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.

Circular dichroism and fluorescence spectroscopy were used to investigate the structure of the p85 alpha subunit of the PI 3‐kinase, a closely related p85 beta protein, and a recombinant SH2 domain‐containing fragment of p85 alpha. Significant spectral changes, indicative of a conformational change, were observed on formation of a complex with a 17 residue peptide containing a phosphorylated tyrosine residue. The sequence of this peptide is identical to the sequence surrounding Tyr751 in the kinase‐insert region of the platelet‐derived growth factor beta‐receptor (beta PDGFR). The rotational correlation times measured by fluorescence anisotropy decay indicated that phosphopeptide binding changed the shape of the SH2 domain‐containing fragment. The CD and fluorescence spectroscopy data support the secondary structure prediction based on sequence analysis and provide evidence for flexible linker regions between the various domains of the p85 proteins. The significance of these results for SH2 domain‐containing proteins is discussed.

Three-dimensional models of the five functional modules in human protein kinase C alpha (PKC alpha) have been generated on the basis of known related structures. The catalytic region at the C-terminus of the sequence and the N-terminal auto-inhibitory pseudo-substrate have been modeled using the crystal structure complex of cAMP-dependent protein kinase (cAPK) and PKI peptide. While the N-terminal helix of the catalytic region of PKC alpha is predicted to be in a different location compared with cAPK, the C-terminal extension is modeled like that in the cAPK. The predicted permissive phosphorylation site of PKC alpha, Thr 497, is found to be entirely consistent with the mutagenesis studies. Basic Lys and Arg residues in the pseudo-substrate make several specific interactions with acidic residues in the catalytic region and may interact with the permissive phosphorylation site. Models of the two zinc-binding modules of PKC alpha are based on nuclear magnetic resonance and crystal structures of such modules in other PKC isoforms while the calcium phospholipid binding module (C2) is based on the crystal structure of a repeating unit in synaptotagmin I. Phorbol ester binding regions in zinc-binding modules and the calcium binding region in the C2 domain are similar to those in the basis structures. A hypothetical model of the relative positions of all five modules has the putative lipid binding ends of the C2 and the two zinc-binding domains pointing in the same direction and may serve as a basis for further experiments.

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