RESUMO
The protein kinase C (PKC) family, discovered in the late 1970s, is composed of at least 10 serine/threonine kinases, divided into three groups based on their molecular architecture and cofactor requirements. PKC enzymes have been conserved throughout evolution and are expressed in virtually all cell types; they represent critical signal transducers regulating cell activation, differentiation, proliferation, death, and effector functions. PKC family members play important roles in a diverse array of hematopoietic and immune responses. This review covers the discovery and history of this enzyme family, discusses the roles of PKC enzymes in the development and effector functions of major hematopoietic and immune cell types, and points out gaps in our knowledge, which should ignite interest and further exploration, ultimately leading to better understanding of this enzyme family and, above all, its role in the many facets of the immune system.
Assuntos
Hematopoese , Sistema Imunitário , Proteína Quinase C/metabolismo , Animais , Coenzimas/metabolismo , Ativação Enzimática/imunologia , Humanos , Proteína Quinase C/imunologia , Transdução de SinaisRESUMO
The interferon (IFN) pathway is critical for cytotoxic T cell activation, which is central to tumor immunosurveillance and successful immunotherapy. We demonstrate here that PKCλ/ι inactivation results in the hyper-stimulation of the IFN cascade and the enhanced recruitment of CD8+ T cells that impaired the growth of intestinal tumors. PKCλ/ι directly phosphorylates and represses the activity of ULK2, promoting its degradation through an endosomal microautophagy-driven ubiquitin-dependent mechanism. Loss of PKCλ/ι results in increased levels of enzymatically active ULK2, which, by direct phosphorylation, activates TBK1 to foster the activation of the STING-mediated IFN response. PKCλ/ι inactivation also triggers autophagy, which prevents STING degradation by chaperone-mediated autophagy. Thus, PKCλ/ι is a hub regulating the IFN pathway and three autophagic mechanisms that serve to maintain its homeostatic control. Importantly, single-cell multiplex imaging and bioinformatics analysis demonstrated that low PKCλ/ι levels correlate with enhanced IFN signaling and good prognosis in colorectal cancer patients.
Assuntos
Neoplasias Colorretais/metabolismo , Interferons/metabolismo , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de Sinais , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Autofagia , Linfócitos T CD8-Positivos/metabolismo , Carcinogênese , Transformação Celular Neoplásica , Neoplasias Colorretais/mortalidade , Cicloeximida/química , Feminino , Células HEK293 , Humanos , Imunofenotipagem , Fator Regulador 3 de Interferon/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Pessoa de Meia-Idade , Transplante de Neoplasias , Fosforilação , Prognóstico , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição , Regulação para CimaRESUMO
Serrated adenocarcinoma, an alternative pathway for colorectal cancer (CRC) development, accounts for 15%-30% of all CRCs and is aggressive and treatment resistant. We show that the expression of atypical protein kinase C ζ (PKCζ) and PKCλ/ι was reduced in human serrated tumors. Simultaneous inactivation of the encoding genes in the mouse intestinal epithelium resulted in spontaneous serrated tumorigenesis that progressed to advanced cancer with a strongly reactive and immunosuppressive stroma. Whereas epithelial PKCλ/ι deficiency led to immunogenic cell death and the infiltration of CD8+ T cells, which repressed tumor initiation, PKCζ loss impaired interferon and CD8+ T cell responses, which resulted in tumorigenesis. Combined treatment with a TGF-ß receptor inhibitor plus anti-PD-L1 checkpoint blockade showed synergistic curative activity. Analysis of human samples supported the relevance of these kinases in the immunosurveillance defects of human serrated CRC. These findings provide insight into avenues for the detection and treatment of this poor-prognosis subtype of CRC.
