scholarly journals mTORC2 controls the activity of PKC and Akt by phosphorylating a conserved TOR interaction motif

2021 ◽  
Vol 14 (678) ◽  
pp. eabe4509
Author(s):  
Timothy R. Baffi ◽  
Gema Lordén ◽  
Jacob M. Wozniak ◽  
Andreas Feichtner ◽  
Wayland Yeung ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Activation Loop ◽  
Dimer Interface ◽  
Agc Kinases ◽  
Evolutionarily Conserved ◽  
Phosphorylation Event ◽  
Hydrophobic Motif

The complex mTORC2 is accepted to be the kinase that controls the phosphorylation of the hydrophobic motif, a key regulatory switch for AGC kinases, although whether mTOR directly phosphorylates this motif remains controversial. Here, we identified an mTOR-mediated phosphorylation site that we termed the TOR interaction motif (TIM; F-x3-F-pT), which controls the phosphorylation of the hydrophobic motif of PKC and Akt and the activity of these kinases. The TIM is invariant in mTORC2-dependent AGC kinases, is evolutionarily conserved, and coevolved with mTORC2 components. Mutation of this motif in Akt1 and PKCβII abolished cellular kinase activity by impairing activation loop and hydrophobic motif phosphorylation. mTORC2 directly phosphorylated the PKC TIM in vitro, and this phosphorylation event was detected in mouse brain. Overexpression of PDK1 in mTORC2-deficient cells rescued hydrophobic motif phosphorylation of PKC and Akt by a mechanism dependent on their intrinsic catalytic activity, revealing that mTORC2 facilitates the PDK1 phosphorylation step, which, in turn, enables autophosphorylation. Structural analysis revealed that PKC homodimerization is driven by a TIM-containing helix, and biophysical proximity assays showed that newly synthesized, unphosphorylated PKC dimerizes in cells. Furthermore, disruption of the dimer interface by stapled peptides promoted hydrophobic motif phosphorylation. Our data support a model in which mTORC2 relieves nascent PKC dimerization through TIM phosphorylation, recruiting PDK1 to phosphorylate the activation loop and triggering intramolecular hydrophobic motif autophosphorylation. Identification of TIM phosphorylation and its role in the regulation of PKC provides the basis for AGC kinase regulation by mTORC2.

scholarly journals Negative regulation of Raf-1 by phosphorylation of serine 621.

1996 ◽  
Vol 16 (10) ◽  
pp. 5409-5418 ◽  
Author(s):  
H Mischak ◽  
T Seitz ◽  
P Janosch ◽  
M Eulitz ◽  
H Steen ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Kinase Domain ◽  
Negative Regulation ◽  
Catalytic Function ◽  
Dependent Protein ◽  
The One

The elevation of cyclic AMP (cAMP) levels in the cell downregulates the activity of the Raf-1 kinase. It has been suggested that this effect is due to the activation of cAMP-dependent protein kinase (PKA), which can directly phosphorylate Raf-1 in vitro. In this study, we confirmed this hypothesis by coexpressing Raf-1 with the constitutively active catalytic subunit of PKA, which could fully reproduce the inhibition previously achieved by cAMP. PKA-phosphorylated Raf-1 exhibits a reduced affinity for GTP-loaded Ras as well as impaired catalytic activity. As the binding to GTP-loaded Ras induces Raf-1 activation in the cell, we examined which mechanism is required for PKA-mediated Raf-1 inhibition in vivo. A Raf-1 point mutant (RafR89L), which is unable to bind Ras, as well as the isolated Raf-1 kinase domain were still fully susceptible to inhibition by PKA, demonstrating that the phosphorylation of the Raf-1 kinase suffices for inhibition. By the use of mass spectroscopy and point mutants, PKA phosphorylation site was mapped to a single site in the Raf-1 kinase domain, serine 621. Replacement of serine 621 by alanine or cysteine or destruction of the PKA consensus motif by changing arginine 618 resulted in the loss of catalytic activity. Notably, a mutation of serine 619 to alanine did not significantly affect kinase activity or regulation by activators or PKA. Changing serine 621 to aspartic acid yielded a Raf-1 protein which, when expressed to high levels in Sf-9 insect cells, retained a very low inducible kinase activity that was resistant to PKA downregulation. The purified Raf-1 kinase domain displayed slow autophosphorylation of serine 621, which correlated with a decrease in catalytic function. The Raf-1 kinase domain activated by tyrosine phosphorylation could be downregulated by PKA. Specific removal of the phosphate residue at serine 621 reactivated the catalytic activity. These results are most consistent with a dual role of serine 621. On the one hand, serine 621 appears essential for catalytic activity; on the other hand, it serves as a phosphorylation site which confers negative regulation.

