Molecular Mechanism for the Regulation of Protein Kinase B/Akt by Hydrophobic Motif Phosphorylation

Yang, Jing; Cron, Peter; Thompson, Vivienne; Good, Valerie M.; Heß, Daniel; Hemmings, Brian A.; Barford, David

doi:10.1016/s1097-2765(02)00550-6

Cited by 406 publications

(433 citation statements)

References 41 publications

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“…For a kinase reaction, we added 2 ml (100 ng) of the activated or inactivated recombinant PKBb (Yang et al, 2002) to a reaction mix containing 70 mM of the corresponding peptide (RKRRSSRRSAGG, S42/S45; RKRRSARRSAGG, S42A; RKRRSSRRAAGG, S45A; RERQRTQSLNEA, T121/S123; RERQRAQSLNEA, T121A; RERQRTQALNEA, S123A), 2 ml (2 mCi) of g-32 P-ATP and 20 mM ATP in 20 ml of kinase reaction buffer (30 mM HEPES/KOH (pH 7.4), 25 mM b-glycerophosphate, 2 mM DTT, 20 mM MgCl 2 , 0.1 mM sodium vanadate). After incubation for 30 min at 30 1C, kinase reactions were stopped with 50 mM EDTA, transferred to phosphocellulose P11 paper (Whatman, Bottmingen, Switzerland), fixed and washed four times in 1% phosphoric acid and once with acetone, dried and assayed by scintillation counting.…”

Section: Discussionmentioning

confidence: 99%

PKB/AKT phosphorylation of the transcription factor Twist-1 at Ser42 inhibits p53 activity in response to DNA damage

et al. 2010

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Protein kinase B (PKB/Akt) is ubiquitously expressed in cells. Phosphorylation of its multiple targets in response to various stimuli, including growth factors or cytokines, promotes cell survival and inhibits apoptosis. PKB is upregulated in many different cancers and a significant amount of the enzyme is present in its activated form. Here we show that PKB phosphorylates one of the anti-apoptotic proteins-transcription factor Twist-1 at Ser42. Cells expressing Twist-1 displayed inefficient p53 upregulation in response to DNA damage induced by c-irradiation or the genotoxic drug adriamycin. This influenced the activation of p53 target genes such as p21 Waf1 and Bax and led to aberrant cell-cycle regulation and the inhibition of apoptosis. The impaired induction of these p53 effector molecules is likely to be mediated by PKB-dependent phosphorylation of Twist-1 because, unlike the wild-type mutant, the Twist-1 S42A mutant did not confer cell resistance to DNA damage. Moreover, phosphorylation of Twist-1 at Ser42 was shown in vivo in various human cancer tissues, suggesting that this posttranslational modification ensures functional activation of Twist-1 after promotion of survival during carcinogenesis.

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Section: Discussionmentioning

confidence: 99%

PKB/AKT phosphorylation of the transcription factor Twist-1 at Ser42 inhibits p53 activity in response to DNA damage

et al. 2010

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“…13,14 Phosphorylation of Akt on both S473 and T308 are required for Akt activity. 33,34 Notably, there is evidence suggesting otherwise, with differences in substrate preferences associated with phosphorylation on the serine or threonine residue of the kinase. 23,25,35 In our experiments, Ala substitution of S473 abrogated antiapoptotic activity, although this was not the case with T308.…”

Section: Discussionmentioning

confidence: 99%

A hierarchical cascade activated by non-canonical Notch signaling and the mTOR–Rictor complex regulates neglect-induced death in mammalian cells

et al. 2009

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The regulation of cellular metabolism and survival by trophic factors is not completely understood. Here, we describe a signaling cascade activated by the developmental regulator Notch, which inhibits apoptosis triggered by neglect in mammalian cells. In this pathway, the Notch intracellular domain (NIC), which is released after interaction with ligand, converges on the kinase mammalian target of rapamycin (mTOR) and the substrate-defining protein rapamycin independent companion of mTOR (Rictor), culminating in the activation of the kinase Akt/PKB. Biochemical and molecular approaches using site-directed mutants identified AktS473 as a key downstream target in the antiapoptotic pathway activated by NIC. Despite the demonstrated requirement for Notch processing and its predominant nuclear localization, NIC function was independent of CBF1/RBP-J, an essential DNA-binding component required for canonical signaling. In experiments that placed spatial constraints on NIC, enforced nuclear retention abrogated antiapoptotic activity and a membrane-anchored form of NIC-blocked apoptosis through mTOR, Rictor and Akt-dependent signaling. We show that the NIC-mTORC2-Akt cascade blocks the apoptotic response triggered by removal of medium or serum deprivation. Consistently, membrane-tethered NIC, and AktS473 inhibited apoptosis triggered by cytokine deprivation in activated T cells. Thus, this study identifies a non-canonical signaling cascade wherein NIC integrates with multiple pathways to regulate cell survival.

