Reduced phosphorylation of mitogen-activated protein kinase kinase in response to insulin in cells with truncated C-terminal domain of insulin receptor.
Insulin-stimulated activity of Raf-1 kinase was examined in Rat-1 fibroblasts transfected with wild-type and mutant human insulin receptors. Insulin stimulated Raf-1 binding to p21Ras in HIRc (wild-type), delta CT (insulin receptor lacking a 43-amino acid C-terminal domain), and Y/F2 (tyrosine 1316 and 1322 replaced by phenylalanine) cells. Despite equal binding to p21Ras, the activity of Raf-1 kinase (measured by phosphorylation of its downstream substrate, mitogen-activated protein/extracellular receptor kinase (MEK) was significantly reduced in the delta CT cells. As an association of Raf-1 with p21Ras does not activate Raf-1 kinase, but merely targets Raf-1 to the plasma membrane, we examined the binding of Raf-1 to 14-3-3 proteins and to the insulin receptor itself. Raf-1 was detected in both 14-3-3 and insulin receptor immunoprecipitates. Association of Raf-1 with either 14-3-3 protein or insulin receptor was not influenced by insulin and was similar in all control and insulin-treated cell lines. These results indicate that the delta CT cells are deficient in stimulating Raf-1 activity despite normal binding of Raf-1 to p21Ras. Thus, an unidentified mechanism of Raf-1 activation at the plasma membrane must be impaired in these cells.
Carel
K
K
Medical Research Service, Veterans Affairs Medical Center, Denver, Colorado 80220, USA.
DePaolo
D
D
Reusch
J E
JE
Leitner
J W
JW
Draznin
B
B
D016475:3T3 Cells; D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007163:Immunosorbent Techniques; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism*
D007328:Insulin; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D006160:Guanosine Triphosphate; D011494:Protein Kinases; D011972:Receptor, Insulin; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D020929:Mitogen-Activated Protein Kinase Kinases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)