Regulation of the p85/p110 Phosphatidylinositol 3′-Kinase: Stabilization and Inhibition of the p110α Catalytic Subunit by the p85 Regulatory Subunit

Abstract

We propose a novel model for the regulation of the p85/p110␣ phosphatidylinositol 3-kinase. In insect cells, the p110␣ catalytic subunit is active as a monomer but its activity is decreased by coexpression with the p85 regulatory subunit. Similarly, the lipid kinase activity of recombinant glutathione S-transferase (GST)-p110␣ is reduced by 65 to 85% upon in vitro reconstitution with p85. Incubation of p110␣/p85 dimers with phosphotyrosyl peptides restored activity, but only to the level of monomeric p110␣. These data show that the binding of phosphoproteins to the SH2 domains of p85 activates the p85/p110␣ dimers by inducing a transition from an inhibited to a disinhibited state. In contrast, monomeric p110 had little activity in HEK 293T cells, and its activity was increased 15-to 20-fold by coexpression with p85. However, this apparent requirement for p85 was eliminated by the addition of a bulky tag to the N terminus of p110␣ or by the growth of the HEK 293T cells at 30°C. These nonspecific interventions mimicked the effects of p85 on p110␣, suggesting that the regulatory subunit acts by stabilizing the overall conformation of the catalytic subunit rather than by inducing a specific activated conformation. This stabilization was directly demonstrated in metabolically labeled HEK 293T cells, in which p85 increased the half-life of p110. Furthermore, p85 protected p110 from thermal inactivation in vitro. Importantly, when we examined the effect of p85 on GST-p110␣ in mammalian cells at 30°C, culture conditions that stabilize the catalytic subunit and that are similar to the conditions used for insect cells, we found that p85 inhibited p110␣. Thus, we have experimentally distinguished two effects of p85 on p110␣: conformational stabilization of the catalytic subunit and inhibition of its lipid kinase activity. Our data reconcile the apparent conflict between previous studies of insect versus mammalian cells and show that p110␣ is both stabilized and inhibited by dimerization with p85.Phosphatidylinositol (PI) 3Ј-kinases constitute a family of enzymes that mediate intracellular signaling initiated by receptor tyrosine kinases and heterotrimeric G-protein-coupled receptors. Activation of PI 3Ј-kinase leads to increases in the intracellular levels of PI[3,4]P 2 and PI[3,4,5]P 3 , which are presumed second messengers (4). PI 3Ј-kinases have been implicated in the control of proliferation, cytoskeletal organization, apoptosis, and vesicular trafficking (6,16,20,31,46).A classification of PI 3Ј-kinases has been described by Zvelebil and coworkers (48). The class I enzymes are heterodimeric proteins that are composed of separate regulatory and catalytic subunits and that utilize PI, PI[4]P, and PI[4,5]P 2 as substrates. Class I enzymes include the p85/p110 PI 3Ј-kinase, which is activated by binding to phosphotyrosyl proteins, and the p101/ p120 PI 3-kinase-␥ isoform, which is activated by ␤␥ subunits from trimeric G proteins (11,13,38,39). Class II PI 3Ј-kinases contain C-terminal C2 domains and preferentially utiliz...

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“…The observed reduction of p110β expression may also be attributed to the reduced expression of p85. Previous studies have suggested that the interaction between the catalytic and regulatory subunits is important for the stability of the p110 catalytic subunit [25,26].…”

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“…In p110*, the iSH2 domain of p85a is fused to the N-terminus of p110a, 26 thus stabilizing monomeric p110 against thermal denaturation at 371. 27 Its in vivo activity is much weaker than the membrane-localizing forms, 26 but is ideally suited for our purpose, which was to use p110* to augment PI3-kinase activity in DKI cells stimulated by CSF-1. Four independent DKI clones transfected with p110* were selected for further analysis.…”

Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110α and are disrupted in oncogenic p85 mutants

“…3). The regulatory subunits have two major functions: they stabilize the catalytic subunits against thermal denaturation, and they maintain the catalytic subunit in an inhibited, low activity state (4,5).…”

“…Class IA PI 3-kinases, which produce PI [3,4,5]P3 in intact cells (2), are obligate heterodimers of a regulatory subunit (p85␣, p85␤, p55␣, p50␣, or p55␥) and a catalytic subunit (p110␣, p110␤, or p110␦) (reviewed in ref. 3).…”