Assuntos
Mucosa Intestinal/imunologia , Neoplasias Intestinais/imunologia , Isoenzimas/imunologia , Proteína Quinase C/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/imunologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/metabolismo , Feminino , Humanos , Vigilância Imunológica/genética , Vigilância Imunológica/imunologia , Mucosa Intestinal/enzimologia , Mucosa Intestinal/patologia , Neoplasias Intestinais/enzimologia , Neoplasias Intestinais/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismoRESUMO
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/metabolismoRESUMO
The inflammatory response mediated by nuclear factor κB (NF-κB) signaling is essential for host defense against pathogens. Although the regulatory mechanism of NF-κB signaling has been well studied, the molecular basis for epigenetic regulation of the inflammatory response is poorly understood. Here we identify a new signaling axis of PKCα-LSD1-NF-κB, which is critical for activation and amplification of the inflammatory response. In response to excessive inflammatory stimuli, PKCα translocates to the nucleus and phosphorylates LSD1. LSD1 phosphorylation is required for p65 binding and facilitates p65 demethylation, leading to enhanced stability. In vivo genetic analysis using Lsd1SA/SA mice with ablation of LSD1 phosphorylation and chemical approaches in wild-type mice with inhibition of PKCα or LSD1 activity show attenuated sepsis-induced inflammatory lung injury and mortality. Together, we demonstrate that the PKCα-LSD1-NF-κB signaling cascade is crucial for epigenetic control of the inflammatory response, and targeting this signaling could be a powerful therapeutic strategy for systemic inflammatory diseases, including sepsis.
Assuntos
Histona Desmetilases/metabolismo , Proteína Quinase C/metabolismo , Animais , Núcleo Celular/metabolismo , Epigênese Genética/genética , Histona Desmetilases/genética , Inflamação/metabolismo , Metilação , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Fosforilação , Proteína Quinase C/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genética , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Protein kinase C (PKC) functions are essential for synaptic plasticity, learning, and memory. However, the roles of specific members of the PKC family in synaptic function, learning, and memory are poorly understood. Here, we investigated the role of individual PKC homologs for synaptic plasticity in Caenorhabditis elegans and found a differential role for pkc-2 and tpa-1, but not pkc-1 and pkc-3 in associative olfactory learning and memory. More specifically we show that PKC-2 is essential for associative learning and TPA-1 for short-term associative memory (STAM). Using endogenous labeling and cell-specific rescues, we show that TPA-1 and PKC-2 are required in AVA for their functions. Previous studies demonstrated that olfactory learning and memory in C. elegans are tied to proper synaptic content and trafficking of AMPA-type ionotropic glutamate receptor homolog GLR-1 in the AVA command interneurons. Therefore, we quantified synaptic content, transport, and delivery of GLR-1 in AVA and showed that loss of pkc-2 and tpa-1 leads to decreased transport and delivery but only a subtle decrease in GLR-1 levels at synapses. AVA-specific expression of both PKC-2 and TPA-1 rescued these defects. Finally, genetic epistasis showed that PKC-2 and TPA-1 likely act in the same pathway to control GLR-1 transport and delivery, while regulating different aspects of olfactory learning and STAM. Thus, our data tie together cell-specific functions of 2 PKCs to neuronal and behavioral outcomes in C. elegans, enabling comparative approaches to understand the evolutionarily conserved role of PKC in synaptic plasticity, learning, and memory.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Aprendizagem , Proteína Quinase C/metabolismo , Receptores de AMPA/metabolismo , Sinapses/metabolismoRESUMO
Inhibition of protein kinase C (PKC) efficiently promoted the self-renewal of embryonic stem cells (ESCs). However, information about the function of PKC inhibition remains lacking. Here, RNA-sequencing showed that the addition of Go6983 significantly inhibited the expression of de novo methyltransferases (Dnmt3a and Dnmt3b) and their regulator Dnmt3l, resulting in global hypomethylation of DNA in mouse ESCs. Mechanistically, PR domain-containing 14 (Prdm14), a site-specific transcriptional activator, partially contributed to Go6983-mediated repression of Dnmt3 genes. Administration of Go6983 increased Prdm14 expression mainly through the inhibition of PKCδ. High constitutive expression of Prdm14 phenocopied the ability of Go6983 to maintain` mouse ESC stemness in the absence of self-renewal-promoting cytokines. In contrast, the knockdown of Prdm14 eliminated the response to PKC inhibition and substantially impaired the Go6983-induced resistance of mouse ESCs to differentiation. Furthermore, liquid chromatography-mass spectrometry profiling and Western blotting revealed low levels of Suv39h1 and Suv39h2 in Go6983-treated mouse ESCs. Suv39h enzymes are histone methyltransferases that recognize dimethylated and trimethylated histone H3K9 specifically and usually function as transcriptional repressors. Consistently, the inhibition of Suv39h1 by RNA interference or the addition of the selective inhibitor chaetocin increased Prdm14 expression. Moreover, chromatin immunoprecipitation assay showed that Go6983 treatment led to decreased enrichment of dimethylation and trimethylation of H3K9 at the Prdm14 promoter but increased RNA polymerase ⠡ binding affinity. Together, our results provide novel insights into the pivotal association between PKC inhibition-mediated self-renewal and epigenetic changes, which will help us better understand the regulatory network of stem cell pluripotency.
