RESUMO
The p38 MAPK (mitogen-activated protein kinase)/MK2 [MAPKAP (MAPK-activated protein) kinase-2] signalling pathway is a major regulator of stress- and cytokine-induced gene expression at the transcriptional and post-transcriptional level. Using phosphoproteomics we identified the ER (endoplasmic reticulum)-associated ubiquitin-conjugating enzyme Ube2j1 as a potential substrate of MK2. We demonstrate that Ube2j1 is phosphorylated in a cytokine-, cytosolic stress- and LPS (lipopolysaccharide)-induced manner. The cytosolic stress-induced phosphorylation of Ube2j1 proceeds at Ser(184), a site described previously to be phosphorylated in response to ER stress, which is located in a perfect MK2 consensus motif. The cytosolic stress-induced phosphorylation of Ube2j1, but not its ER-stress-induced phosphorylation is sensitive to p38/MK2 inhibitors and abrogated in MK2/MK3-deficient cells. In a pull-down assay we demonstrate the interaction of MK2 with Ube2j1 in HEK (human embryonic kidney)-293T cells. Furthermore, MK2 is able to phosphorylate recombinant Ube2j1, but not the S184A mutant in an in vitro kinase assay. These findings strongly suggest that MK2 directly phosphorylates Ube2j1 at Ser(184) upon p38-activating stress in vivo. However, ectopically expressed Ube2j1-S184A mutant displays ubiquitinating activity towards the model substrate ER-synthesized T-cell receptor-α similar to that of the wild-type protein. Interestingly, Ube2j1 is phosphorylated in response to LPS also in macrophages and contributes to MK2-dependent TNFα biosynthesis by a so far unknown mechanism.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação , Animais , Linhagem Celular , Estresse do Retículo Endoplasmático , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteólise , Estresse Fisiológico , Receptores Toll-Like/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Receptor-interacting protein kinase-1 (RIPK1), a master regulator of cell fate decisions, was identified as a direct substrate of MAPKAP kinase-2 (MK2) by phosphoproteomic screens using LPS-treated macrophages and stress-stimulated embryonic fibroblasts. p38MAPK/MK2 interact with RIPK1 in a cytoplasmic complex and MK2 phosphorylates mouse RIPK1 at Ser321/336 in response to pro-inflammatory stimuli, such as TNF and LPS, and infection with the pathogen Yersinia enterocolitica. MK2 phosphorylation inhibits RIPK1 autophosphorylation, curtails RIPK1 integration into cytoplasmic cytotoxic complexes, and suppresses RIPK1-dependent apoptosis and necroptosis. In Yersinia-infected macrophages, RIPK1 phosphorylation by MK2 protects against infection-induced apoptosis, a process targeted by Yersinia outer protein P (YopP). YopP suppresses p38MAPK/MK2 activation to increase Yersinia-driven apoptosis. Hence, MK2 phosphorylation of RIPK1 is a crucial checkpoint for cell fate in inflammation and infection that determines the outcome of bacteria-host cell interaction.
Assuntos
Apoptose , Inflamação/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Yersiniose/enzimologia , Yersinia enterocolitica/patogenicidade , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Citosol/enzimologia , Citosol/microbiologia , Feminino , Genótipo , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Quinase I-kappa B/metabolismo , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , MAP Quinase Quinase Quinases/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Macrófagos/patologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos Knockout , Necrose , Fenótipo , Fosforilação , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Serina , Transdução de Sinais , Fatores de Tempo , Transfecção , Fator de Necrose Tumoral alfa/toxicidade , Yersiniose/microbiologia , Yersiniose/patologia , Yersinia enterocolitica/metabolismoRESUMO
Myocardin-related transcription factor A (MRTF-A) is a known actin-regulated transcriptional coactivator of serum response factor (SRF). Stimulation of actin polymerization activates MRTF-A by releasing it from G-actin and thus allowing it to bind to and activate SRF. Here, we compared protein phosphorylation in MK2/3-deficient cells rescued or not by ectopic expression of MK2 in two independent phosphoproteomic approaches using anisomycin-treated MEF cells and LPS-stimulated mouse macrophages, respectively. Two MRTF-A sites, Ser(351) (corresponding to Ser(312) in human) and Ser(371) (Ser(333) in human), showed significantly stronger phosphorylation (12-fold and 6-fold increase) in the cells expressing MK2. MRTF-A is phosphorylated at these sites in a stress-, but not in a mitogen-induced manner, and p38(MAPK)/MK2 catalytic activities are indispensable for this phosphorylation. MK2-mediated phosphorylation of MRTF-A at Ser(312) and Ser(333) was further confirmed in an in vitro kinase assay and using the phospho-protein kinase-D (PKD)-consensus motif antibody (anti-LXRXXpS/pT), the p38(MAPK) inhibitor BIRB-796, MK2/3-deficient cells and MRTF-A phospho-site mutants. Unexpectedly, dimerization, subcellular localization and translocation, interaction with actin, SRF or SMAD3 and transactivating potential of MRTF-A seem to be unaffected by manipulating the p38(MAPK)/MK2-dependent phosphorylations. Hence, MRTF-A is stress-dependently phosphorylated by MK2 at Ser(312) and Ser(333) with so far undetected functional and physiological consequences.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/genética , Proteômica/métodos , Estresse Fisiológico , Transativadores/química , Transativadores/metabolismo , Animais , Anisomicina/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Mutação , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Serina/metabolismo , Transdução de SinaisRESUMO
Septin 7 (SEPT7) has been described to be essential for successful completion of cytokinesis in mouse fibroblasts, and Sept7-deficiency in fibroblasts constitutively results in multinucleated cells which stop proliferation. Using Sept7(flox/flox)fibroblasts we generated a cellular system, where the cytokinetic defects of Cre-mediated deletion of the Sept7 gene can be rescued by ectopically expressed doxycycline-inducible wild type SEPT7. Using this system, we analyzed the ability of SEPT7-mutants with alterations in their GTPase domain-dependent dimerization to prevent multinucleation and rescue proliferation. Although biochemical analysis of the mutants demonstrates differences in homo- and/or hetero-polymerization, in GTP-binding and/or GTPase activities, all analyzed mutants were able to rescue the cytokinesis phenotype of Sept7(flox/flox)fibroblasts associated with Cre-mediated deletion of endogenous Sept7. These findings indicate that the ability of septins to assemble into well-defined SEPT7-dimerization dependent native filaments is dispensable for cytokinesis in fibroblasts and opens the way to search for other mechanisms of the involvement of SEPT7 in cytokinesis.
