Dynamic regulation of intact and C-terminal truncated insulin receptor phosphorylation in permeabilized cells.
Using digitonin-permeabilized Chinese hamster ovary (CHO) cells that were transfected with intact human insulin receptors (CHO/HIRc cells), we examined insulin receptor phosphorylation and dephosphorylation using pulse-chase techniques. Insulin activated receptor autophosphorylation on tyrosyl residues to a level severalfold over basal, reaching maximal levels after 2, 5, and 10 min of stimulation at 34, 18, and 6 degrees C, respectively. Phosphopeptide analysis revealed that the triply phosphorylated form of the 1146-kinase domain of the insulin receptor was the major species, which is characteristic of the fully active tyrosine kinase function. The dephosphorylation reaction was time- and temperature-dependent with t1/2 values of 0.67 and 2 min at 18 and 6 degrees C, respectively. Vanadate completely inhibited dephosphorylation. Under similar permeabilization conditions when compared with CHO/HIRc cells, CHO/delta CT cells (CHO cells overexpressing a mutated form of the receptor with a 43 amino acid deletion at the C-terminus) stimulated with insulin exhibited larger increases in receptor autophosphorylation levels and in tyrosine kinase activity toward a synthetic peptide substrate; the rate of CHO/delta CT receptor dephosphorylation was not reduced. There was near-complete absence of insulin receptor substrate 1 (IRS-1) in the cell ghosts after permeabilization. We therefore examined the pattern of tyrosine phosphorylation and dephosphorylation of residual cellular proteins in permeabilized CHO/HIRc cells by Western blot analysis. In addition to the 95-kDa receptor beta-subunit, we detected the phosphorylation of two glycoproteins which included the commonly found 120-kDa protein and a novel 195-kDa protein whose dephosphorylation rate is slower than that of receptor beta-subunit.(ABSTRACT TRUNCATED AT 250 WORDS)
Bernier
M
M
Diabetes Unit, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224.
Liotta
A S
AS
Kole
H K
HK
Shock
D D
DD
Roth
J
J
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002463:Cell Membrane Permeability / Q000187:drug effects; D006224:Cricetinae; D004072:Digitonin; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D011972:Receptor, Insulin; D004072:Digitonin