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1.
Sci Signal ; 15(753): eabk1147, 2022 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-36166510

RESUMO

Spinocerebellar ataxia type 14 (SCA14) is a neurodegenerative disease caused by germline variants in the diacylglycerol (DAG)/Ca2+-regulated protein kinase Cγ (PKCγ), leading to Purkinje cell degeneration and progressive cerebellar dysfunction. Most of the identified mutations cluster in the DAG-sensing C1 domains. Here, we found with a FRET-based activity reporter that SCA14-associated PKCγ mutations, including a previously undescribed variant, D115Y, enhanced the basal activity of the kinase by compromising its autoinhibition. Unlike other mutations in PKC that impair its autoinhibition but lead to its degradation, the C1 domain mutations protected PKCγ from such down-regulation. This enhanced basal signaling rewired the brain phosphoproteome, as revealed by phosphoproteomic analysis of cerebella from mice expressing a human SCA14-associated H101Y mutant PKCγ transgene. Mutations that induced a high basal activity in vitro were associated with earlier average age of onset in patients. Furthermore, the extent of disrupted autoinhibition, but not agonist-stimulated activity, correlated with disease severity. Molecular modeling indicated that almost all SCA14 variants not within the C1 domain were located at interfaces with the C1B domain, suggesting that mutations in and proximal to the C1B domain are a susceptibility for SCA14 because they uniquely enhance PKCγ basal activity while protecting the enzyme from down-regulation. These results provide insight into how PKCγ activation is modulated and how deregulation of the cerebellar phosphoproteome by SCA14-associated mutations affects disease progression.


Assuntos
Diglicerídeos , Ataxias Espinocerebelares , Animais , Diglicerídeos/metabolismo , Humanos , Camundongos , Mutação , Proteína Quinase C , Células de Purkinje/metabolismo , Ataxias Espinocerebelares/genética
2.
Trends Biochem Sci ; 47(6): 518-530, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35361526

RESUMO

Protein kinase C (PKC) isozymes are maintained in a 'ready-to-go' but 'safe' autoinhibited conformation until second messenger binding unleashes an autoinhibitory pseudosubstrate to allow substrate phosphorylation. However, to gain this 'ready-to-go' conformation, PKC must be processed by a series of complex priming phosphorylations, the mechanism of which was enigmatic until now. Recent findings snapped the pieces of the phosphorylation puzzle into place to unveil a process that involves a newly described motif (TOR interaction motif, TIM), a well-described kinase [mechanistic target of rapamycin complex 2 (mTORC2)], and an often-used mechanism (autophosphorylation) to prime PKC to signal. This review highlights new insights into how phosphorylation controls PKC and discusses them in the context of common mechanisms for AGC kinase regulation by phosphorylation and autophosphorylation.


Assuntos
Proteína Quinase C , Quarentena , Isoenzimas/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Fosforilação , Proteína Quinase C/metabolismo
3.
Mol Pharmacol ; 101(4): 213-218, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34155089

RESUMO

The family of AGC kinases not only regulates cellular biology by phosphorylating substrates but is itself controlled by phosphorylation. Phosphorylation generally occurs at two conserved regions in these kinases: a loop near the entrance to the active site, termed the activation loop, that correctly aligns residues for catalysis, and a C-terminal tail whose phosphorylation at a site termed the hydrophobic motif stabilizes the active conformation. Whereas phosphorylation of the activation loop is well established to be catalyzed by the phosphoinositide-dependent kinase 1, the mechanism of phosphorylation of the C-tail hydrophobic motif has been controversial. For a subset of AGC kinases, which include most protein kinase C (PKC) isozymes and Akt, phosphorylation of the hydrophobic motif in cells was shown to depend on mTORC2 over 15 years ago, yet whether this was by direct phosphorylation or by another mechanism has remained elusive. The recent identification of a novel and evolutionarily conserved phosphorylation site on the C-tail, termed the TOR interaction motif (TIM), has finally unraveled the mystery of how mTORC2 regulates its client kinases. mTORC2 does not directly phosphorylate the hydrophobic motif; instead, it converts kinases such as PKC and Akt into a conformation that can ultimately autophosphorylate at the hydrophobic motif. Identification of the direct mTOR phosphorylation that facilitates autoregulation of the C-tail hydrophobic motif revises the activation mechanisms of mTOR-regulated AGC kinases. This new twist to an old tail opens avenues for therapeutic intervention. SIGNIFICANCE STATEMENT: The enzyme mTORC2 has been an enigmatic regulator of AGC kinases such as protein kinase C (PKC) and Akt. The recent discovery of a motif named the TOR interaction motif in the C-tail of these kinases solves the mystery: mTORC2 marks these kinases for maturity by, ultimately, facilitating autophosphorylation of another C-tail site, the hydrophobic motif.


