TY - JOUR
T1 - Requirement of phospholipase Cγ, the tyrosine phosphatase Syp and the adaptor proteins Shc and Nck for PDGF-induced DNA synthesis
T2 - Evidence for the existence of Ras-dependent and Ras-independent pathways
AU - Roche, Serge
AU - McGlade, Jane
AU - Jones, Margaret
AU - Gish, Gerald D.
AU - Pawson, Tony
AU - Courtneidge, Sara A.
PY - 1996/9/16
Y1 - 1996/9/16
N2 - We have investigated the roles of the phosphotyrosine phosphatase Syp (also called SH-PTP2), phospholipase C (PLC) γ1, 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 PLCγ, 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 β-galactosidase under the control of a c-fos promoter, we showed that PLCγ, Syp and Shc were necessary for ligand-induced cFos expression whereas Nck and phosphatidylinositol 3-kinase α were not. From these results we concluded that PDGF generates Ras-dependent and Ras-independent pathways important for DNA synthesis.
AB - We have investigated the roles of the phosphotyrosine phosphatase Syp (also called SH-PTP2), phospholipase C (PLC) γ1, 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 PLCγ, 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 β-galactosidase under the control of a c-fos promoter, we showed that PLCγ, Syp and Shc were necessary for ligand-induced cFos expression whereas Nck and phosphatidylinositol 3-kinase α were not. From these results we concluded that PDGF generates Ras-dependent and Ras-independent pathways important for DNA synthesis.
KW - DNA synthesis
KW - PDGF receptor
KW - Ras
KW - Signalling
KW - Tyrosine kinase
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U2 - 10.1002/j.1460-2075.1996.tb00874.x
DO - 10.1002/j.1460-2075.1996.tb00874.x
M3 - Article
C2 - 8890167
AN - SCOPUS:0029814709
VL - 15
SP - 4940
EP - 4948
JO - EMBO Journal
JF - EMBO Journal
SN - 0261-4189
IS - 18
ER -