Wheadon Helen, Edmead Christine, Welham Melanie J
Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK.
Biochem J. 2003 Nov 15;376(Pt 1):147-57. doi: 10.1042/BJ20031160.
The cytosolic SHP-2 (Src homology protein tyrosine phosphatase 2) has previously been implicated in IL-3 (interleukin-3) signalling [Bone, Dechert, Jirik, Schrader and Welham (1997) J. Biol. Chem. 272, 14470 -14476; Craddock and Welham (1997) J. Biol. Chem. 272, 29281-29289; Welham, Dechert, Leslie, Jirik and Schrader (1994) J. Biol. Chem. 269, 23764-23768; Qu, Nguyen, Chen and Feng (2001) Blood 97, 911-914]. To investigate the role of SHP-2 in IL-3 signalling in greater detail, we have inducibly expressed WT (wild-type) or two potentially substrate-trapping mutant forms of SHP-2, generated by mutation of Asp-425 to Ala (D425A) or Cyst-459 to Ser (C459S), in IL-3-dependent BaF/3 cells. Effects on IL-3-induced tyrosine phosphorylation, signal transduction and functional responses were examined. Expression of C459S SHP-2 protected the beta-chain of the murine IL-3R (IL-3 receptor), the adaptor protein Gab2 (Grb2-associated binder 2), and a cytosolic protein of 48 kDa from tyrosine dephosphorylation, consistent with them being bona fide substrates of SHP-2 in IL-3 signalling. The tyrosine phosphorylation of a 135 kDa transmembrane protein was also protected upon expression of C459S SHP-2. We have identified the inhibitory immunoreceptor PECAM-1 (platelet endothelial cell adhesion molecule-1)/CD31 (cluster determinant 31) as a component of this 135 kDa substrate and also show that IL-3 can induce tyrosine phosphorylation of PECAM-1. Expression of WT, C459S and D425A forms of SHP-2 had little effect on IL-3-driven proliferation or STAT5 (signal transduction and activators of transcription) phosphorylation or activation of protein kinase B. However, expression of WT SHP-2 increased ERK (extracellular-signal-regulated kinase) activation. Interestingly, expression of C459S SHP-2 decreased ERK activation at later times after IL-3 stimulation, but potentiated IL-3-induced activation of Jun N-terminal kinases. In addition, expression of C459S SHP-2 decreased cell survival in suboptimal IL-3 and upon IL-3 withdrawal. These findings indicate that SHP-2 plays an important role in mediating the anti-apoptotic effect of IL-3 and raises the possibility that PECAM-1 participates in the modulation of cytokine-induced signals.
胞质中的SHP-2（Src同源蛋白酪氨酸磷酸酶2）先前已被证明参与白细胞介素-3（IL-3）信号传导[Bone、Dechert、Jirik、Schrader和Welham（1997年）《生物化学杂志》272卷，第14470 - 14476页；Craddock和Welham（1997年）《生物化学杂志》272卷，第29281 - 29289页；Welham、Dechert、Leslie、Jirik和Schrader（1994年）《生物化学杂志》269卷，第23764 - 23768页；Qu、Nguyen、Chen和Feng（2001年）《血液》97卷，第911 - 914页]。为了更详细地研究SHP-2在IL-3信号传导中的作用，我们在依赖IL-3的BaF/3细胞中诱导表达了野生型（WT）或通过将天冬氨酸-425突变为丙氨酸（D425A）或将半胱氨酸-459突变为丝氨酸（C459S）产生的两种潜在底物捕获突变形式的SHP-2。检测了其对IL-3诱导的酪氨酸磷酸化、信号转导和功能反应的影响。C459S SHP-2的表达保护了小鼠IL-3受体（IL-3R）的β链、衔接蛋白Gab2（Grb2相关结合蛋白2）以及一种48 kDa的胞质蛋白不被酪氨酸去磷酸化，这与它们是IL-3信号传导中SHP-2的真正底物一致。C459S SHP-2表达后，一种135 kDa跨膜蛋白的酪氨酸磷酸化也受到保护。我们已确定抑制性免疫受体PECAM-1（血小板内皮细胞黏附分子-1）/CD31（簇分化抗原31）是这种135 kDa底物的一个组成部分，并且还表明IL-3可诱导PECAM-1的酪氨酸磷酸化。野生型、C459S和D425A形式的SHP-2的表达对IL-3驱动的增殖、信号转导和转录激活因子5（STAT5）磷酸化或蛋白激酶B的激活影响很小。然而，野生型SHP-2的表达增加了细胞外信号调节激酶（ERK）的激活。有趣的是，C459S SHP-2的表达在IL-3刺激后的后期降低了ERK的激活，但增强了IL-3诱导的Jun N末端激酶的激活。此外，C459S SHP-2的表达降低了在次优IL-3条件下以及IL-3撤除后的细胞存活率。这些发现表明SHP-2在介导IL-3的抗凋亡作用中起重要作用，并增加了PECAM-1参与细胞因子诱导信号调节的可能性。