Assuntos
Proteínas Proto-Oncogênicas c-akt , Serina-Treonina Quinases TOR , Humanos , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Fosforilação/fisiologia , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo
4.
Sci Signal ; 14(678)2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33850054

RESUMO

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 PKCßII 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.


Assuntos
Alvo Mecanístico do Complexo 2 de Rapamicina , Peptídeos , Proteína Quinase C , Proteínas Proto-Oncogênicas c-akt , Motivos de Aminoácidos , Animais , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Camundongos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo
5.
J Biol Chem ; 296: 100445, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33617877

RESUMO

Within the AGC kinase superfamily, gene fusions resulting from chromosomal rearrangements have been most frequently described for protein kinase C (PKC), with gene fragments encoding either the C-terminal catalytic domain or the N-terminal regulatory moiety fused to other genes. Kinase fusions that eliminate regulatory domains are typically gain of function and often oncogenic. However, several quality control pathways prevent accumulation of aberrant PKC, suggesting that PKC fusions may paradoxically be loss of function. To explore this topic, we used biochemical, cellular, and genome editing approaches to investigate the function of fusions that retain the portion of the gene encoding either the catalytic domain or regulatory domain of PKC. Overexpression studies revealed that PKC catalytic domain fusions were constitutively active but vulnerable to degradation. Genome editing of endogenous genes to generate a cancer-associated PKC fusion resulted in cells with detectable levels of fusion transcript but no detectable protein. Hence, PKC catalytic domain fusions are paradoxically loss of function as a result of their instability, preventing appreciable accumulation of protein in cells. Overexpression of a PKC regulatory domain fusion suppressed both basal and agonist-induced endogenous PKC activity, acting in a dominant-negative manner by competing for diacylglycerol. For both catalytic and regulatory domain fusions, the PKC component of the fusion proteins mediated the effects of the full-length fusions on the parameters examined, suggesting that the partner protein is dispensable in these contexts. Taken together, our findings reveal that PKC gene fusions are distinct from oncogenic fusions and present a mechanism by which loss of PKC function occurs in cancer.


Assuntos
Neoplasias/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Animais , Sítios de Ligação , Células COS , Domínio Catalítico , Linhagem Celular Tumoral , Chlorocebus aethiops , Diglicerídeos/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Mutação com Perda de Função/genética , Fosforilação , Domínios Proteicos , Proteína Quinase C-alfa/genética , Proteína Quinase C-alfa/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
6.
Annu Rev Pharmacol Toxicol ; 61: 723-743, 2021 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-32997603

RESUMO

Whereas protein kinases have been successfully targeted for a variety of diseases, protein phosphatases remain an underutilized therapeutic target, in part because of incomplete characterization of their effects on signaling networks. The pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) is a relatively new player in the cell signaling field, and new roles in controlling the balance among cell survival, proliferation, and apoptosis are being increasingly identified. Originally characterized for its tumor-suppressive function in deactivating the prosurvival kinase Akt, PHLPP may have an opposing role in promoting survival, as recent evidence suggests. Additionally, identification of the transcription factor STAT1 as a substrate unveils a role for PHLPP as a critical mediator of transcriptional programs in cancer and the inflammatory response. This review summarizes the current knowledge of PHLPP as both a tumor suppressor and an oncogene and highlights emerging functions in regulating gene expression and the immune system. Understanding the context-dependent functions of PHLPP is essential for appropriate therapeutic intervention.


Assuntos
Neoplasias , Proteínas Proto-Oncogênicas c-akt , Humanos , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
7.
Mol Cell ; 74(2): 378-392.e5, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30904392

RESUMO

Protein kinase C (PKC) isozymes function as tumor suppressors in increasing contexts. In contrast to oncogenic kinases, whose function is acutely regulated by transient phosphorylation, PKC is constitutively phosphorylated following biosynthesis to yield a stable, autoinhibited enzyme that is reversibly activated by second messengers. Here, we report that the phosphatase PHLPP1 opposes PKC phosphorylation during maturation, leading to the degradation of aberrantly active species that do not become autoinhibited. Cancer-associated hotspot mutations in the pseudosubstrate of PKCß that impair autoinhibition result in dephosphorylated and unstable enzymes. Protein-level analysis reveals that PKCα is fully phosphorylated at the PHLPP site in over 5,000 patient tumors, with higher PKC levels correlating (1) inversely with PHLPP1 levels and (2) positively with improved survival in pancreatic adenocarcinoma. Thus, PHLPP1 provides a proofreading step that maintains the fidelity of PKC autoinhibition and reveals a prominent loss-of-function mechanism in cancer by suppressing the steady-state levels of PKC.