scholarly journals TOR Complex 2- independent mutations in the regulatory PIF pocket of Gad8AKT1/SGK1 define separate branches of the stress response mechanisms in fission yeast

PLoS Genetics ◽  
2020 ◽  
Vol 16 (11) ◽  
pp. e1009196
Author(s):  
Emese Pataki ◽  
Luba Simhaev ◽  
Hamutal Engel ◽  
Adiel Cohen ◽  
Martin Kupiec ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Stress Responses ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Genotoxic Stress ◽  
Stress Conditions ◽  
Developmental Processes ◽  
Agc Kinases ◽  
Separate Branch

The Target of rapamycin (TOR) protein kinase forms part of TOR complex 1 (TORC1) and TOR complex 2 (TORC2), two multi-subunit protein complexes that regulate growth, proliferation, survival and developmental processes by phosphorylation and activation of AGC-family kinases. In the fission yeast, Schizosaccharomyces pombe, TORC2 and its target, the AGC kinase Gad8 (an orthologue of human AKT or SGK1) are required for viability under stress conditions and for developmental processes in response to starvation cues. In this study, we describe the isolation of gad8 mutant alleles that bypass the requirement for TORC2 and reveal a separation of function of TORC2 and Gad8 under stress conditions. In particular, osmotic and nutritional stress responses appear to form a separate branch from genotoxic stress responses downstream of TORC2-Gad8. Interestingly, TORC2-independent mutations map into the regulatory PIF pocket of Gad8, a highly conserved motif in AGC kinases that regulates substrate binding in PDK1 (phosphoinositide dependent kinase-1) and kinase activity in several AGC kinases. Gad8 activation is thought to require a two-step mechanism, in which phosphorylation by TORC2 allows further phosphorylation and activation by Ksg1 (an orthologue of PDK1). We focus on the Gad8-K263C mutation and demonstrate that it renders the Gad8 kinase activity independent of TORC2 in vitro and independent of the phosphorylation sites of TORC2 in vivo. Molecular dynamics simulations of Gad8-K263C revealed abnormal high flexibility at T387, the phosphorylation site for Ksg1, suggesting a mechanism for the TORC2-independent Gad8 activity. Significantly, the K263 residue is highly conserved in the family of AGC-kinases, which may suggest a general way of keeping their activity in check when acting downstream of TOR complexes.

scholarly journals Activation of Jak2 catalytic activity requires phosphorylation of Y1007 in the kinase activation loop.

1997 ◽  
Vol 17 (5) ◽  
pp. 2497-2501 ◽  
Author(s):  
J Feng ◽  
B A Witthuhn ◽  
T Matsuda ◽  
F Kohlhuber ◽  
I M Kerr ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Kinase Activity ◽  
Critical Role ◽  
Regulatory Region ◽  
Kinase Domain ◽  
Detectable Effect ◽  
Activation Loop ◽  
Receptor Complexes ◽  
Induced Aggregation

