Protein Kinase C-α and Protein Kinase C-ε Are Required for Grb2-associated Binder-1 Tyrosine Phosphorylation in Response to Platelet-derived Growth Factor

Yuji Saito,Yukihiro Hojo,Tatsuo Tanimoto,Jun-Ichi Abe,Bradford C Berk

doi:10.1074/jbc.m200605200

Yuji Saito, Yukihiro Hojo + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m200605200

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Abstract

Grb2-associated binder-1 (Gab1) is an adapter protein related to the insulin receptor substrate family. It is a substrate for the insulin receptor as well as the epidermal growth factor (EGF) receptor and other receptor-tyrosine kinases. To investigate the role of Gab1 in signaling pathways downstream of growth factor receptors, we stimulated rat aortic vascular smooth muscle cells (VSMC) with EGF and platelet-derived growth factor (PDGF). Gab1 was tyrosine-phosphorylated by EGF and PDGF within 1 min. AG1478 (an EGF receptor kinase-specific inhibitor) failed to block PDGF-induced Gab1 tyrosine phosphorylation, suggesting that transactivated EGF receptor is not responsible for this signaling event. Because Gab1 associates with phospholipase Cgamma (PLCgamma), we studied the role of the PLCgamma pathway in Gab1 tyrosine phosphorylation. Gab1 tyrosine phosphorylation by PDGF was impaired in Chinese hamster ovary cells expressing mutant PDGFbeta receptor (Y977F/Y989F: lacking the binding site for PLCgamma). Pretreatment of VSMC with (a specific PLCgamma inhibitor) inhibited Gab1 tyrosine phosphorylation as well, indicating the importance of the PLCgamma pathway. Gab1 was tyrosine-phosphorylated by phorbol ester to the same extent as PDGF stimulation. Studies using antisense protein kinase C (PKC) oligonucleotides and specific inhibitors showed that PKCalpha and PKCepsilon are required for Gab1 tyrosine phosphorylation. Binding of Gab1 to the protein-tyrosine phosphatase SHP2 and phosphatidylinositol 3-kinase was significantly decreased by PLCgamma and/or PKC inhibition, suggesting the importance of the PLCgamma/PKC-dependent Gab1 tyrosine phosphorylation for the interaction with other signaling molecules. Because PDGF-mediated ERK activation is enhanced in Chinese hamster ovary cells that overexpress Gab1, Gab1 serves as an important link between PKC and ERK activation by PDGFbeta receptors in VSMC.

Highlights

Grb2-associated binder-1 (Gab1) tyrosine phosphorylation was inhibited by the protein-tyrosine kinase inhibitor genistein, showing the imporsmooth muscle cells; PDGF, platelet-derived growth factor; PLC, phospholipase C; CHO, Chinese hamster ovary; PKC, protein kinase C; SH, Src homology; HGF, hepatocyte growth factor; IL, interleukin; PI3-K, phosphatidylinositol 3-kinase; WT, wild type; cPKC, conventional PKC; nPKC, novel PKC; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; phorbol 12-myristate acetate (PMA), phorbol 12-myristate 13-acetate; PDBu, phorbol 12,13-dibutyrate; DMEM, Dulbecco’s modified Eagle’s medium; HA, hemagglutinin; Protein-Tyrosine Kinases (PTK), protein-tyrosine kinase; EGFR, epidermal growth factor (EGF) receptor; PDGF␤R, PDGF␤ receptor
These results show that EGF receptor kinase activity is required for Gab1 tyrosine phosphorylation when vascular smooth muscle cells (VSMC) is stimulated by EGF, but transactivated EGF receptor kinase activity is not required when stimulated by PDGF
The major findings of this study are that Gab1 is tyrosinephosphorylated by PDGF via a pathway that requires PLC␥, PKC␣, and PKC⑀ activity and genistein-sensitive tyrosine kinases (Fig. 8)

Summary

EXPERIMENTAL PROCEDURES

Reagents—Reagents and other supplies were obtained from the following sources. Cell culture medium was from Invitrogen. Rabbit polyclonal anti-human PDGF␤ receptor, rabbit anti-PI3-K p85, and mouse monoclonal antiphosphotyrosine antibody (4G10) were from Upstate Biotechnology (Lake Placid, NY). VSMC of passage 8 to 14 at 70 – 80% confluence were growth-arrested by incubation in DMEM without serum for 48 h before use. After incubation in blocking solution (5% bovine serum albumin, phosphate-buffered saline, pH 7.5, 0.1% Tween 20), membranes were incubated with primary antibodies (1:1000 dilution in blocking solution) for 2 h at room temperature. After washing the membranes six times (5 min each) with washing buffer (phosphatebuffered saline, pH 7.5, 0.1% Tween 20), the blots were incubated with the appropriate secondary antibodies (1:5000 dilution in blocking solution) for 1 h at room temperature. After a 48-h incubation, cells were serum-starved for 24 h and stimulated with PDGF for 5 min. Significant differences (p Ͻ 0.01) were determined by Student’s t test

RESULTS

It has been reported that PKC is essential for erythropoietin

DISCUSSION