Role of a Novel PH-Kinase Domain Interface in PKB/Akt Regulation: Structural Mechanism for Allosteric Inhibition

Calleja, Véronique; Laguerre, Michel S.; Parker, Peter J.; Larijani, Banafshé

doi:10.1371/journal.pbio.1000017

Cited by 218 publications

(234 citation statements)

References 25 publications

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“…Previous studies have shown that AKT allosteric inhibitors require an intact PH-KD interface, because such inhibitors preferentially bind the closed PH-in conformation (16,17,38). Consistent with this result, we found that mutations in AKT that favor an open (PH-out) conformation show reduced sensitivity to allosteric AKT inhibitors, although they retain sensitivity to ATP-competitive inhibitors.…”

Section: Discussionsupporting

confidence: 90%

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

Parikh

Janakiraman

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

126

160

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The protein kinase v-akt murine thymoma viral oncogene homolog (AKT), a key regulator of cell survival and proliferation, is frequently hyperactivated in human cancers. Intramolecular pleckstrin homology (PH) domain-kinase domain (KD) interactions are important in maintaining AKT in an inactive state. AKT activation proceeds after a conformational change that dislodges the PH from the KD. To understand these autoinhibitory interactions, we generated mutations at the PH-KD interface and found that most of them lead to constitutive activation of AKT. Such mutations are likely another mechanism by which activation may occur in human cancers and other diseases. In support of this likelihood, we found somatic mutations in AKT1 at the PH-KD interface that have not been previously described in human cancers. Furthermore, we show that the AKT1 somatic mutants are constitutively active, leading to oncogenic signaling. Additionally, our studies show that the AKT1 mutants are not effectively inhibited by allosteric AKT inhibitors, consistent with the requirement for an intact PH-KD interface for allosteric inhibition. These results have important implications for therapeutic intervention in patients with AKT mutations at the PH-KD interface.interdomain | AKT-targeting | PI3K-pathway | next generation sequencing | BaF3

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Section: Discussionsupporting

confidence: 90%

“…Recent molecular modeling and structure-based studies suggest that, under basal conditions, interactions between the PH and KD maintain AKT in a closed conformation (PH-in) (15)(16)(17). In this state, PDK1 is unable to access and phosphorylate T308.…”

mentioning

confidence: 99%

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

Parikh

Janakiraman

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

126

160

View full text Add to dashboard Cite

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“…Specifically, we tested whether rapamycininduced phosphorylation of the hydrophobic motif site in Sin1 Ϫ/Ϫ MEFs was prevented by 1) an inhibitor of PI3K (wortmannin) or 2) an allosteric inhibitor of Akt (Akti VIII) that locks Akt in an inactive conformation and prevents the PH domain from disengaging and binding to membranes (17). As reported above, treatment with rapamycin rescued Ser-473 phosphorylation of Akt (Fig.…”

Section: Rescue Of Ser-473 Phosphorylation In Sin1 ϫ/ϫ Mefs Is Dependmentioning

confidence: 87%

“…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 ).…”

mentioning

confidence: 99%

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

Warfel

Niederst

Newton

2011

Journal of Biological Chemistry

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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,...

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“…We used Akt Inhibitor VIII, a selective, reversible Akt inhibitor that blocks PH (pleckstrin homology) and kinase domains interactions. This restricts phosphorylation of Akt at Thr308 and Ser473, reduces the levels of active Akt in cells, and blocks the phosphorylation of known Akt substrates (26). SUIT-2 cells were treated with Akt Inhibitor VIII at 5 M or 20 M or with dimethyl sulfoxide (DMSO) prior to infection with wild-type VV, and a standard internalization assay was performed as detailed in Materials and Methods (Fig.…”

Section: Resultsmentioning

confidence: 99%

Vascular Endothelial Growth Factor A Promotes Vaccinia Virus Entry into Host Cells via Activation of the Akt Pathway

Hiley

Chard

Gangeswaran

et al. 2013

J Virol

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Vaccinia virus (VV) is an enveloped DNA virus from the poxvirus family and has played a crucial role in the eradication of smallpox. It continues to be used in immunotherapy for the prevention of infectious diseases and treatment of cancer. However, the mechanisms of poxvirus entry, the host factors that affect viral virulence, and the reasons for its natural tropism for tumor cells are incompletely understood. By studying the effect of hypoxia on VV infection, we found that vascular endothelial growth factor A (VEGF-A) augments oncolytic VV cytotoxicity. VEGF derived from tumor cells acts to increase VV internalization, resulting in increased replication and cytotoxicity in an AKT-dependent manner in both tumor cells and normal respiratory epithelial cells. Overexpression of VEGF also enhances VV infection within tumor tissue in vivo after systemic delivery. These results highlight the importance of VEGF expression in VV infection and have potential implications for the design of new strategies to prevent poxvirus infection and the development of future generations of oncolytic VV in combination with conventional or biological therapies.

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Role of a Novel PH-Kinase Domain Interface in PKB/Akt Regulation: Structural Mechanism for Allosteric Inhibition

Cited by 218 publications

References 25 publications

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

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

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

Vascular Endothelial Growth Factor A Promotes Vaccinia Virus Entry into Host Cells via Activation of the Akt Pathway

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