Regulation of phosphatidylinositol 3′-kinase by tyrosyl phosphoproteins

Full activation requires occupancy of both SH2 domains in the 85-kDa regulatory subunit

Tamara Rordorf-Nikolic, Debra J. Van Horn, Daxin Chen, Morris F. White, Jonathan M. Backer

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Abstract

Phosphatidylinositol 3′-kinase (PI 3′-kinase) is activated in insulin-stimulated cells by the binding of the SH2 domains in its 85-kDa regulatory subunit to insulin receptor substrate-1 (IRS-1). We have previously shown that both tyrosyl-phosphorylated IRS-1 and mono-phosphopeptides containing a single YXXM motif activate PI 3′-kinase in vitro. However, activation by the monophosphopeptides was significantly less potent than activation by the multiply phosphorylated IRS-1. We now show that the increased potency of PI 3′-kinase activation by IRS-1 relative to phosphopeptide is not due to tertiary structural features IRS-1, as PI 3′-kinase is activated normally by denatured, reduced, and carboxymethylated IRS-1. Furthermore, activation of PI 3′-kinase by bis-phosphorylated peptides containing two YXXM motifs is 100-fold more potent than the corresponding mono-phosphopeptides and similar to activation by IRS-1. These data suggest that tyrosyl-phosphorylated IRS-1 or bis-phosphorylated peptides bind simultaneously to both SH2 domains of p85. However, these data cannot differentiate between an activation mechanism that requires two-site occupancy for maximal activity as opposed to one in which bivalent binding enhances the occupancy of a single activating site. To distinguish between these possibilities, we produced recombinant PI 3′-kinase containing either wild-type p85 or p85 mutated in its N-terminal, C-terminal, or both SH2 domains. We find that mutation of either SH2 domains significantly reduced phosphopeptide binding and decreased PI 3′-kinase activation by 50%, whereas mutation of both SH2 domains completely blocked binding and activation. These data provide the first direct evidence that full activation of PI 3′-kinase by tyrosyl-phosphorylated proteins requires occupancy of both SH2 domains in p85.

Original languageEnglish (US)
Pages (from-to)3662-3666
Number of pages5
JournalJournal of Biological Chemistry
Volume270
Issue number8
StatePublished - Feb 24 1995

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Phosphatidylinositol 3-Kinase
Insulin Receptor Substrate Proteins
src Homology Domains
Phosphoproteins
Chemical activation
Phosphopeptides
Peptides
Mutation
Insulin

ASJC Scopus subject areas

  • Biochemistry

Cite this

Regulation of phosphatidylinositol 3′-kinase by tyrosyl phosphoproteins : Full activation requires occupancy of both SH2 domains in the 85-kDa regulatory subunit. / Rordorf-Nikolic, Tamara; Van Horn, Debra J.; Chen, Daxin; White, Morris F.; Backer, Jonathan M.

In: Journal of Biological Chemistry, Vol. 270, No. 8, 24.02.1995, p. 3662-3666.

Research output: Contribution to journalArticle

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abstract = "Phosphatidylinositol 3′-kinase (PI 3′-kinase) is activated in insulin-stimulated cells by the binding of the SH2 domains in its 85-kDa regulatory subunit to insulin receptor substrate-1 (IRS-1). We have previously shown that both tyrosyl-phosphorylated IRS-1 and mono-phosphopeptides containing a single YXXM motif activate PI 3′-kinase in vitro. However, activation by the monophosphopeptides was significantly less potent than activation by the multiply phosphorylated IRS-1. We now show that the increased potency of PI 3′-kinase activation by IRS-1 relative to phosphopeptide is not due to tertiary structural features IRS-1, as PI 3′-kinase is activated normally by denatured, reduced, and carboxymethylated IRS-1. Furthermore, activation of PI 3′-kinase by bis-phosphorylated peptides containing two YXXM motifs is 100-fold more potent than the corresponding mono-phosphopeptides and similar to activation by IRS-1. These data suggest that tyrosyl-phosphorylated IRS-1 or bis-phosphorylated peptides bind simultaneously to both SH2 domains of p85. However, these data cannot differentiate between an activation mechanism that requires two-site occupancy for maximal activity as opposed to one in which bivalent binding enhances the occupancy of a single activating site. To distinguish between these possibilities, we produced recombinant PI 3′-kinase containing either wild-type p85 or p85 mutated in its N-terminal, C-terminal, or both SH2 domains. We find that mutation of either SH2 domains significantly reduced phosphopeptide binding and decreased PI 3′-kinase activation by 50{\%}, whereas mutation of both SH2 domains completely blocked binding and activation. These data provide the first direct evidence that full activation of PI 3′-kinase by tyrosyl-phosphorylated proteins requires occupancy of both SH2 domains in p85.",
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