The structure of the inter-SH2 domain of class IA phosphoinositide 3-kinase determined by site-directed spin labeling EPR and homology modeling

Abstract

Phosphoinositide (PI) 3-kinases catalyze the phosphorylation of the D3 position of the inositol ring of PI, and its phosphorylated derivatives and play important roles in many intracellular signal transducing pathways. Class IA PI3-kinases contain distinct regulatory (p85) and catalytic (p110) subunits. p110 is stabilized and inhibited by constitutive association with p85, and is disinhibited when the SH2 domains of p85 bind to tyrosyl-phosphorylated proteins. Because the two subunits do not dissociate, disinhibition of p110 presumably occurs by an allosteric mechanism. To explore the means by which p85 regulates the activity of p110, structures of the inter-SH2 domain of p85 were determined with and without phosphopeptide by using a combination of site directed spin labeling EPR and homology modeling and molecular dynamics. The inter-SH2 domain is assigned as a rigid anti-parallel coiled-coil whose primary function is to bind p110, facilitating inhibition of p110 by the N-terminal SH2 domain of p85.nitroxide ͉ distance measurement ͉ SDSL-EPR ͉ p85 ͉ p110 P hosphoinositide (PI) 3-kinases are important regulators of proliferation, apoptosis, motility, and vesicular trafficking (1). Class IA PI3-kinases contain distinct regulatory (p85) and catalytic (p110) subunits. It has been shown previously that p85 plays two roles with regard to regulation of p110: stabilization of p110 against thermal denaturation and degradation, and inhibition of p110 catalytic activity (2). The N-terminal half of p85 contains an SH3 domain and a Rac͞CDC42-binding domain, which is flanked by two proline-rich domains (3, 4). These domains are important for cytoskeletal regulation but not mitogenic signaling (5), and are not present in three of the regulatory subunit variants (p50␣, p55␣, and p55␥). The Cterminal half of p85 contains two src homology 2 (SH2) domains (the N-terminal or nSH2 domain and the C-terminal or cSH2 domain) separated by the inter-SH2 (iSH2) domain, a putative coiled-coil that contains the binding site for the p110 catalytic subunit (6). Although CD studies of the iSH2 domain show it to be highly helical (7), its structure has not been further characterized.The binding of tyrosine-phosphorylated proteins or peptides to the SH2 domains of p85 releases its inhibition of p110, leading to an activation of p85͞p110 heterodimers (8, 9) through unknown conformational changes. The minimal fragment of p85 capable of regulating p110 is the nSH2 domain linked to the iSH2 domain (10) (Fig. 1a). Although the iSH2 domain is sufficient to bind p110 (6,11,12) it has minimal effects on p110 activity (10). In contrast, the nSH2-iSH2 fragment (heretofore referred to as p85ni) inhibits p110, and p85ni͞p110 complexes are activated on tyrosine phosphopeptide binding. Given that the nSH2 domain is the sole phosphopeptide binding domain in p85ni, several explanations for inhibition͞disinhibition regulation are possible. These include phosphopeptide-induced conformational changes in the nSH2 domain, the iSH2 domain, the adjoining segment, or ...

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Breast Cancer–Associated PIK3CA Mutations Are Oncogenic in Mammary Epithelial Cells

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“…In the currently accepted model of class IA activation, p110 subunits are constitutively bound to p85 and kept inactive in the cytosol. Upon receptor activation, p85 is recruited to tyrosine-phosphorylated motifs and this event not only liberates the p85-mediated constraint on PI3K catalytic activity (Fu et al 2003) but also localizes the enzyme next to the plasma membrane in the proximity of its lipid substrate (Yu et al 1998). In addition to controlling activity and localization of PI3K, p85 family members possess specific activities: for example, p85 can control cell cycle in a p110-independent way (Garcia et al 2006).…”