The Janus protein tyrosine kinases (Jaks) play critical roles in transducing growth and differentiation signals emanating from ligand-activated cytokine receptor complexes. The activation of the Jaks is hypothesized to occur as a consequence of auto- or transphosphorylation on tyrosine residues associated with ligand-induced aggregation of the receptor chains and the associated Jaks. In many kinases, regulation of catalytic activity by phosphorylation occurs on residues within the activation loop of the kinase domain. Within the Jak2 kinase domain, there is a region that has considerable sequence homology to the regulatory region of the insulin receptor and contains two tyrosines, Y1007 and Y1008, that are potential regulatory sites. In the studies presented here, we demonstrate that among a variety of sites, Y1007 and Y1008 are sites of trans- or autophosphorylation in vivo and in in vitro kinase reactions. Mutation of Y1007, or both Y1007 and Y1008, to phenylalanine essentially eliminated kinase activity, whereas mutation of Y1008 to phenylalanine had no detectable effect on kinase activity. The mutants were also examined for the ability to reconstitute erythropoietin signaling in gamma2 cells, which lack Jak2. Consistent with the kinase activity, mutation of Y1007 to phenylalanine eliminated the ability to restore signaling. Moreover, phosphorylation of a kinase-inactive mutant (K882E) was not detected, indicating that Jak2 activation during receptor aggregation is dependent on Jak2 and not another receptor-associated kinase. The results demonstrate the critical role of phosphorylation of Y1007 in Jak2 regulation and function.

The hydrophobic motif of ROCK2 requires association with the N-terminal extension for kinase activity

2009 ◽  
Vol 419 (1) ◽  
pp. 141-148 ◽  
Author(s):  
Amber L. Couzens ◽  
Vivian Saridakis ◽  
Michael P. Scheid
Keyword(s):  
Growth Factor ◽  
Kinase Activity ◽  
Coiled Coil ◽  
Kinase Domain ◽  
Agc Kinases ◽  
Threonine Residue ◽  
Substrate Phosphorylation ◽  
Hydrophobic Motif

ROCK (Rho-associated coiled-coil kinase) 2 is a member of the AGC kinase family that plays an essential role downstream of Rho in actin cytoskeleton assembly and contractility. The process of ROCK2 activation is complex and requires suppression of an autoinhibitory mechanism that is facilitated by Rho binding. ROCK2 harbours a C-terminal extension within the kinase domain that contains a hydrophobic cluster of phenylalanine and tyrosine residues surrounding a key threonine residue. In growth-factor-stimulated AGC kinases, the hydrophobic motif is important for the transition of the kinase from inactive to active complex and requires phosphorylation of the conserved serine/threonine residue. Less is understood about the contribution that the hydrophobic motif plays in the activation of ROCK, and the role of the hydrophobic motif threonine at position 405. In the present study, we show that this residue of ROCK is essential for substrate phosphorylation and kinase domain dimerization. However, in contrast with the growth-factor-activated AGC kinases, a phosphomimetic residue at position 405 was inhibitory for ROCK2 activity and dimerization. A soluble hydrophobic motif peptide allosterically activated ROCK2 In vitro, but not the equivalent peptide with Asp405 substitution. Mechanistically, both ROCK2 activity and dimerization were dependent upon the interaction between Thr405 of the hydrophobic motif and Asp39 of the N-terminal extension. The reciprocal exchange of these residues was permissive for kinase activity, but dimerization was lost. These results support the rationale for development of small-molecule inhibitors designed to block ROCK activation by selectively interfering with hydrophobic motif-mediated activation-state transition and dimer formation.

scholarly journals Characterization of Rictor Phosphorylation Sites Reveals Direct Regulation of mTOR Complex 2 by S6K1

2009 ◽  
Vol 29 (21) ◽  
pp. 5657-5670 ◽  
Author(s):  
Christian C. Dibble ◽  
John M. Asara ◽  
Brendan D. Manning
Keyword(s):  
Control Cell ◽  
Mammalian Target Of Rapamycin ◽  
Phosphorylation Site ◽  
Phosphorylation Sites ◽  
Mediated Effects ◽  
Phosphorylation Event ◽  
Liquid Chromatography Tandem Mass ◽  
Precise Molecular Mechanism

