Assembly and Molecular Architecture of the Phosphoinositide 3-Kinase p85α Homodimer

Jaclyn Lopiccolo,Seung Joong Kim,Yi Shi,Bin Wu,Haiyan Wu,Brian T Chait,Robert H Singer,Andrej Sali,Michael Brenowitz,Anne R Bresnick,Jonathan M Backer

doi:10.1074/jbc.m115.689604

Abstract

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are activated by growth factor and G-protein-coupled receptors and propagate intracellular signals for growth, survival, proliferation, and metabolism. p85α, a modular protein consisting of five domains, binds and inhibits the enzymatic activity of class IA PI3K catalytic subunits. Here, we describe the structural states of the p85α dimer, based on data from in vivo and in vitro solution characterization. Our in vitro assembly and structural analyses have been enabled by the creation of cysteine-free p85α that is functionally equivalent to native p85α. Analytical ultracentrifugation studies showed that p85α undergoes rapidly reversible monomer-dimer assembly that is highly exothermic in nature. In addition to the documented SH3-PR1 dimerization interaction, we identified a second intermolecular interaction mediated by cSH2 domains at the C-terminal end of the polypeptide. We have demonstrated in vivo concentration-dependent dimerization of p85α using fluorescence fluctuation spectroscopy. Finally, we have defined solution conditions under which the protein is predominantly monomeric or dimeric, providing the basis for small angle x-ray scattering and chemical cross-linking structural analysis of the discrete dimer. These experimental data have been used for the integrative structure determination of the p85α dimer. Our study provides new insight into the structure and assembly of the p85α homodimer and suggests that this protein is a highly dynamic molecule whose conformational flexibility allows it to transiently associate with multiple binding proteins.

Highlights

The class IA PI 3-kinase regulatory subunit p85␣ dimerizes by mechanisms not fully understood
Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are activated by growth factor and G-protein-coupled receptors and propagate intracellular signals for growth, survival, proliferation, and metabolism. p85␣, a modular protein consisting of five domains, binds and inhibits the enzymatic activity of class IA PI3K catalytic subunits
We mutated each of the six cysteines in p85␣ to serine, leucine, or valine (Fig. 1A), depending on whether the cysteines were predicted to participate in hydrophobic interactions based on crystal structures of isolated domains

Summary

Background

The class IA PI 3-kinase regulatory subunit p85␣ dimerizes by mechanisms not fully understood. The lipid phosphatase PTEN antagonizes PI3K signaling by dephosphorylating phosphatidylinositol 3,4,5-trisphosphate [3] Given their modular structure, it is not surprising that p85 subunits participate in intra- and intermolecular interactions in addition to binding p110. Dimerization of p85␣ as well as the SH3-PR1-BCR fragment of p85␣ (residues 1–333; Fig. 1B) has been reported, and peptides derived from the PR1 motif disrupt p85␣ dimerization [13] These results show that intermolecular SH3-PR1 interactions in the native protein are involved in p85␣ dimerization. We have defined solution conditions under which the protein is monomeric or predominantly dimeric and used these conditions for small angle x-ray scattering (SAXS) and chemical cross-linking studies that have informed the structural modeling of the p85␣ dimer. Our study provides new insight into p85␣ dimerization, suggesting that p85␣ is a highly dynamic molecule whose conformational flexibility allows it to efficiently exchange among multiple binding partners

Experimental Procedures

Results

Discussion