Molecular Mechanism of an Oncogenic Mutation That Alters Membrane Targeting: Glu17Lys Modifies the PIP Lipid Specificity of the AKT1 PH Domain

Abstract

The protein kinase AKT1 regulates multiple signaling pathways essential for cell function. Its Nterminal PH domain (AKT1 PH) binds the rare signaling phospholipid phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ], resulting in plasma membrane targeting and phosphoactivation of AKT1 by a membrane-bound kinase. Recently, it was discovered that the Glu17Lys mutation in the AKT1 PH domain is associated with multiple human cancers. This mutation constitutively targets the AKT1 PH domain to the plasma membrane by an unknown mechanism, thereby promoting constitutive AKT1 activation and oncogenesis. To elucidate the molecular mechanism underlying constitutive plasma membrane targeting, this work compares the membrane docking reactions of the isolated wild-type and E17K AKT1 PH domains. In vitro studies reveal that the E17K mutation dramatically increases the affinity for the constitutive plasma membrane lipid PI(4,5)P 2 . The resulting PI(4,5)P 2 equilibrium affinity is indistinguishable from that of the standard PI(4,5)P 2 sensor, PLCδ1 PH domain. Kinetic studies indicate that the effects of E17K on PIP lipid binding arise largely from electrostatic modulation of the dissociation rate. Membrane targeting analysis in live cells confirms that the constitutive targeting of E17K AKT1 PH to plasma membrane, like PLCδ1 PH, stems from PI(4,5)P 2 binding. Overall, the evidence indicates that the molecular mechanism underlying E17K oncogenesis is a broadened target lipid selectivity that allows high-affinity binding to PI(4,5)P 2 . Moreover, the findings strongly implicate the native Glu17 side chain as a key element of PIP lipid specificity in the wild-type AKT1 PH domain. Other PH domains may employ an analogous anionic residue to control PIP specificity.Many cellular signaling processes are controlled by events occurring at the surface of intracellular membranes. Phosphatidylinositiol phosphates (PIPs) comprise an important class of signaling phospholipids that are highly regulated second messengers and can drive the reversible localization of key signaling proteins from the cytoplasm to the inner leaflet of the plasma membrane (1-5). Such PIP-dependent localization events play essential roles in many signaling pathways. For example, on the cytoplasmic surface of the plasma membrane, the phosphatidylinositol 3-kinase (PI3K) family of lipid kinases convert a constitutive PIP lipid, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ], 1 into the rare second-messenger lipid phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ] (1,6,7). This second messenger, in turn, recruits a wide array of important downstream protein effectors † Supported by NIH Grant R01 GM-063235 (to J.J. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 August 10. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript containing pleckstrin homology (PH) domains that specifically bind to the PI(3,4,5)P 3 headgroup (8-10). The resulting plasma membrane targeting activates signali...

Disruption of PH–kinase domain interactions leads to oncogenic activation of AKT in human cancers

“…Consistent with this finding, both L52R and D323H weaken PH-KD binding in two-hybrid assays. Previously, the mechanism of activation of E17K has been attributed to an altered lipid-binding specificity (10,11). Our results indicate perturbation of interdomain interactions to be an additional mechanism underlying E17K activation.…”

“…Recently, a somatic mutation in the PH domain of AKT1 was identified in a subset of human carcinomas (10). This mutation results in the substitution of glutamic acid at codon 17 of AKT1 with lysine (E17K) and alters the lipid-binding specificity of AKT, leading to pathological membrane association and constitutive signaling (10,11). Other than human cancers, germ-line and somatic E17K mutations in AKT have been identified in Proteus syndrome, human hypoglycemia, and hemimegalencephaly (12)(13)(14).…”

AKT1 mutations in bladder cancer: identification of a novel oncogenic mutation that can co-operate with E17K

“…It has been shown that the E17K mutation results in enhanced translocation of AKT1 to the membrane in which phosphorylation by PDK1 occurs (Carpten et al, 2007). Recent results indicate that the effect of E17K is to broaden the lipid-binding specificity of AKT such that it can bind physiological concentrations PIP2 found in the plasma membrane (Landgraf et al, 2008). It has also been shown that in the inactive state, AKT is bound to PDK1, but activation is suppressed through interaction of the PH domain with the kinase domain, preventing PDK1 access to Thr308 and preventing phosphorylation (Calleja et al, 2007).…”

Tumors with AKT1E17K Mutations Are Rational Targets for Single Agent or Combination Therapy with AKT Inhibitors

“…MK-2206 binds to the region that interacts with both the plekstrin homology and kinase domains, and prevents translocation of AKT to the membrane and activation. AKT1 E17K mutation alters the electrostatic interactions of the lipid binding pocket and dramatically increases the affinity of the protein for the constitutive membrane lipid PI (4,5)P2 (24), which probably explains increased membrane localization compared with wild-type AKT1. The allosteric inhibitor VIII has previously been reported to be less effective than several different catalytic inhibitors at inhibiting purified AKT1 E17K protein and proliferation of AKT1 E17K transfected fibroblasts (15), but presumably because membrane translocation is still a necessary step and/or because it is structurally distinct from inhibitor VIII, MK-2206 is still able to inhibit colony formation in vitro and inhibit tumor growth in vivo.…”