Assuntos
Neoplasias/genética , Proteínas Nucleares/genética , Fosfoproteínas Fosfatases/genética , Proteína Quinase C beta/genética , Proteína Quinase C-alfa/genética , Humanos , Isoenzimas/genética , Mutação com Perda de Função/genética , Neoplasias/patologia , Fosforilação , Proteólise , Proteínas Proto-Oncogênicas c-akt/genética , Controle de Qualidade , Transdução de Sinais/genética
8.
Sci Rep ; 8(1): 6518, 2018 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-29695735

RESUMO

Many bioinformatics resources with unique perspectives on the protein landscape are currently available. However, generating new knowledge from these resources requires interoperable workflows that support cross-resource queries. In this study, we employ federated queries linking information from the Protein Kinase Ontology, iPTMnet, Protein Ontology, neXtProt, and the Mouse Genome Informatics to identify key knowledge gaps in the functional coverage of the human kinome and prioritize understudied kinases, cancer variants and post-translational modifications (PTMs) for functional studies. We identify 32 functional domains enriched in cancer variants and PTMs and generate mechanistic hypotheses on overlapping variant and PTM sites by aggregating information at the residue, protein, pathway and species level from these resources. We experimentally test the hypothesis that S768 phosphorylation in the C-helix of EGFR is inhibitory by showing that oncogenic variants altering S768 phosphorylation increase basal EGFR activity. In contrast, oncogenic variants altering conserved phosphorylation sites in the 'hydrophobic motif' of PKCßII (S660F and S660C) are loss-of-function in that they reduce kinase activity and enhance membrane translocation. Our studies provide a framework for integrative, consistent, and reproducible annotation of the cancer kinomes.


Assuntos
Mutação/genética , Neoplasias/genética , Proteínas Quinases/genética , Processamento de Proteína Pós-Traducional/genética , Proteínas/genética , Animais , Células CHO , Células COS , Linhagem Celular , Chlorocebus aethiops , Biologia Computacional/métodos , Cricetulus , Ontologia Genética , Variação Genética/genética , Humanos , Camundongos , Fosforilação/genética
9.
Mol Biosyst ; 12(12): 3651-3665, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27731453

RESUMO

Multiple sequence alignments (MSAs) are a fundamental analysis tool used throughout biology to investigate relationships between protein sequence, structure, function, evolutionary history, and patterns of disease-associated variants. However, their widespread application in systems biology research is currently hindered by the lack of user-friendly tools to simultaneously visualize, manipulate and query the information conceptualized in large sequence alignments, and the challenges in integrating MSAs with multiple orthogonal data such as cancer variants and post-translational modifications, which are often stored in heterogeneous data sources and formats. Here, we present the Multiple Sequence Alignment Ontology (MSAOnt), which represents a profile or consensus alignment in an ontological format. Subsets of the alignment are easily selected through the SPARQL Protocol and RDF Query Language for downstream statistical analysis or visualization. We have also created the Kinome Viewer (KinView), an interactive integrative visualization that places eukaryotic protein kinase cancer variants in the context of natural sequence variation and experimentally determined post-translational modifications, which play central roles in the regulation of cellular signaling pathways. Using KinView, we identified differential phosphorylation patterns between tyrosine and serine/threonine kinases in the activation segment, a major kinase regulatory region that is often mutated in proliferative diseases. We discuss cancer variants that disrupt phosphorylation sites in the activation segment, and show how KinView can be used as a comparative tool to identify differences and similarities in natural variation, cancer variants and post-translational modifications between kinase groups, families and subfamilies. Based on KinView comparisons, we identify and experimentally characterize a regulatory tyrosine (Y177PLK4) in the PLK4 C-terminal activation segment region termed the P+1 loop. To further demonstrate the application of KinView in hypothesis generation and testing, we formulate and validate a hypothesis explaining a novel predicted loss-of-function variant (D523NPKCß) in the regulatory spine of PKCß, a recently identified tumor suppressor kinase. KinView provides a novel, extensible interface for performing comparative analyses between subsets of kinases and for integrating multiple types of residue specific annotations in user friendly formats.


Assuntos
Biologia Computacional/métodos , Proteínas Quinases/química , Proteínas Quinases/genética , Análise de Sequência/métodos , Software , Sequência de Aminoácidos , Mutação , Fosforilação , Matrizes de Pontuação de Posição Específica , Domínios e Motivos de Interação entre Proteínas , Proteína Quinase C beta/genética , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional , Receptores de Fatores de Crescimento de Fibroblastos/química , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/química , Receptores do Fator de Crescimento Derivado de Plaquetas/genética
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