The iSH2 domain of PI 3-kinase is a rigid tether for p110 and not a conformational switch

Zheng Fu, Eliah Aronoff-Spencer, Haiyan Wu, Gary J. Gerfen, Jonathan M. Backer

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Class IA PI 3-kinases are heterodimeric proteins with distinct catalytic (p110) and regulatory (p85) subunits. The minimal fragment of p85 capable of regulating p110 activity (p85ni) is the N-terminal SH2 domain linked to the iSH2 coiled-coil domain. We used cysteine mutagenesis and 14C-NEM- labeling to show that the p110-binding site in the iSH2 domain includes two regions: residues 482-484 and 532-541. These regions are adjacent to each other in the three-dimensional structural model of the iSH2 domain, and define a coherent binding site. We then used spin labeling and EPR spectroscopy to demonstrate that the conformation of the iSH2 domain is unaffected by binding to the N-terminal fragment of p110 (residues 1-108), and/or by phosphopeptide binding to p85ni/p110(1-108) heterodimers. Finally, we show that the cSH2 domain cannot substitute for the nSH2 domain with regard to inhibition of p110. These data support a model in which the iSH2 domain is a rigid tether for p110, and regulation of p85/p110 is mediated by nSH2-p110 contacts.

Original languageEnglish (US)
Pages (from-to)244-251
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume432
Issue number2
DOIs
StatePublished - Dec 15 2004

Fingerprint

Phosphatidylinositol 3-Kinases
Labeling
Binding Sites
Switches
Phosphopeptides
Mutagenesis
src Homology Domains
Structural Models
Cysteine
Paramagnetic resonance
Conformations
Spectrum Analysis
Spectroscopy
Proteins
IA 3

Keywords

  • Coiled-coil
  • iSH2 domain
  • p110
  • p85
  • Phosphoinositides
  • PI 3-kinase
  • SH2 domain

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

The iSH2 domain of PI 3-kinase is a rigid tether for p110 and not a conformational switch. / Fu, Zheng; Aronoff-Spencer, Eliah; Wu, Haiyan; Gerfen, Gary J.; Backer, Jonathan M.

In: Archives of Biochemistry and Biophysics, Vol. 432, No. 2, 15.12.2004, p. 244-251.

Research output: Contribution to journalArticle

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