Dynamic regulation of intact and C-terminal truncated insulin receptor phosphorylation in permeabilized cells

Biochemistry. 1994 Apr 12;33(14):4343-51. doi: 10.1021/bi00180a031.

Abstract

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)

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Blotting, Western
  • CHO Cells
  • Cell Membrane Permeability / drug effects
  • Cricetinae
  • Digitonin
  • Humans
  • Insulin Receptor Substrate Proteins
  • Molecular Sequence Data
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Receptor, Insulin / genetics
  • Receptor, Insulin / metabolism*
  • Recombinant Proteins / metabolism

Substances

  • IRS1 protein, human
  • Insulin Receptor Substrate Proteins
  • Phosphoproteins
  • Recombinant Proteins
  • Receptor, Insulin
  • Digitonin