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“…The B helix structure is conserved among the PKA, PDK1, and PKB structures (39,40), all members of the AGC family; therefore, the B helix is likely Proximal to the conserved Gly-rich loop (residues 50-55, red), the B helix (residues 76-80, red) and the C-terminal tail (residues 330-334, red) are variable between the two protein kinases and may account for the difference in inhibition potency of the analogues. In PKA, a stretch of Glu residues in the C-terminal tail is not present in PKC.…”

Section: Discussionmentioning

confidence: 99%

Balanol Analogues Probe Specificity Determinants and the Conformational Malleability of the Cyclic 3‘,5‘-Adenosine Monophosphate-Dependent Protein Kinase Catalytic Subunit^,

et al. 2003

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The protein kinase family is a prime target for therapeutic agents, since unregulated protein kinase activities are linked to myriad diseases. Balanol, a fungal metabolite consisting of four rings, potently inhibits Ser/Thr protein kinases and can be modified to yield potent inhibitors that are selectives characteristics of a desirable pharmaceutical compound. Here, we characterize three balanol analogues that inhibit cyclic 3′,5′-adenosine monophosphate-dependent protein kinase (PKA) more specifically and potently than calcium-and phospholipid-dependent protein kinase (PKC). Correlation of thermostability and inhibition potency suggests that better inhibitors confer enhanced protection against thermal denaturation. Crystal structures of the PKA catalytic (C) subunit complexed to each analogue show the Gly-rich loop stabilized in an "intermediate" conformation, disengaged from important phosphoryl transfer residues. An analogue that perturbs the PKA C-terminal tail has slightly weaker inhibition potency. The malleability of the PKA C subunit is illustrated by active site residues that adopt alternate rotamers depending on the ligand bound. On the basis of sequence homology to PKA, a preliminary model of the PKC active site is described. The balanol analogues serve to test the model and to highlight differences in the active site local environment of PKA and PKC. The PKA C subunit appears to tolerate balanol analogues with D-ring modifications; PKC does not. We attribute this difference in preference to the variable B helix and C-terminal tail. By understanding the details of ligand binding, more specific and potent inhibitors may be designed that differentiate among closely related AGC protein kinase family members.As cellular processes are better understood at the molecular level, especially in the context of recent genomic information, there has been an increased effort to target specific proteins that are linked to disease. Protein kinases are a diverse family of enzymes that have various regulatory roles yet function similarly by catalyzing the phosphoryl transfer of the γ-phosphate of ATP 1 to an enzyme-specific protein substrate. Since many diseases, including cancer, autoimmune disorders, cardiac disease, and diabetes, are associated with defects in protein phosphorylation, and there are an estimated ∼500 protein kinases in the human genome (1), this family is a major target in the design of pharmaceutical agents and inhibitors. A major challenge for the development of therapeutics is the identification of compounds that have high selectivity. To better understand binding diversity and how this correlates with specificity for protein kinases, three analogues of balanol were studied that specifically inhibit † Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory, a national user facility operated by Stanford University on behalf of the Office of Basic Energy Sciences, U.S.

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Molecular Mechanism for the Regulation of Protein Kinase B/Akt by Hydrophobic Motif Phosphorylation

Cited by 406 publications

References 41 publications

PKB/AKT phosphorylation of the transcription factor Twist-1 at Ser42 inhibits p53 activity in response to DNA damage

PKB/AKT phosphorylation of the transcription factor Twist-1 at Ser42 inhibits p53 activity in response to DNA damage

A hierarchical cascade activated by non-canonical Notch signaling and the mTOR–Rictor complex regulates neglect-induced death in mammalian cells

Balanol Analogues Probe Specificity Determinants and the Conformational Malleability of the Cyclic 3‘,5‘-Adenosine Monophosphate-Dependent Protein Kinase Catalytic Subunit^,

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Molecular Mechanism for the Regulation of Protein Kinase B/Akt by Hydrophobic Motif Phosphorylation

Cited by 406 publications

References 41 publications

PKB/AKT phosphorylation of the transcription factor Twist-1 at Ser42 inhibits p53 activity in response to DNA damage

PKB/AKT phosphorylation of the transcription factor Twist-1 at Ser42 inhibits p53 activity in response to DNA damage

A hierarchical cascade activated by non-canonical Notch signaling and the mTOR–Rictor complex regulates neglect-induced death in mammalian cells

Balanol Analogues Probe Specificity Determinants and the Conformational Malleability of the Cyclic 3‘,5‘-Adenosine Monophosphate-Dependent Protein Kinase Catalytic Subunit,

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Balanol Analogues Probe Specificity Determinants and the Conformational Malleability of the Cyclic 3‘,5‘-Adenosine Monophosphate-Dependent Protein Kinase Catalytic Subunit^,