Requirement of phospholipase C gamma, the tyrosine phosphatase Syp and the adaptor proteins Shc and Nck for PDGF-induced DNA synthesis: evidence for the existence of Ras-dependent and Ras-independent pathways.
We have investigated the roles of the phosphotyrosine phosphatase Syp (also called SH-PTP2), phospholipase C (PLC) gamma1, rasGTPase Activating Protein (rasGAP) and the adapter molecules Nck and Shc in the mitogenic response induced by PDGF in fibroblasts. Two separate approaches were used to inhibit the biological activity of these signalling proteins in vivo. Either glutathione S-transferase (GST) fusion proteins containing the SH2 domains of these proteins, or antibodies specific for these polypeptides, were microinjected into cells. GST-SH2 fusion proteins are expected to act as dominant inhibitors by competing for physiological SH2-mediated interactions, while microinjected antibodies can directly block protein functions. Inhibition of PLCgamma, Syp, Shc and Nck signals blocked PDGF-stimulated cells in G1 showing a requirement for these proteins for S-phase entry. Inhibition of rasGAP, in contrast, had no effect on S-phase entry. We next examined which of these signals were required for PDGF-induced cFos expression, a Ras-dependent event important for signalling. By using the same approaches with cells expressing beta-galactosidase under the control of a c-fos promoter, we showed that PLCgamma, Syp and Shc were necessary for ligand-induced cFos expression whereas Nck and phosphatidylinositol 3-kinase alpha were not. From these results we concluded that PDGF generates Ras-dependent and Ras-independent pathways important for DNA synthesis.
Differentiation Programme, European Molecular Biology Laboratory, Heidelberg, Germany.
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Roche
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McGlade
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Jones
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Gish
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Pawson
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SA
Courtneidge
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D004261:DNA Replication / Q000187:drug effects*; D016193:G1 Phase; D020690:GTPase-Activating Proteins; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D016762:Genes, fos; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism*; D051379:Mice; D015513:Oncogene Proteins / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases; D051966:Phospholipase C gamma; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D016196:S Phase; D010738:Type C Phospholipases / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism; D020703:ras GTPase-Activating Proteins
D048868:Adaptor Proteins, Signal Transducing; D020690:GTPase-Activating Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; C078332:Nck protein; D015513:Oncogene Proteins; D010982:Platelet-Derived Growth Factor; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; C516725:Ptpn11 protein, mouse; C509248:Ptpn6 protein, mouse; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D001616:beta-Galactosidase