Reduced cell attachment and phosphorylation of focal adhesion kinase associated with expression of a mutant insulin receptor.
Insulin signaling results in rapid changes to the cell cytoskeleton, and it has recently been shown that insulin stimulates the dephosphorylation of the cytoskeletal-associated tyrosine kinase, focal adhesion kinase (pp125(FAK)). We report here that mutation of two tryptic cleavage sites (Lys164 and Lys582 --> Asn; 2N) in the insulin receptor alpha-subunit results in a cell-line (CHO.2N-10) with altered morphology associated with an increase in cell size, a decrease in cell adhesiveness, and a decrease in pp125(FAK) tyrosine phosphorylation in the absence of insulin (45.2 +/- 9.7% compared to nontransfected Chinese hamster ovary (CHO) cells). In contrast to pp125(FAK), paxillin phosphorylation was similar in all cell lines despite lower levels (61.0 +/- 10.4% compared to CHO cells) of paxillin protein in CHO.2N-10 cells. We observed comparable protein levels of pp125(FAK) and the structural focal adhesion protein, vinculin, in all cell lines. Despite underphosphorylation of pp125(FAK) in the basal state, insulin stimulation of CHO.2N-10 cells still resulted in dephosphorylation of pp125(FAK). CHO.2N-10 and CHO.T (overexpress wild-type insulin receptor) cells have similar insulin binding characteristics, insulin-stimulated autokinase and peptide phosphorylation, and insulin-stimulated pp185/IRS-1 phosphorylation. Our results suggest that the insulin receptor may play an important role in cell-matrix interactions, such as modulating cell adhesion and inducing cell architecture changes.
Konstantopoulos
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University of Melbourne, Department of Medicine, P. O. Royal Melbourne Hospital, Parkville 3050, Australia. stellac@deakin.edu.au
Clark
S
S
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion / Q000235:genetics*; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D006224:Cricetinae; D046508:Culture Techniques; D003598:Cytoskeletal Proteins / Q000378:metabolism; D018076:DNA, Complementary; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D007328:Insulin / Q000378:metabolism; D008969:Molecular Sequence Data; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000235:genetics* / Q000378:metabolism; D016596:Vinculin / Q000378:metabolism
D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D007328:Insulin; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; C496063:Pxn protein, rat; D016596:Vinculin; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; C496052:Ptk2 protein, rat