Assuntos
Proteínas de Ligação a DNA , Células-Tronco Embrionárias Murinas , Proteína Quinase C , Animais , Camundongos , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Indóis/farmacologia , Maleimidas/farmacologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/enzimologia , Células-Tronco Embrionárias Murinas/fisiologia , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Rab proteins are small GTPases that regulate a myriad of intracellular membrane trafficking events. Rab29 is one of the Rab proteins phosphorylated by leucine-rich repeat kinase 2 (LRRK2), a Parkinson's disease-associated kinase. Recent studies suggest that Rab29 regulates LRRK2, whereas the mechanism by which Rab29 is regulated remained unclear. Here, we report a novel phosphorylation in Rab29 that is not mediated by LRRK2 and occurs under lysosomal overload stress. Mass spectrometry analysis identified the phosphorylation site of Rab29 as Ser185, and cellular expression studies of phosphomimetic mutants of Rab29 at Ser185 unveiled the involvement of this phosphorylation in counteracting lysosomal enlargement. PKCα and PKCδ were deemed to be involved in this phosphorylation and control the lysosomal localization of Rab29 in concert with LRRK2. These results implicate PKCs in the lysosomal stress response pathway comprised of Rab29 and LRRK2, and further underscore the importance of this pathway in the mechanisms underlying lysosomal homeostasis.
Assuntos
Lisossomos , Proteínas rab de Ligação ao GTP , Fosforilação , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Lisossomos/metabolismo , MutaçãoRESUMO
Apico-basolateral polarization is essential for epithelial cells to function as selective barriers and transporters, and to provide mechanical resilience to organs. Epithelial polarity is established locally, within individual cells to establish distinct apical, junctional and basolateral domains, and globally, within a tissue where cells coordinately orient their apico-basolateral axes. Using live imaging of endogenously tagged proteins and tissue-specific protein depletion in the Caenorhabditiselegans embryonic intestine, we found that local and global polarity establishment are temporally and genetically separable. Local polarity is initiated prior to global polarity and is robust to perturbation. PAR-3 is required for global polarization across the intestine but local polarity can arise in its absence, as small groups of cells eventually established polarized domains in PAR-3-depleted intestines in a HMR-1 (E-cadherin)-dependent manner. Despite the role of PAR-3 in localizing PKC-3 to the apical surface, we additionally found that PAR-3 and PKC-3/aPKC have distinct roles in the establishment and maintenance of local and global polarity. Taken together, our results indicate that different mechanisms are required for local and global polarity establishment in vivo.
Assuntos
Polaridade Celular , Células Epiteliais , Células Epiteliais/metabolismo , Junções Intercelulares , Mucosa Intestinal , Intestinos , EpitélioRESUMO
Influenza viruses remain a major public health concern causing contagious respiratory illnesses that result in around 290,000-650,000 global deaths every year. Their ability to constantly evolve through antigenic shifts and drifts leads to the emergence of newer strains and resistance to existing drugs and vaccines. To combat this, there is a critical need for novel antiviral drugs through the introduction of host-targeted therapeutics. Influenza viruses encode only 14 gene products that get extensively modified through phosphorylation by a diverse array of host kinases. Reversible phosphorylation at serine, threonine, or tyrosine residues dynamically regulates the structure, function, and subcellular localization of viral proteins at different stages of their life cycle. In addition, kinases influence a plethora of signaling pathways that also regulate virus propagation by modulating the host cell environment thus establishing a critical virus-host relationship that is indispensable for executing successful infection. This dependence on host kinases opens up exciting possibilities for developing kinase inhibitors as next-generation anti-influenza therapy. To fully capitalize on this potential, extensive mapping of the influenza virus-host kinase interaction network is essential. The key focus of this review is to outline the molecular mechanisms by which host kinases regulate different steps of the influenza A virus life cycle, starting from attachment-entry to assembly-budding. By assessing the contributions of different host kinases and their specific phosphorylation events during the virus life cycle, we aim to develop a holistic overview of the virus-host kinase interaction network that may shed light on potential targets for novel antiviral interventions.