ABSTRACT The mammalian target of rapamycin (mTOR) functions within two distinct complexes (mTORC1 and mTORC2) to control cell growth, proliferation, survival, and metabolism. While there has been great progress in our understanding of mTORC1 regulation, the signaling mechanisms that regulate mTORC2 have not been defined. In this study, we use liquid chromatography-tandem mass spectrometry analyses to identify 21 phosphorylation sites on the core mTORC2 component Rictor. We find that one site, T1135, undergoes growth factor-responsive phosphorylation that is acutely sensitive to rapamycin and is phosphorylated downstream of mTORC1. We find that Rictor-T1135 is directly phosphorylated by the mTORC1-dependent kinase S6K1. Although this phosphorylation event does not affect mTORC2 integrity or in vitro kinase activity, expression of a phosphorylation site mutant of Rictor (T1135A) in either wild-type or Rictor null cells causes an increase in the mTORC2-dependent phosphorylation of Akt on S473. However, Rictor-T1135 phosphorylation does not appear to regulate mTORC2-mediated effects on SGK1 or PKCα. While the precise molecular mechanism affecting Akt is unknown, phosphorylation of T1135 stimulates binding of Rictor to 14-3-3 proteins. We provide evidence that Rictor-T1135 phosphorylation acts in parallel with other mTORC1-dependent feedback mechanisms, such as those affecting IRS-1 signaling to PI3K, to regulate the response of Akt to insulin.

scholarly journals Discovery of catalytically active orthologues of the Parkinson's disease kinase PINK1: analysis of substrate specificity and impact of mutations

Open Biology ◽  
2011 ◽  
Vol 1 (3) ◽  
pp. 110012 ◽  
Author(s):  
Helen I. Woodroof ◽  
Joe H. Pogson ◽  
Mike Begley ◽  
Lewis C. Cantley ◽  
Maria Deak ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Substrate Specificity ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Kinase Domain ◽  
Missense Mutations ◽  
Pediculus Humanus ◽  
Tensin Homolog

Missense mutations of the phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) gene cause autosomal-recessive Parkinson's disease. To date, little is known about the intrinsic catalytic properties of PINK1 since the human enzyme displays such low kinase activity in vitro . We have discovered that, in contrast to mammalian PINK1, insect orthologues of PINK1 we have investigated—namely Drosophila melanogaster (dPINK1) , Tribolium castaneum (TcPINK1) and Pediculus humanus corporis (PhcPINK1)—are active as judged by their ability to phosphorylate the generic substrate myelin basic protein. We have exploited the most active orthologue, TcPINK1, to assess its substrate specificity and elaborated a peptide substrate (PINKtide, KKWIpYRRSPRRR) that can be employed to quantify PINK1 kinase activity. Analysis of PINKtide variants reveal that PINK1 phosphorylates serine or threonine, but not tyrosine, and we show that PINK1 exhibits a preference for a proline at the +1 position relative to the phosphorylation site. We have also, for the first time, been able to investigate the effect of Parkinson's disease-associated PINK1 missense mutations, and found that nearly all those located within the kinase domain, as well as the C-terminal non-catalytic region, markedly suppress kinase activity. This emphasizes the crucial importance of PINK1 kinase activity in preventing the development of Parkinson's disease. Our findings will aid future studies aimed at understanding how the activity of PINK1 is regulated and the identification of physiological substrates.

scholarly journals BRCA1 Is Phosphorylated at Serine 1497 In Vivo at a Cyclin-Dependent Kinase 2 Phosphorylation Site

1999 ◽  
Vol 19 (7) ◽  
pp. 4843-4854 ◽  
Author(s):  
Heinz Ruffner ◽  
Wei Jiang ◽  
A. Grey Craig ◽  
Tony Hunter ◽  
Inder M. Verma
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Cyclin A ◽  
Dominant Negative ◽  
Protein Kinase Activity ◽  
Cyclin Dependent Kinase

