p110α Hot Spot Mutations E545K and H1047R Exert Metabolic Reprogramming Independently of p110α Kinase Activity

Abstract

eThe phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) catalytic subunit p110␣ is the most frequently mutated kinase in human cancer, and the hot spot mutations E542K, E545K, and H1047R are the most common mutations in p110␣. Very little is known about the metabolic consequences of the hot spot mutations of p110␣ in vivo. In this study, we used adenoviral gene transfer in mice to investigate the effects of the E545K and H1047R mutations on hepatic and whole-body glucose metabolism. We show that hepatic expression of these hot spot mutations results in rapid hepatic steatosis, paradoxically accompanied by increased glucose tolerance, and marked glycogen accumulation. In contrast, wild-type p110␣ expression does not lead to hepatic accumulation of lipids or glycogen despite similar degrees of upregulated glycolysis and expression of lipogenic genes. The reprogrammed metabolism of the E545K and H1047R p110␣ mutants was surprisingly not dependent on altered p110␣ lipid kinase activity. Class IA phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) is an integral mediator of several signaling pathways, including insulin-like growth factor 1 (IGF-1) and insulin signaling (1). Upon activation by IGF-1, insulin, or other growth factors, it catalyzes the formation of phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ), which has a high affinity for the pleckstrin homology (PH) domain of the downstream target Akt/protein kinase B (Akt/ PKB). The interaction of PIP 3 with the PH domain enables phosphorylation of Akt/PKB by phosphoinositide-dependent kinase 1 (PDK 1) and PDK 2. PI3K and Akt/PKB are both critical nodes in many intracellular signaling pathways and are imperative for the actions of IGF-1, insulin, and other growth factors (2).Class IA PI3Ks consist of two subunits. The catalytic subunit, p110, contains the kinase domain responsible for the formation of PIP 3 . The regulatory subunit, usually designated p85, binds to phosphorylated tyrosine residues in other proteins via its SH2 domain, leading to subsequent activation of the kinase. Both subunits exist as several different isoforms. There are four known human catalytic subunit isoforms: p110␣, p110␤, p110␦, and p37␦. p110␣, p110␤, and p110␦ are encoded by three different genes, i.e., PIK3CA, PIK3CB, and PIK3CD, respectively (reviewed in reference 1), whereas p37␦ (PIK3CD_v2) is a splice variant of p110␦ (3). We and others have shown that among the catalytic subunits, p110␣ is the major contributor to transmission of the insulin signal (4-6), whereas p110␤ becomes active primarily in response to G-protein-coupled receptor signaling and plays a role in proliferation (7,8). p110␦ is more cell specific than p110␣ and p110␤ and plays an important role in immune cells and the embryonic nervous system (9-11).p110␣ is the most frequently mutated kinase in human cancer; three mutations in particular, often referred to as the hot spot mutations, namely, E542K, E545K, and H1047R, are the most common in p110␣ (PIK3CA) and comprise 80% of all mutations (reviewed in ref...

Hepatic deletion of p110α and p85α results in insulin resistance despite sustained IRS1-associated phosphatidylinositol kinase activity

“…As expected, p110α kinase activity, as assessed by the ability to add the 3’-phosphate group to phosphoinositides in p110α immunoprecipitates, was markedly decreased both in the basal- and insulin-stimulated states of the L-DKO mice compared to controls ( Figure 2A ). Overall p110β kinase activity was much lower than p110α kinase activity in control mice, consistent with previous studies 7 , 27 , and was not changed in the basal state between controls and L-DKO mice ( Figure 2B ). However, p110β kinase activity was significantly decreased in the insulin-stimulated state of the L-DKO mice ( Figure 2B ), even though similar amounts of p110β protein were associated with IRS1 in controls and L-DKO mice ( Figure 1K and 1M ).…”

Hepatic deletion of p110α and p85α results in insulin resistance despite sustained IRS1-associated phosphatidylinositol kinase activity

“…Overall p110β kinase activity was much lower than p110α kinase activity in control mice, consistent with previous studies 7, 27 , and was not changed in the basal state between controls and L-DKO mice ( Figure 2B). However, p110β kinase activity was significantly decreased in the insulin-stimulated state of the L-DKO mice ( Figure 2B), even though similar amounts of p110β protein were associated with IRS1 in controls and L-DKO mice ( Figure 1K and 1M).…”

Phosphoinositide 3-kinase p110α negatively regulates thrombopoietin-mediated platelet activation and thrombus formation

“…Indeed, we found that the platelet p110α phenotype could not be mimicked by the presence of the p110α inhibitor PIK-75. Kinase-independent functions of p110α have recently been described in cells with gain-of-function mutations in the p110α gene (PIK3CA; hot spot mutations in E545K/H1047R) [51]. Lipid kinase-independent functions have also been demonstrated for the Class I PI3K isoforms p110β and p110δ, which contribute to insulin signalling and cell survival respectively [52,53].…”