Assuntos
Interações Hospedeiro-Patógeno , Influenza Humana , Proteínas Quinases , Transdução de Sinais , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , Replicação Viral , Proteínas Quinases/metabolismo , FosforilaçãoRESUMO
The nematode Caenorhabditis elegans learns the concentration of NaCl and moves toward the previously experienced concentration. In this behavior, the history of NaCl concentration change is reflected in the level of diacylglycerol and the activity of protein kinase C, PKC-1, in the gustatory sensory neuron ASER and determines the direction of migration. Here, through a genetic screen, we found that the activation of Gq protein compensates for the behavioral defect of the loss-of-function mutant of pkc-1 We found that Gq activation results in hyperproduction of diacylglycerol in ASER sensory neuron, which leads to recruitment of TPA-1, an nPKC isotype closely related to PKC-1. Unlike the pkc-1 mutants, loss of tpa-1 did not obviously affect migration directions in the conventional learning assay. This difference was suggested to be due to cooperative functions of the C1 and C2-like domains of the nPKC isotypes. Furthermore, we investigated how the compensatory capability of tpa-1 contributes to learning and found that learning was less robust in the context of cognitive decline or environmental perturbation in tpa-1 mutants. These results highlight how two nPKC isotypes contribute to the learning system.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Aprendizagem/fisiologia , Proteína Quinase C/metabolismo , Cloreto de Sódio/metabolismo , Animais , Caenorhabditis elegans/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Mutação , Proteína Quinase C/genética , Células Receptoras Sensoriais , Transdução de Sinais/fisiologiaRESUMO
The activity-dependent plasticity of synapses is believed to be the cellular basis of learning. These synaptic changes are mediated through the coordination of local biochemical reactions in synapses and changes in gene transcription in the nucleus to modulate neuronal circuits and behavior. The protein kinase C (PKC) family of isozymes has long been established as critical for synaptic plasticity. However, because of a lack of suitable isozyme-specific tools, the role of the novel subfamily of PKC isozymes is largely unknown. Here, through the development of fluorescence lifetime imaging-fluorescence resonance energy transfer activity sensors, we investigate novel PKC isozymes in synaptic plasticity in CA1 pyramidal neurons of mice of either sex. We find that PKCδ is activated downstream of TrkB and DAG production, and that the spatiotemporal nature of its activation depends on the plasticity stimulation. In response to single-spine plasticity, PKCδ is activated primarily in the stimulated spine and is required for local expression of plasticity. However, in response to multispine stimulation, a long-lasting and spreading activation of PKCδ scales with the number of spines stimulated and, by regulating cAMP response-element binding protein activity, couples spine plasticity to transcription in the nucleus. Thus, PKCδ plays a dual functional role in facilitating synaptic plasticity.SIGNIFICANCE STATEMENT Synaptic plasticity, or the ability to change the strength of the connections between neurons, underlies learning and memory and is critical for brain health. The protein kinase C (PKC) family is central to this process. However, understanding how these kinases work to mediate plasticity has been limited by a lack of tools to visualize and perturb their activity. Here, we introduce and use new tools to reveal a dual role for PKCδ in facilitating local synaptic plasticity and stabilizing this plasticity through spine-to-nucleus signaling to regulate transcription. This work provides new tools to overcome limitations in studying isozyme-specific PKC function and provides insight into molecular mechanisms of synaptic plasticity.