ABSTRACT BRCA1 is a cell cycle-regulated nuclear protein that is phosphorylated mainly on serine and to a lesser extent on threonine residues. Changes in phosphorylation occur in response to cell cycle progression and DNA damage. Specifically, BRCA1 undergoes hyperphosphorylation during late G1 and S phases of the cell cycle. Here we report that BRCA1 is phosphorylated in vivo at serine 1497 (S1497), which is part of a cyclin-dependent kinase (CDK) consensus site. S1497 can be phosphorylated in vitro by CDK2-cyclin A or E. BRCA1 coimmunoprecipitates with an endogenous serine-threonine protein kinase activity that phosphorylates S1497 in vitro. This cellular kinase activity is sensitive to transfection of a dominant negative form of CDK2 as well as the application of the CDK inhibitors p21 and butyrolactone I but not p16. Furthermore, BRCA1 coimmunoprecipitates with CDK2 and cyclin A. These results suggest that the endogenous kinase activity is composed of CDK2-cyclin complexes, at least in part, concordant with the G1/S-specific increase in BRCA1 phosphorylation.

scholarly journals Autophosphorylation in the Activation Loop Is Required for Full Kinase Activity In Vivo of Human and Yeast Eukaryotic Initiation Factor 2α Kinases PKR and GCN2

1998 ◽  
Vol 18 (4) ◽  
pp. 2282-2297 ◽  
Author(s):  
Patrick R. Romano ◽  
Minerva T. Garcia-Barrio ◽  
Xiaolong Zhang ◽  
Qizhi Wang ◽  
Deborah R. Taylor ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Regulatory Region ◽  
Yeast Cells ◽  
Mass Spectrometry Analysis ◽  
Initiation Factor ◽  
Interferon Response ◽  
Activation Loop ◽  
Eukaryotic Initiation Factor

ABSTRACT The human double-stranded RNA-dependent protein kinase (PKR) is an important component of the interferon response to virus infection. The activation of PKR is accompanied by autophosphorylation at multiple sites, including one in the N-terminal regulatory region (Thr-258) that is required for full kinase activity. Several protein kinases are activated by phosphorylation in the region between kinase subdomains VII and VIII, referred to as the activation loop. We show that Thr-446 and Thr-451 in the PKR activation loop are required in vivo and in vitro for high-level kinase activity. Mutation of either residue to Ala impaired translational control by PKR in yeast cells and COS1 cells and led to tumor formation in mice. These mutations also impaired autophosphorylation and eukaryotic initiation factor 2 subunit α (eIF2α) phosphorylation by PKR in vitro. Whereas the Ala-446 substitution substantially reduced PKR function, the mutant kinase containing Ala-451 was completely inactive. PKR specifically phosphorylated Thr-446 and Thr-451 in synthetic peptides in vitro, and mass spectrometry analysis of PKR phosphopeptides confirmed that Thr-446 is an autophosphorylation site in vivo. Substitution of Glu-490 in subdomain X of PKR partially restored kinase activity when combined with the Ala-451 mutation. This finding suggests that the interaction between subdomain X and the activation loop, described previously for MAP kinase, is a regulatory feature conserved in PKR. We found that the yeast eIF2α kinase GCN2 autophosphorylates at Thr-882 and Thr-887, located in the activation loop at exactly the same positions as Thr-446 and Thr-451 in PKR. Thr-887 was more critically required than was Thr-882 for GCN2 kinase activity, paralleling the relative importance of Thr-446 and Thr-451 in PKR. These results indicate striking similarities between GCN2 and PKR in the importance of autophosphorylation and the conserved Thr residues in the activation loop.

scholarly journals Complex Effects of Naturally Occurring Mutations in the JAK3 Pseudokinase Domain: Evidence for Interactions between the Kinase and Pseudokinase Domains

