Threonine 1336 of the Human Insulin Receptor Is a Major Target for Phosphorylation by Protein Kinase C

Robert E. Lewis, David Perregaux, Michael P. Czech, Lin Cao

Research output: Contribution to journalArticle

67 Citations (Scopus)

Abstract

The ability of tumor-promoting phorbol diesters to inhibit both insulin receptor tyrosine kinase activity and its intracellular signaling correlates with the phosphorylation of the insulin receptor β subunit on serine and threonine residues. In the present studies, mouse 3T3 fibroblasts transfected with a human insulin receptor cDNA and expressing greater than one million of these receptors per cell were labeled with [32P] phosphate and treated with or without 100 nM 4β-phorbol 12β-myristate 13α-acetate (PMA). Phosphorylated insulin receptors were immunoprecipitated and digested with trypsin. Alternatively, insulin receptors affinity purified from human term placenta were phosphorylated by protein kinase C prior to trypsin digestion of the 32P-labeled β subunit. Analysis of the tryptic phosphopeptides from both the in vivo and in vitro labeled receptors by reversed-phase HPLC and two-dimensional thin-layer separation revealed that PMA and protein kinase C enhanced the phosphorylation of a peptide with identical chromatographic properties. Partial hydrolysis and radiosequence analysis of the phosphopeptide derived from insulin receptor phosphorylated by protein kinase C indicated that the phosphorylation of this tryptic peptide occurred specifically on a threonine, three amino acids from the amino terminus of the tryptic fragment. Comparison of these data with the known, deduced receptor sequence suggested that the receptor-derived tryptic phosphopeptide might be Ile-Leu-Thr(P)-Leu-Pro-Arg. Comigration of a phosphorylated synthetic peptide containing this sequence with the receptor-derived phosphopeptide confirmed the identity of the tryptic fragment. The phosphorylation site corresponds to threonine 1336 in the human insulin receptor β subunit. This threonine, which resides in a receptor domain also containing tyrosine phosphorylation sites, is located eight amino acids from the carboxyl terminus of the β subunit and may play a role in the protein kinase C induced inhibition of insulin receptor tyrosine kinase activity.

Original languageEnglish (US)
Pages (from-to)1807-1813
Number of pages7
JournalBiochemistry
Volume29
Issue number7
DOIs
StatePublished - Feb 1 1990
Externally publishedYes

Fingerprint

Phosphorylation
Threonine
Phosphopeptides
Protein Kinase C
Insulin Receptor
Trypsin
Peptides
leucylproline
Acetate Kinase
Amino Acids
Phorbol Esters
Fibroblasts
Serine
Placenta
Tyrosine
Tumors
Digestion
Hydrolysis
Acetates
Complementary DNA

ASJC Scopus subject areas

  • Biochemistry

Cite this

Threonine 1336 of the Human Insulin Receptor Is a Major Target for Phosphorylation by Protein Kinase C. / Lewis, Robert E.; Perregaux, David; Czech, Michael P.; Cao, Lin.

In: Biochemistry, Vol. 29, No. 7, 01.02.1990, p. 1807-1813.

Research output: Contribution to journalArticle

Lewis, Robert E. ; Perregaux, David ; Czech, Michael P. ; Cao, Lin. / Threonine 1336 of the Human Insulin Receptor Is a Major Target for Phosphorylation by Protein Kinase C. In: Biochemistry. 1990 ; Vol. 29, No. 7. pp. 1807-1813.
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