Disruption of the Interface between the Pleckstrin Homology (PH) and Kinase Domains of Akt Protein Is Sufficient for Hydrophobic Motif Site Phosphorylation in the Absence of mTORC2

Abstract

The pro-survival kinase Akt requires phosphorylation at two conserved residues, the activation loop site (Thr-308) and the hydrophobic motif site (Ser-473), for maximal activation. Previous reports indicate that mTORC2 is necessary for phosphorylation of the hydrophobic motif and that this site is not phosphorylated in cells lacking components of the mTORC2 complex, such as Sin1. Here The Akt/protein kinase B Ser/Thr protein kinases play a central role in signaling downstream of phosphatidylinositol 3-kinase (PI3K) 2 (1). The three isoforms (Akt1, Akt2, and Akt3) share a similar domain structure: an N-terminal pleckstrin homology (PH) domain, followed by an ␣-helical linker, and a C-terminal kinase domain that is controlled by phosphorylation (2). Once activated by phosphorylation, Akt phosphorylates defined substrates throughout the cell, ultimately inducing pro-proliferation and anti-apoptotic signaling pathways (1). The activation state of Akt is tightly controlled, and its dysregulation is implicated in the development of a variety of diseases, most notably cancer (3).One key regulator of Akt phosphorylation is the mammalian target of rapamycin (mTOR), an evolutionarily conserved Ser/ Thr protein kinase that forms two distinct protein complexes in cells. These complexes are differentially regulated and perform distinct cellular functions (4). The mTOR complex 1 (mTORC1) is composed of mTOR, Raptor, and mLST8 and is sensitive to inhibition by rapamycin. mTORC1 is a crucial regulator of cell growth in response to nutrients, stress, and growth factors (5, 6). A second complex, mTOR complex 2 (mTORC2), consists of mTOR, Rictor, mLST8, and Sin1 and is generally considered to be rapamycin-insensitive (7,8). It is this complex, mTORC2, that regulates Akt and, additionally, protein kinase C (9, 10).Upon biosynthesis, the nascent Akt polypeptide is phosphorylated at the ribosome by mTORC2 (10 -12) at a conserved C-terminal site originally identified in protein kinase C and named the turn motif site (13). Phosphorylation of this residue (Thr-450 in Akt1) (14) is important for the stability of AGC kinases (15,16). Thus, Akt is heavily ubiquitinated and degraded in cells lacking mTORC2 because the turn motif site is not phosphorylated (10, 11). This processing phosphorylation of Akt is constitutive and its dephosphorylation in cells has not been reported.Once processed by phosphorylation at the turn motif site, Akt localizes to the cytosol, where it is maintained in an inactive conformation through the interaction between its PH and kinase domains (17). In the presence of proliferative signals, PI3K is activated and generates the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ). Akt is subsequently recruited from the cytosol to the plasma membrane through binding of its PH domain to PIP 3 , resulting in a conformational change that separates the PH and kinase domains and unmasks two key regulatory residues whose phosphorylations are required for maximal activation of the kinase. The first site,...

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

“…These results indicate that disrupting the PH-KD contacts leads to constitutive phosphorylation of AKT. In agreement with this result, a recent study showed that deletion of AKT1 PH domain led to its constitutive S473 phopshorylation (22). breast, 48 nonsmall-cell lung (NSCLC) adenocarcinoma (adeno), 43 NSCLC (squamous), 43 renal carcinoma, 37 melanoma, 33 gastric, 32 ovarian, 15 esophageal, 11 hepatocellular, 10 small-cell lung cancer, and 6 others (5 lung large cell and 1 lung cancer other) (Dataset S2).…”

Mammalian Target of Rapamycin Complex 1 (mTORC1) and 2 (mTORC2) Control the Dendritic Arbor Morphology of Hippocampal Neurons

“…Moreover, when the myrAkt mutant was compared in experiments with phospho-mimicking non-myristoylated Akt mutants, their ability to rescue the phenotypic effects of Rictor knock-out was similar (48). Another potential explanation for mTORC2-independent activation of myr-Akt was provided by work of Warfel et al (39). The authors showed that Ser-473 of Akt mutants lacking pleckstrin homology domain, like our myr-Akt, can be still phosphorylated in the absence of functional mTORC2.…”

“…phosphorylates itself (39). Thus, although myr-Akt mutant is a good tool to provide evidence for sufficiency of Akt to rescue dendritic arbor simplification upon Rictor knockdown, more experiments are needed in the future with use of additional Akt mutants to fully describe the mechanism of Akt regulation by mTORC2 in neurons.…”

Regulation of OSR1 and the sodium, potassium, two chloride cotransporter by convergent signals

“…DNA-PK and casein kinase II are predicted to phosphorylate OSR1 S339 (http://scansite.mit.edu). Akt, like the Ste20 kinase noted above, can autophosphorylate its own hydrophobic motif site; this mechanism is enhanced by mutation of the pleckstrin homology domain or prolonged inhibition of mTORC1 (30,49,52). Autophosphorylation is an intriguing possibility because OSR1 can exist as an activation loop-swapped dimer (53).…”

“…It seems likely that one or more other kinases can also phosphorylate the PF1 site on OSR1 following prolonged mTOR inhibition. Enzymes other than mTORC2 phosphorylate the Akt hydrophobic motif under certain circumstances; these include Akt itself, the DNA-dependent protein kinase (DNA-PK), and the Tank binding kinase (30,(49)(50)(51). DNA-PK and casein kinase II are predicted to phosphorylate OSR1 S339 (http://scansite.mit.edu).…”