2000 ◽  
Vol 20 (3) ◽  
pp. 947-956 ◽  
Author(s):  
Min Chen ◽  
Alan Cheng ◽  
Fabio Candotti ◽  
Yong-Jie Zhou ◽  
Anka Hymel ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Intramolecular Interaction ◽  
Severe Combined Immunodeficiency ◽  
Kinase Domain ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT The structure of Janus kinases (JAKs) is unique among protein tyrosine kinases in having tandem, nonidentical kinase and pseudokinase domains. Despite its conservation in evolution, however, the function of the pseudokinase domain remains poorly understood. Lack of JAK3 expression results in severe combined immunodeficiency (SCID). In this study, we analyze two SCID patients with mutations in the JAK3 pseudokinase domain, which allows for protein expression but disrupts the regulation of the kinase activity. Specifically, these mutant forms of JAK3 had undetectable kinase activity in vitro but were hyperphosphorylated both in patients' Epstein-Barr virus-transformed B cells and when overexpressed in COS7 cells. Moreover, reconstitution of cells with these mutants demonstrated that, although they were constitutively phosphorylated basally, they were unable to transmit cytokine-dependent signals. Further analysis showed that the isolated catalytic domain of JAK3 was functional whereas either the addition of the pseudokinase domain or its deletion from the full-length molecule reduced catalytic activity. Through coimmunoprecipitation of the isolated pseudokinase domain with the isolated catalytic domain, we provide the first evidence that these two domains interact. Furthermore, whereas the wild-type pseudokinase domain modestly inhibited kinase domain-mediated STAT5 phosphorylation, the patient-derived mutants markedly inhibited this phosphorylation. We thus conclude that the JAK3 pseudokinase domain is essential for JAK3 function by regulating its catalytic activity and autophosphorylation. We propose a model in which this occurs via intramolecular interaction with the kinase domain and that increased inhibition of kinase activity by the pseudokinase domain likely contributes to the disease pathogenesis in these two patients.

Structural Positioning of the SH2 Domain Is Critical for Bcr-Abl Kinase Activity, Signal Transduction and Oncogenic Transformation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 569-569
Author(s):  
Oliver D. Hantschel ◽  
Florian Grebien ◽  
Ines Kaupe ◽  
Giulio Superti-Furga
Keyword(s):  
Signal Transduction ◽  
Catalytic Activity ◽  
Kinase Activity ◽  
Critical Factor ◽  
Kinase Domain ◽  
Sh2 Domain ◽  
Pharmacological Intervention ◽  
Single Point Mutation ◽  
Mouse Bone Marrow

Abstract We have recently shown that the SH2 domain stimulates c-Abl catalytic activity and substrate phosphorylation. This effect is exerted directly through the establishment of a tight SH2-kinase domain interface in the active conformation of c-Abl (Filippakopoulos et al. (2008) Cell, in press, scheduled to be published on September 5, 2008). Mutations in the SH2 domain that presumably disrupt this SH2-kinase domain interface, such as the Ile164Glu mutation, result in severe impairment of Abl catalytic activity. Thus, correct positioning of the SH2 and kinase domain modules appears to be critical for efficient activation of cytoplasmic tyrosine kinases. Here, we present data showing that the same structural coupling of the SH2 and kinase domain is also a critical factor for full activation of the oncogenic fusion kinase Bcr-Abl. A single point mutation in the SH2 domain (Ile164Glu) led to a dramatic reduction in Bcr-Abl in vitro tyrosine kinase activity and Bcr-Abl autophosphorylation, both on the activation loop (pTyr-412) and the SH2-kinase domain linker (pTyr-245). This resulted in a strong decrease in global cellular tyrosine phosphorylation, as well as decreased phosphorylation of critical downstream mediators of Bcr-Abl signaling. Both wildtype Bcr-Abl, as well as the Bcr-Abl Ile164Glu mutant were able to confer factor independent growth to Ba/F3- and UT-7 cell lines, although to a different extent. Detailed data on the properties of the Ile164Glu mutation in vitro, in imatinib inhibition assays, transformation assays and mouse bone marrow transplant models will be presented. We propose that the structural positioning of the SH2 domain is a crucial factor for constitutive activity, signal transduction and transforming capacity of Bcr-Abl. Besides oligomerization via the N-terminal coiled-coiled domain and loss of the auto-inhibitory N-terminal myristoyl group, the proper positioning of the SH2 domain appears to be another critical factor that is required for constitutive activation of Bcr- Abl, which is the prerequisite for its ability to induce chronic myeloid leukemia (CML). Inhibitors of the SH2-kinase domain interface of Bcr-Abl may comprise alternative or additional points of pharmacological intervention for the treatment of imatinib-sensitive or -resistant CML or Ph+ acute lymphocytic leukemia.

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