Regulation of the p85/p110α Phosphatidylinositol 3′-Kinase

Abstract

Our previous studies on the p85/p110 phosphatidyli-nositol 3-kinase showed that the p85 regulatory subunit inhibits the p110 catalytic subunit, and that phos-phopeptide activation of p85/p110 dimers reflects a dis-inhibition of p110 (Yu, J., Zhang, Y., McIlroy, J., Ror-dorf-Nikolic, T., Orr, G. A., and Backer, J. M. (1998) Mol. Cell. Biol. 18, 1379-1387). We now define the domains of p85 required for inhibition of p110. The iSH2 domain of p85 is sufficient to bind p110 but does not inhibit it. Inhibition of p110 requires the presence of the nSH2 domain linked to the iSH2 domain. Phosphopeptides increase the activity of nSH2/iSH2-p110 dimers, demonstrating that the nSH2 domain mediates both inhibition of p110 and disinhibition by phosphopeptides. In contrast, phosphopeptides did not increase the activity of iSH2/cSH2-p110 dimers, or dimers composed of p110 and an nSH2/iSH2/cSH2 construct containing a mutant nSH2 domain. Phosphopeptide binding to the cSH2 domain increased p110 activity only in the context of an intact p85 containing both the nSH2 domain and residues 1-322 (the SH3, proline-rich and break-point cluster region-homolgy domains). These data suggest that the nSH2 domain of p85 is a direct regulator of p110 activity. Regulation of p110 by phosphopeptide binding to the cSH2 domain occurs by a mechanism that requires the additional presence of the nSH2 domain and residues 1-322 of p85. PI 1 3-kinases form a diverse family of lipid kinases that phosphorylate phosphatidylinositol at the D3-position (1). The regulation of the p85/p110 PI 3-kinase is particularly complex. The p85 regulatory subunit contains an N-terminal SH3 domain followed by a proline rich domain, a breakpoint cluster region-homology domain, a second proline-rich domain, and two SH2 domains linked by a putative coiled coil domain (the inter-SH2 or iSH2 domain) that binds to the N terminus of the p110 catalytic subunit (2-4). The binding of proteins such as CDC42 (to the BCR homology domain), Fyn and Lyn (to the proline-rich domains), and p21-ras (to p110) increases the activity of p85/p110 dimers in vitro (5-7). p85/p110 activity is also increased when the two SH2 domains bind to phosphopro-teins containing appropriate phosphotyrosyl motifs (8-10). We previously examined the effect of p85 on p110 in mam-malian cells and in vitro (11), and experimentally distinguished two effects of p85 on p110: inhibition of its lipid kinase activity and stabilization against thermal inactivation. The inhibition of p110 by the p85 subunit is clearly seen during in vitro reconstitution experiments, where dimerization with p85 decreases p110 activity by 80%. The activity of p85/p110 dimers is increased when phosphotyrosyl peptides bind to the p85 SH2 domains, but to a level no greater than that of the corresponding amount of monomeric p110. These data suggest that phos-phopeptide activation of p85/p110 dimers reflects a transition between inhibited and disinhibited states. In addition to its inhibition of p110, p85 stabilizes p110 against thermal denaturation. Recombinant p110 monomers lose activity rapidly when incubated at 37 °C, whereas p85/ p110 dimers are stable, and coexpression of p85 with p110 in mammalian cells significantly increases the half-life of p110 (11). The rapid inactivation of p110 at 37 °C explains a long-standing discrepancy between mammalian and insect cells (12): monomeric p110 is active in insect cells but not mam-malian cells because of differences in their culture temperatures (27 versus 37 °C). However, the stabilization of p110 by p85 in mammalian cells is mimicked by addition of bulky epitope tags to the N terminus of p110 (11). Thus, the stabilization by p85 appears to involve the overall conformation of p110 rather than the induction of a specific activated state. In this paper, we examine the regulation of p110 by p85 in detail. We find that the iSH2 domain of p85 is sufficient to bind p110 but does not affect its activity. Inhibition of p110 requires the presence of the nSH2 domain linked to the iSH2 domain. Phosphopeptide binding to the nSH2 domain can directly modulate p110 activity. In contrast, phosphopeptide binding to the cSH2 domain modulates p110 activity by a mechanism that requires residues 1-322 of p85 (the SH3, BCR homology and proline-rich domains) and the nSH2 domain. These data suggest that the nSH2 domain is the principle regulator of p85/p110 activity. EXPERIMENTAL PROCEDURES Construction of Mutant p85 and p85 Fragments-The GST-nSH2/ iSH2/cSH2 construct (in pGEX-3X) has been previously described (8). The GST-iSH2 construct was created by PCR amplification of residues 431-600 of human p85, followed by subcloning into pGEX-2T. The nSH2/iSH2 construct was created by introducing two STOP codons after the codon corresponding to p85 residue 600 in the GST-nSH2/ iSH2/cSH2 construct. The GST-iSH2/cSH2 construct was produced by PCR amplification of residues 431-724 of p85, followed by subcloning into pGEX-2T. Full-length N-terminal HA-tagged p85 (10) was directly subcloned into pGEX-2T, and brought into frame by digestion with BamHI (present in the pGEX multiple cloning site) and EagI (present in the HA tag), filling in with Klenow polymerase, and the blunt ligation. Point mutations in the nSH2 domain (R358A) and cSH2 domain (R659A) of the pGEX-nSH2/iSH2/cSH2 construct were introduced , and XhoI/EcoRI fragments from these mutated constructs were