Assuntos
Citocinese , Fibroblastos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Septinas/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , Expressão Gênica , Técnicas de Inativação de Genes , Hidrólise , Camundongos , Modelos Moleculares , Mutação , Nucleotídeos/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Septinas/química , Septinas/genéticaRESUMO
The neural cell adhesion molecule (NCAM) mediates cell-cell and cell-matrix adhesion. It is broadly expressed in the nervous system and regulates neurite outgrowth, synaptogenesis, and synaptic plasticity. Previous in vitro studies revealed that palmitoylation of NCAM is required for fibroblast growth factor 2 (FGF2)-stimulated neurite outgrowth and identified the zinc finger DHHC (Asp-His-His-Cys)-containing proteins ZDHHC3 and ZDHHC7 as specific NCAM-palmitoylating enzymes. Here, we verified that FGF2 controlled NCAM palmitoylation in vivo and investigated molecular mechanisms regulating NCAM palmitoylation by ZDHHC3. Experiments with overexpression and pharmacological inhibition of FGF receptor (FGFR) and Src revealed that these kinases control tyrosine phosphorylation of ZDHHC3 and that ZDHHC3 is phosphorylated by endogenously expressed FGFR and Src proteins. By site-directed mutagenesis, we found that Tyr18 is an FGFR1-specific ZDHHC3 phosphorylation site, while Tyr295 and Tyr297 are specifically phosphorylated by Src kinase in cell-based and cell-free assays. Abrogation of tyrosine phosphorylation increased ZDHHC3 autopalmitoylation, enhanced interaction with NCAM, and upregulated NCAM palmitoylation. Expression of ZDHHC3 with tyrosine mutated in cultured hippocampal neurons promoted neurite outgrowth. Our findings for the first time highlight that FGFR- and Src-mediated tyrosine phosphorylation of ZDHHC3 modulates ZDHHC3 enzymatic activity and plays a role in neuronal morphogenesis.
Assuntos
Aciltransferases/metabolismo , Proteínas de Membrana/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Tirosina/metabolismo , Quinases da Família src/metabolismo , Aciltransferases/genética , Animais , Células Cultivadas , Fator 2 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Lipoilação , Proteínas de Membrana/genética , Camundongos , Mutação , Neuritos/metabolismo , Fosforilação , Tirosina/genéticaRESUMO
MK5 (MAPK-activated protein kinase 5) or PRAK (p38-regulated and -activated kinase) are alternative names for a serine/threonine protein kinase downstream to ERK3/4 and p38 MAPK. A previous gene targeting approach for MK5/PRAK (termed here MK5/PRAK-Δex8) revealed a seemingly tumor-suppressive role of MK5/PRAK in DMBA-induced one step skin carcinogenesis and Ras-induced transformation. Here we demonstrate that an alternative targeting strategy of MK5/PRAK (termed MK5/PRAK-Δex6) increased neither tumor incidence in the one step skin carcinogenesis model, nor Ras-induced transformation in primary cells. Interestingly, due to the targeting strategies and exon skipping both knockouts do not completely abolish the generation of MK5/PRAK protein, but express MK5/PRAK deletion mutants with different biochemical properties depending on the exon targeted: Targeting of exon 6 leads to expression of an unstable cytoplasmic catalytically inactive MK5/PRAK-Δex6 mutant while targeting of exon 8 results in a more stable nuclear MK5/PRAK-Δex8 mutant with residual catalytic activity. The different properties of the MK5/PRAK deletion mutants could be responsible for the observed discrepancy between the knockout strains and challenge the role of MK5/PRAK in p53-dependent tumor suppression. Further MK5/PRAK knockout and knock-in mouse strains will be necessary to assign a physiological function to MK5/PRAK in this model organism.