Regulation of interleukin-3-induced substrate phosphorylation and cell survival by SHP-2 (Src-homology protein tyrosine phosphatase 2).
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.
Wheadon
Helen
H
Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK. m.j.welham@bath.ac.uk
Edmead
Christine
C
Welham
Melanie J
MJ
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D004268:DNA-Binding Proteins / Q000378:metabolism; D007377:Interleukin-3 / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008894:Milk Proteins; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009154:Mutation; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D054648:Protein Phosphatase 2; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D016185:Receptors, Interleukin-3 / Q000378:metabolism; D050799:STAT5 Transcription Factor; D015534:Trans-Activators / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; C490894:Gab2 protein, mouse; D007377:Interleukin-3; D047908:Intracellular Signaling Peptides and Proteins; D008894:Milk Proteins; D010750:Phosphoproteins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D016185:Receptors, Interleukin-3; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; C072185:Csf2rb2 protein, mouse; D014443:Tyrosine; D020928:Mitogen-Activated Protein Kinases; D054648:Protein Phosphatase 2; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases; C516725:Ptpn11 protein, mouse