PI 3-kinase: structural and functional analysis of intersubunit interactions.

Abstract

Phosphatidylinositol (PI) 3-kinase has an 85 kDa subunit (p85a) which mediates its association with activated protein tyrosine kinase receptors through SH2 domains, and an 110 kDa subunit (p110) which has intrinsic catalytic activity. Here p85a and a related protein p853 are shown to form stable complexes with recombinant pllO in vivo and in vitro. Using a panel of glutathione S-transferase (GST) fusion proteins of the inter-SH2 region of p85, 104 amino acids were found to bind directly the pllO protein, while deletion mutants within this region further defined the binding site to a sequence of 35 amino acids. Transient expression of the mutant p85ct protein in mouse L cells showed it was unable to bind PI 3-kinase activity in vivo. Mapping of the complementary site of interaction on the pllO protein defined 88 amino acids in the N-terminal region of pllO which mediate the binding of this subunit to either the p85a or the p853 proteins. The inter-SH2 region of p85 is predicted to be an independently folded module of a coiled-coil of two long anti-parallel c-helices. The predicted structure of p85 suggests a basis for the intersubunit interaction and the relevance of this interaction with respect to the regulation of the PI 3-kinase complex is discussed.

Characterizing the Interactions between the Two Subunits of the p101/p110γ Phosphoinositide 3-Kinase and Their Role in the Activation of This Enzyme by Gβγ Subunits

“…The binding site for p101 in p110␥, although primarily involving the N terminus of the protein, is a far larger region extending deeper into the protein. Considering the fact that several parts of p101 are involved in binding to p110␥, it would seem reasonable that a substantial piece of p110␥ is involved in binding p101 (note that in p110␣ a relatively short region of the N terminus is thought to bind to a short inter-SH2 domain segment of p85 (17,18)). In contrast to the situation with p101, where deletions affecting the total contact area and strength of binding to p110␥ also affected the sensitivity of the heterodimer to G ␤␥ , deletions through the N terminus of p110␥, that reduced the efficiency of binding to p101, had no effect on the activation of heterodimeric enzyme that could be recovered.…”

An oncogenic fusion product of the phosphatidylinositol 3-kinase p85β subunit and HUMORF8, a putative deubiquitinating enzyme

“…Regarding p85b, only the carboxy-terminal SH2 domain and part of the inter-SH2 sequence are deleted in the chimeras. In fact, the 40 amino acids (residue 445 ± 485) in the inter-SH2 domain, that are required for the binding of the catalytic PI 3-kinase p110 subunit are una ected (Dhand et al, 1994a). Therefore, signaling of PI 3-kinase through its speci®c phosphoinositide lipid products may remain intact and activate various downstream targets such as protein kinase C isomers, ras and akt (Carpenter and Cantley, 1996;Hemmings, 1997;Toker and Cantley, 1997).…”

PI3K is negatively regulated by PIK3IP1, a novel p110 interacting protein

“…Altogether, these data indicate that PIK3IP1 does not prevent the interaction of p85 and p110 in cells that either have enhanced or reduced PIK3IP1 protein expression suggesting that the binding of p85 and PIK3IP1 to p110 is not mutually exclusive; rather, it appears that they exist in a ternary complex. The fact that PIK3IP1 does not disrupt p85-p110 coupling to any substantial degree is not surprising since p85 and p110 associate with very high affinity [13] through multiple contact sites [10,14].…”

“…the interSH2 or iSH2 domain) that the p85-like domain of PIK3IP1 resembles is critical to the association of p85 and p110 [10,11], we wanted to determine if PIK3IP1, like p85, interacts with p110. Accordingly, we performed binding assays using mycor hemagglutinin (HA)-tagged-PIK3IP1, p85 or p110 as described in the Supplemental Materials and Methods.…”