Zhang Y, Xie Y, Zheng Y, Xu H, Qian C, Zhou J, Yu Y, Liu S, Feng G, Cao X. SHP-2 phosphatase negatively regulates the TRIF adaptor protein-dependent type Ⅰ interferon and proinflammatory cytokine production. Immunity.2006;25:919-928.
61. Alberola-Ila J, Takaki S, Kerner JD, Perlmutter RM. Differential signaling by lymphocyte antigen receptors. Annu Rev Immunol.1997;15:125-154.
62. Healy JI, Goodnow CC. Positive versus negative signaling by lymphocyte antigen receptors. Annu Rev Immunol.1998;16:645-670.
63. van Oers NS. T cell receptor-mediated signs and signals governing T cell development. Semin Immunol.1999;11:227-237.
64. Campbell KS. Signal transduction from the B cell antigen-receptor. Curr Opin Immunol. 1999;11:256-264.
65. Green MC, Shultz LD. Motheaten, an immunodeficient mutant of the mouse. I. Genetics and pathology. J Hered.1975;66:250-258.
66. Bignon JS, Siminovitch KA. Identification of PTPIC mutation as the genetic defect in motheaten and viable motheaten mice:a step toward defining the roles of protein tyrosine phosphatases in the regulation of hemopoietic cell differentiation and function. Clin Immunol Immunopathol. 1994;73:168-179.
67. Koo GC, Rosen H, Sirotina A, Ma XD, Shultz LD. Anti-CD11b antibody prevents immunopathologic changes in viable moth-eaten bone marrow chimeric mice. J Immunol. 1993;151:6733-6741.
68. Pani G, Kozlowski M, Cambier JC, Mills GB, Siminovitch KA. Identification of the tyrosine phosphatase PTP1C as a B cell antigen receptor-associated protein involved in the regulation of B cell signaling. J Exp Med.1995;181:2077-2084.
69. Ono M, Okada H, Bolland S, Yanagi S, Kurosaki T, Ravetch JV. Deletion of SHIP or SHP-1 reveals two distinct pathways for inhibitory signaling. Cell.1997;90:293-301.
70. Nitschke L, Carsetti R, Ocker B, Kohler G, Lamers MC. CD22 is a negative regulator of B-cell receptor signalling. Curr Biol.1997;7:133-143.
71. Fujimoto M, Bradney AP, Poe JC, Steeber DA, Tedder TF. Modulation of B lymphocyte antigen receptor signal transduction by a CD19/CD22 regulatory loop. Immunity.1999;11:191-200.
72. Tsubata T. Co-receptors on B lymphocytes. Curr Opin Immunol.1999;11:249-255.
73. Maeda A, Kurosaki M, Ono M, Takai T, Kurosaki T. Requirement of SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 for paired immunoglobulin-like receptor B (PIR-B)-mediated inhibitory signal. J Exp Med.1998;187:1355-1360.
74. Dustin LB, Plas DR, Wong J, Hu YT, Soto C, Chan AC, Thomas ML. Expression of dominant-negative src-homology domain 2-containing protein tyrosine phosphatase-1 results in increased Syk tyrosine kinase activity and B cell activation. J Immunol.1999; 162:2717-2724.
75. Lorenz U, Ravichandran KS, Burakoff SJ, Neel BG. Lack of SHPTP1 results in src-family kinase hyperactivation and thymocyte hyperresponsiveness. Proc Natl Acad Sci USA.1996;93:9624-9629.
76. Tarakhovsky A, Kanner SB, Hombach J, Ledbetter JA, Muller W, Killeen N, Rajewsky K. A role for CD5 in TCR-mediated signal transduction and thymocyte selection. Science.1995;269:535-537.
77. Johnson KG, LeRoy FG, Borysiewicz LK, Matthews RJ. TCR signaling thresholds regulating T cell development and activation are dependent upon SHP-1. J Immunol.1999;162:3802-3813.
78. Zhang J, Somani AK, Watt S, Mills GB, Siminovitch KA. The Src-homology domain 2-bearing protein tyrosine phosphatase-1 inhibits antigen receptor-induced apoptosis of activated peripheral T cells. J Immunol.1999;162:6359-6367.
79. Cuevas B, Lu Y, Watt S, Kumar R, Zhang J, Siminovitch KA, Mills GB. SHP-1 regulates Lck-induced phosphatidylinositol 3-kinase phosphorylation and activity. J Biol Chem. 1999;274:27583-27589.
80. Bertone S, Schiavetti F, Bellomo R, Vitale C, Ponte M, Moretta L, Mingari MC. Transforming growth factor-beta-induced expression of CD94/NKG2A inhibitory receptors in human T lymphocytes. Eur J Immunol.1999;29:23-29.
81. Ashwell JD, D'Oro U. CD45 and Src-family kinases:and now for something completely different. Immunol Today.1999;20:412-416.
82. Thomas ML. The regulation of antigen-receptor signaling by protein tyrosine phosphatases:a hole in the story. Curr Opin Immunol.1999;11:270-276.
83. Leitenberg D, Boutin Y, Lu DD, Bottomly K. Biochemical association of CD45 with the T cell receptor complex:regulation by CD45 isoform and during T cell activation. Immunity. 1999;10:701-711.
84. D'Oro U, Ashwell JD. Cutting edge:the CD45 tyrosine phosphatase is an inhibitor of Lck activity in thymocytes. J Immunol.1999; 162:1879-1883.
85. Katagiri T, Ogimoto M, Hasegawa K, Arimura Y, Mitomo K, Okada M, Clark MR, Mizuno K, Yakura H. CD45 negatively regulates lyn activity by dephosphorylating both positive and negative regulatory tyrosine residues in immature B cells. J Immunol.1999;163:1321-1326.
86. Shibasaki F, Hallin U, Uchino H. Calcineurin as a multifunctional regulator. J Biochem. 2002;131:1-15.
87. Rao A, Luo C, Hogan PG. Transcription factors of the NFAT family:regulation and function. Annu Rev Immunol.1997;15:707-747.
88. Im SH, Rao A. Activation and deactivation of gene expression by Ca2+/calcineurin-NFAT-mediated signaling. Mol Cells.2004;18:1-9.
89. Kissinger CR, Parge HE, Knighton DR, Lewis CT, Pelletier LA, Tempczyk A, Kalish VJ, Tucker KD, Showalter RE, Moomaw EW, Gastinel LN, Habuka N, Chen X, Maldonado F, Barker JE, Bacquet R, Ernest Villafranca J. Crystal structures of human calcineurin and the human FKBP12- FK506-calcineurin complex. Nature.1995;378:641-644.
90. Huai Q, Kim HY, Liu Y, Zhao Y, Mondragon A, Liu JO, Ke H. Crystal structure of calcineurin-cyclophilin-cyclosporin shows common but distinct recognition of immunophilin-drug complexes. Proc Natl Acad Sci USA.2002;99:12037-12042.
91. Greenblatt MB, Aliprantis A, Hu B, Glimcher LH. Calcineurin regulates innate antifungal immunity in neutrophils. J Exp Med.2010;207:923-931.
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