Assuntos
Isoenzimas , Transdução de Sinais , Animais , Camundongos , Transdução de Sinais/fisiologia , Sinapses/fisiologia , Plasticidade Neuronal/fisiologia , Proteína Quinase C/metabolismoRESUMO
BACKGROUND: Hypothermic ischemia-reperfusion arrhythmia is a common complication of cardiothoracic surgery under cardiopulmonary bypass, but few studies have focused on this type of arrhythmia. Our prior study discovered reduced myocardial Cx43 protein levels may be linked to hypothermic reperfusion arrhythmias. However, more detailed molecular mechanism research is required. METHOD: The microRNA and mRNA expression levels in myocardial tissues were detected by real-time quantitative PCR (RT-qPCR). Besides, the occurrence of hypothermic reperfusion arrhythmias and changes in myocardial electrical conduction were assessed by electrocardiography and ventricular epicardial activation mapping. Furthermore, bioinformatics analysis, applying antagonists of miRNA, western blotting, immunohistochemistry, a dual luciferase assay, and pearson correlation analysis were performed to investigate the underlying molecular mechanisms. RESULTS: The expression level of novel-miR-17 was up-regulated in hypothermic ischemia-reperfusion myocardial tissues. Inhibition of novel-miR-17 upregulation ameliorated cardiomyocyte edema, reduced apoptosis, increased myocardial electrical conduction velocity, and shortened the duration of reperfusion arrhythmias. Mechanistic studies showed that novel-miR-17 reduced the expression of Cx43 by directly targeting Gja1 while mediating the activation of the PKC/c-Jun signaling pathway. CONCLUSION: Up-regulated novel-miR-17 is a newly discovered pro-arrhythmic microRNA that may serve as a potential therapeutic target and biomarker for hypothermic reperfusion arrhythmias.
Assuntos
Arritmias Cardíacas , Conexina 43 , MicroRNAs , Proteína Quinase C , Transdução de Sinais , Animais , Humanos , Masculino , Ratos , Apoptose/genética , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/genética , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/patologia , Conexina 43/metabolismo , Conexina 43/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/etiologia , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Proteína Quinase C/metabolismo , Proteína Quinase C/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Regulação para CimaRESUMO
Pkc53E is the second conventional protein kinase C (PKC) gene expressed in Drosophila photoreceptors; it encodes at least six transcripts generating four distinct protein isoforms including Pkc53E-B whose mRNA is preferentially expressed in photoreceptors. By characterizing transgenic lines expressing Pkc53E-B-GFP, we show Pkc53E-B is localized in the cytosol and rhabdomeres of photoreceptors, and the rhabdomeric localization appears dependent on the diurnal rhythm. A loss of function of pkc53E-B leads to light-dependent retinal degeneration. Interestingly, the knockdown of pkc53E also impacted the actin cytoskeleton of rhabdomeres in a light-independent manner. Here the Actin-GFP reporter is mislocalized and accumulated at the base of the rhabdomere, suggesting that Pkc53E regulates depolymerization of the actin microfilament. We explored the light-dependent regulation of Pkc53E and demonstrated that activation of Pkc53 E can be independent of the phospholipase C PLCß4/NorpA as degeneration of norpAP24 photoreceptors was enhanced by a reduced Pkc53E activity. We further show that the activation of Pkc53E may involve the activation of Plc21C by Gqα. Taken together, Pkc53E-B appears to exert both constitutive and light-regulated activity to promote the maintenance of photoreceptors possibly by regulating the actin cytoskeleton.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Proteína Quinase C , Animais , Citoesqueleto de Actina/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fosfolipases/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismoRESUMO
Tight coordination of growth regulatory signaling is required for intestinal epithelial homeostasis. Protein kinase C α (PKCα) and transforming growth factor ß (TGFß) are negative regulators of proliferation with tumor suppressor properties in the intestine. Here, we identify novel crosstalk between PKCα and TGFß signaling. RNA-Seq analysis of nontransformed intestinal crypt-like cells and colorectal cancer cells identified TGFß receptor 1 (TGFßR1) as a target of PKCα signaling. RT-PCR and immunoblot analysis confirmed that PKCα positively regulates TGFßR1 mRNA and protein expression in these cells. Effects on TGFßR1 were dependent on Ras-extracellular signal-regulated kinase 1/2 (ERK) signaling. Nascent RNA and promoter-reporter analysis indicated that PKCα induces TGFßR1 transcription, and Runx2 was identified as an essential mediator of the effect. PKCα promoted ERK-mediated activating phosphorylation of Runx2, which preceded transcriptional activation of the TGFßR1 gene and induction of Runx2 expression. Thus, we have identified a novel PKCαâERKâRunx2âTGFßR1 signaling axis. In further support of a link between PKCα and TGFß signaling, PKCα knockdown reduced the ability of TGFß to induce SMAD2 phosphorylation and cell cycle arrest, and inhibition of TGFßR1 decreased PKCα-induced upregulation of p21Cip1 and p27Kip1 in intestinal cells. The physiological relevance of these findings is also supported by The Cancer Genome Atlas data showing correlation between PKCα, Runx2, and TGFßR1 mRNA expression in human colorectal cancer. PKCα also regulated TGFßR1 in endometrial cancer cells, and PKCα, Runx2, and TGFßR1 expression correlates in uterine tumors, indicating that crosstalk between PKCα and TGFß signaling may be a common mechanism in diverse epithelial tissues.
