Abstract
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.
Original language | English (US) |
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Pages (from-to) | 4940-4948 |
Number of pages | 9 |
Journal | EMBO Journal |
Volume | 15 |
Issue number | 18 |
DOIs | |
State | Published - Sep 16 1996 |
Externally published | Yes |
Keywords
- DNA synthesis
- PDGF receptor
- Ras
- Signalling
- Tyrosine kinase
ASJC Scopus subject areas
- General Neuroscience
- Molecular Biology
- General Biochemistry, Genetics and Molecular Biology
- General Immunology and Microbiology