Evidence for functional roles of Crk-II in insulin and epidermal growth factor signaling in Rat-1 fibroblasts overexpressing insulin receptors.
We examined the potential role of Crk-II in insulin and epidermal growth factor (EGF) signaling in Rat-1 fibroblasts overexpressing insulin receptors. Crk is an SH2 and SH3 domain-containing adaptor protein that has been reported to associate with p130cas, paxillin, c-cbl, c-abl, Sos, and C3G in vitro. Insulin- and EGF-induced association of Crk-II with these molecules was assessed by immunoblotting of anti-Crk-II precipitates in Rat-1 fibroblasts overexpressing insulin receptors. Neither insulin nor EGF treatment induced Crk-II association with either Sos or C3G. Basal tyrosine phosphorylation of c-abl and its constitutive association with Crk-II were not further increased by insulin or EGF. p130cas and paxillin were heavily tyrosine phosphorylated in the basal state. Both insulin and EGF stimulated their dephosphorylation, followed by p130cas-Crk-II dissociation and paxillin-Crk-II association, although the magnitude of these effects was greater with insulin than with EGF. Interestingly, EGF, but not insulin, stimulated tyrosine phosphorylation of c-cbl and its association with Crk-II. To investigate the functional roles of Crk-II in mitogenesis and cytoskeletal rearrangement, we performed microinjection analysis. Cellular microinjection of anti-Crk-II antibody inhibited EGF-induced, but not insulin-induced, DNA synthesis. Insulin, but not EGF, stimulated cytoskeletal rearrangement in the cells, and microinjection of anti-Crk-II antibody effectively inhibited insulin-induced membrane ruffling, suggesting that Crk-II is involved in insulin-induced cytoskeletal rearrangement. These results indicate that Crk-II functions as a multifunctional adaptor molecule linking insulin and EGF receptors to their downstream signals. The presence of c-cbl-Crk-II association may partly determine the signal specificities initiated by insulin and EGF.
Ishiki
M
M
First Department of Medicine, Toyama Medical and Pharmaceutical University, Japan.
Sasaoka
T
T
Ishihara
H
H
Imamura
T
T
Usui
I
I
Takata
Y
Y
Kobayashi
M
M
D000818:Animals; D002460:Cell Line; D050719:Crk-Associated Substrate Protein; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000502:physiology*; D005347:Fibroblasts / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D007328:Insulin / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000502:physiology*; D016315:Proto-Oncogene Proteins c-abl / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl; D050737:Proto-Oncogene Proteins c-crk; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*; D050717:Retinoblastoma-Like Protein p130; D015398:Signal Transduction / Q000502:physiology*; D020664:Son of Sevenless Proteins; D013552:Swine; D044767:Ubiquitin-Protein Ligases
C493841:BCAR1 protein, human; C493843:Bcar1 protein, rat; C493872:Crk protein, rat; D050719:Crk-Associated Substrate Protein; D003598:Cytoskeletal Proteins; D020662:Guanine Nucleotide Exchange Factors; D007328:Insulin; D008565:Membrane Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; C496063:Pxn protein, rat; D050717:Retinoblastoma-Like Protein p130; D020664:Son of Sevenless Proteins; D004815:Epidermal Growth Factor; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D011972:Receptor, Insulin; D016315:Proto-Oncogene Proteins c-abl; C067419:CBL protein, human