Assuntos
Neoplasias Colorretais , Subunidade alfa 1 de Fator de Ligação ao Core , Proteína Quinase C-alfa , Receptor do Fator de Crescimento Transformador beta Tipo I , Humanos , Neoplasias Colorretais/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Células Epiteliais/metabolismo , Intestinos , Proteína Quinase C-alfa/genética , Proteína Quinase C-alfa/metabolismo , RNA Mensageiro/genética , Fator de Crescimento Transformador beta/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismoRESUMO
The functional association between stimulation of G-protein-coupled receptors (GPCRs) by eicosanoids and actin cytoskeleton reorganization remains largely unexplored. Using a model of human adrenocortical cancer cells, here we established that activation of the GPCR OXER1 by its natural agonist, the eicosanoid 5-oxo-eicosatetraenoic acid, leads to the formation of filopodia-like elongated projections connecting adjacent cells, known as tunneling nanotube (TNT)-like structures. This effect is reduced by pertussis toxin and GUE1654, a biased antagonist for the Gßγ pathway downstream of OXER1 activation. We also observed pertussis toxin-dependent TNT biogenesis in response to lysophosphatidic acid, indicative of a general response driven by Gi/o-coupled GPCRs. TNT generation by either 5-oxo-eicosatetraenoic acid or lysophosphatidic acid is partially dependent on the transactivation of the epidermal growth factor receptor and impaired by phosphoinositide 3-kinase inhibition. Subsequent signaling analysis reveals a strict requirement of phospholipase C ß3 and its downstream effector protein kinase Cα. Consistent with the established role of Rho small GTPases in the formation of actin-rich projecting structures, we identified the phosphoinositide 3-kinase-regulated guanine nucleotide exchange factor FARP1 as a GPCR effector essential for TNT formation, acting via Cdc42. Altogether, our study pioneers a link between Gi/o-coupled GPCRs and TNT development and sheds light into the intricate signaling pathways governing the generation of specialized actin-rich elongated structures in response to bioactive signaling lipids.
Assuntos
Actinas , Ácidos Araquidônicos , Estruturas da Membrana Celular , Neoplasias , Receptores Eicosanoides , Humanos , Actinas/metabolismo , Neoplasias/metabolismo , Toxina Pertussis/farmacologia , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C-alfa/genética , Proteína Quinase C-alfa/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Estruturas da Membrana Celular/metabolismo , Nanotubos , Receptores Eicosanoides/antagonistas & inibidores , Receptores Eicosanoides/metabolismo , Linhagem Celular Tumoral , Ácidos Araquidônicos/metabolismo , Ácidos Araquidônicos/farmacologia , Transdução de SinaisRESUMO
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 SinaisRESUMO
Many cells can pause their growth cycle, a topic much enriched by studies of the stationary phase (SP) of model microorganisms. Although several kinases are implicated in SP onset, whether protein kinase C has a role remains unknown. We show that Dictyostelium discoideum cells lacking pkcA entered SP at a reduced cell density, but only in shaking conditions. Precocious SP entry occurs because levels of extracellular polyphosphate (polyP) reach the threshold needed to induce the SP onset at a lower cell density than seen in wild-type cells; adding exopolyphosphatase to pkcA- cells reverses the effect and mimics wild-type growth. PkcA-mediated regulation of polyP depended on inositol hexakisphosphate kinase and phospholipase D. PkcA- mutants also had higher F-actin levels, higher rates of exocytosis and lower pinocytosis rates. Postlysosomes were smaller and present in fewer pkcA- cells compared to the wild type. Overall, the results suggest that a reduced PkcA level triggers SP primarily because cells do not acquire or retain nutrients as efficiently, thus mimicking, or amplifying, the conditions of actual starvation. This article has an associated First Person interview with the first author of the paper.
