The iSH2 domain of PI 3-kinase is a rigid tether for p110 and not a conformational switch.
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 (14)C-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.
Fu
Zheng
Z
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, USA.
Aronoff-Spencer
Eliah
E
Wu
Haiyan
H
Gerfen
Gary J
GJ
Backer
Jonathan M
JM
D019943:Amino Acid Substitution; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000737:chemistry*; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D013329:Structure-Activity Relationship; D018909:src Homology Domains
D002135:Calcium-Binding Proteins; C062748:NSP1 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D019869:Phosphatidylinositol 3-Kinases