Assuntos
Dictyostelium , Actinas/metabolismo , Dictyostelium/metabolismo , Exocitose , Humanos , Pinocitose , Polifosfatos/metabolismoRESUMO
Fertilization triggers significant cellular remodeling through the oocyte-to-embryo transition. In this transition, the ubiquitin-proteasome system and autophagy are essential for the degradation of maternal components; however, the significance of degradation of cell surface components remains unknown. In this study, we show that multiple maternal plasma membrane proteins, such as the glycine transporter GlyT1a, are selectively internalized from the plasma membrane to endosomes in mouse embryos by the late two-cell stage and then transported to lysosomes for degradation at the later stages. During this process, large amounts of ubiquitylated proteins accumulated on endosomes. Furthermore, the degradation of GlyT1a with mutations in potential ubiquitylation sites was delayed, suggesting that ubiquitylation may be involved in GlyT1a degradation. The clathrin inhibitor blocked GlyT1a internalization. Strikingly, the protein kinase C (PKC) activator triggered the heterochronic internalization of GlyT1a; the PKC inhibitor markedly blocked GlyT1a endocytosis. Lastly, clathrin inhibition completely blocked embryogenesis at the two-cell stage and inhibited cell division after the four-cell stage. These findings demonstrate that PKC-dependent clathrin-mediated endocytosis is essential for the selective degradation of maternal membrane proteins during oocyte-to-embryo transition and early embryogenesis.
Assuntos
Clatrina/metabolismo , Desenvolvimento Embrionário/fisiologia , Endocitose/fisiologia , Proteínas de Membrana/metabolismo , Animais , Membrana Celular/metabolismo , Embrião de Mamíferos , Endossomos/metabolismo , Feminino , Fertilização , Proteínas da Membrana Plasmática de Transporte de Glicina , Masculino , Camundongos , Oócitos , Proteína Quinase C , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Maternal decidual cells are crucial for the maintenance of canine pregnancy as they are the only cells expressing the nuclear progesterone (P4) receptor (PGR) in the placenta. Interfering with P4/PGR signaling adversely affects decidual cells and terminates pregnancy. Although immortalized dog uterine stromal (DUS) cells can be decidualized in vitro using cAMP, the involvement of cAMP-dependent kinases in canine decidualization had not been investigated. Therefore, the present project investigated changes in the kinome of DUS cells following in vitro decidualization, using the serine/threonine kinase (STK) PamChip assay (PamGene). Decidualization led to a predicted activation of 85 STKs in DUS cells, including protein kinase (PK) A, PKC, extracellular signal-regulated kinase (ERK)1/2 and other mitogen-activated protein kinases (MAPKs), calcium/calmodulin-dependent protein kinases (CAMKs), and Akt1/2. In addition, blocking PGR with type 2 antigestagens (aglepristone or mifepristone) decreased the activity of virtually all kinases modulated by decidualization. The underlying transcriptional effects were inferred from comparison with available transcriptomic data on antigestagen-mediated effects in DUS cells. In targeted studies, interfering with PKA or MAPK kinase (MEK)1/2 resulted in downregulation of important decidualization markers (e.g., insulin-like growth factor 1 (IGF1), prostaglandin E2 synthase (PTGES), prolactin receptor (PRLR), PGR, and prostaglandin-endoperoxide synthase 2 (PTGS2/COX2)). Conversely, blocking of PKC decreased the mRNA availability of IGF1, PGR, and PTGS2, but not of PTGES and PRLR. Moreover, suppressing PKA decreased the phosphorylation of the transcription factors cJUN and CREB, whereas blocking of PKC affected only cJUN. This first kinomics analysis to target decidualization showed an increased activity of a wide range of STKs, which could be hindered by disrupting P4/PGR signaling. Decidualization appears to be regulated in a kinase-dependent manner, with PKA and PKC evoking different effects.