Protein kinase C ζ interacts with a novel binding region of Gαq to act as a functional effector

Heterotrimeric G proteins play an essential role in the initiation of G protein-coupled receptor (GPCR) signaling through specific interactions with a variety of cellular effectors. We have recently reported that GPCR activation promotes a direct interaction between Gαq and protein kinase C ζ (PKCζ), leading to the stimulation of the ERK5 pathway independent of the canonical effector PLCβ. We report herein that the activation-dependent Gαq/PKCζ complex involves the basic PB1-type II domain of PKCζ and a novel interaction module in Gαq different from the classical effector-binding site. Point mutations in this Gαq region completely abrogate ERK5 phosphorylation, indicating that Gαq/PKCζ association is required for the activation of the pathway. Indeed, PKCζ was demonstrated to directly bind ERK5 thus acting as a scaffold between Gαq and ERK5 upon GPCR activation. The inhibition of these protein complexes by G proteincoupled receptor kinase 2, a known Gαq modulator, led to a complete abrogation of ERK5 stimulation. Finally, we reveal thatGαq/ PKCζ complexes link Gαq to apoptotic cell death pathways. Our data suggest that the interaction between this novel region in Gαq and the effector PKCζ is a key event in Gαq signaling.Ministerio de Educación y Ciencia (SAF2011-23800, SAF2014-55511-R), Fundación Ramón Areces, The Cardiovascular Diseases Network of Ministerio Sanidad y Consumo-Instituto Carlos III (RD12/0042/0012), Comunidad de Madrid (S-2011/BMD-2332), and Instituto de Salud Carlos III (PI11/00126, PI14/00201) (to F. M. and C. R.). This work was also supported in part by the NIGMS, National Institutes of Health Grant R01-GM088242 (to G. T.), the Canadian Institutes of Health Research (CIHR) (MOP-GMX-231013) (to S. M.), an EMBO Short Fellowship (to G. S. F.), and Fondo Europeo de Desarrollo Regional (FEDER, European Union)Peer Reviewe

Papel deL PKC[zeta] en la estimulación de ERK5 por GPCRs acoplados a proteínas Gq y su repercusión en hipertrofia cardiaca

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 27-06-08In this work we show that PKCζ plays a key role in the activation of the ERK5 pathway by Gqcoupled GPCR in epithelial cells and in cardiomyocytes as well as in the induction of cardiac hypertrophy by agonists acting through Gq-coupled GPCR such as angiotensin II. Several lines of evidence support the notion that a functional interaction between Gαq and PKCζ mediates the triggering of the ERK5 cascade by Gq-coupled GPCR. First, ERK5 stimulation by carbachol or angiotensin II does not appear to require the activity of EGF-Receptors (EGFR) or cytosolic tyrosine kinases, known to participate in ERK5 activation in response to different mitogens, thus suggesting that potential GPCR/EGFR transactivation mechanisms are not involved. Second, overexpression of a constitutively active Gαq subunit mutant promotes “per se” ERK5 stimulation, independently of its ability to interact with the classical Gαq effector PLCβ. Third, Gαq (and not other Gα subunits) associates with PKCζ in cells, and co-immunoprecipitation of these endogenous proteins can be promoted upon Gq-coupled GPCR activation. Moreover, a direct Gαq/PKCζ interaction can be observed using purified proteins. Fourth, Gαq, PKCζ and MEK5 (the upstream ERK5 activator) are present in the same multimolecular complex as assessed by co-immunoprecipitation experiments. Interestingly, Gαq also directly interacts with MEK5, suggesting a scaffold role for this G protein subunit in the stimulation of this pathway. Finally, stimulation of ERK5 by Gq-coupled GPCR is blocked by PKCζ pharmacological inhibitors and absent in MEFs or cardiomyocytes derived from PKCζ-deficient mice. Thus, these data put forward PKCζ as a novel Gαq effector required for ERK5 activation by Gqcoupled GPCR. Moreover, the finding that PKCζ-deficient mice do not develop angiotensin-induced cardiac hypertrophy indicates an important physiopathological role for this novel Gαq/PKCζ/ERK5 signaling axis. In sum, the novel Gαq/PKCζ/ERK5 signal transduction route presented in this study may help to better understand the mechanisms underlying GPCR-induced cardiac hypertrophy and in the design of new therapeutic strategies. On the other hand, it opens new avenues to investigate the potential role of this pathway in other cell types and physiological processes.EL PRESENTE TRABAJO HA SIDO REALIZADO EN EL DEPARTAMENTO BIOLOGÍA MOLECULAR Y EN EL CENTRO DE BIOLOGÍA MOLECULAR SEVERO OCHOA (CSIC-UAM) DE LA FACULTAD DE CIENCIAS DE LA UNIVERSIDAD AUTÓNOMA DE MADRID, CON LA AYUDA DE UNA BECA PREDOCTORAL FPI, DEL FONDO DE INVESTIGACIÓN DEL MINISTERIO DE EDUCACIÓN Y CIENCIA (MEC) Y UN CONTRATO DE LA RED DE INVESTIGACIÓN COOPERATIVA EN ENFERMEDADES CARDIOVASCULARES (RECAVA) DEL INSTITUTO DE SALUD CARLOS III. PARTE DEL TRABAJO PRESENTADO HA SIDO REALIZADO EN EL DEPARTAMENTO DE BIOMEDICINA DE LA UNIVERSIDAD DE BERGEN, CON LA AYUDA DE UNA BECA DE ESTANCIA BREVE DEL FONDO DE INVESTIGACIÓN DEL MINISTERIO DE EDUCACIÓN Y CIENCIA (MEC) Y CON LA AYUDA DE UNA BECA DE ESTANCIA BREVE EMBO CONCEDIDA POR LA EUROPEAN MOLECULAR BIOLOGY ORGANIZATIONPeer reviewe

ERK5 Activation by Gq-Coupled Muscarinic Receptors is Independent of Receptors Iternalization and beta-Arrestin Recruitment

This work was funded by the Ministerio de Ciencia e Innovacion (MICINN) grants (SAF2008-00211; PIE- 200820I166), and a grant from the Spanish Comunidad de Madrid CCG08-CSIC/SAL-3464. G-protein-coupled receptors (GPCRs) are known to activate both G protein- and β -arrestin-dependent signalling cascades. The initiation of mitogen-activated protein kinase (MAPK) pathways is a key downstream event in the control of cellular functions including proliferation, differentiation, migration and apoptosis. Both G proteins and β-arrestins have been reported to mediate context-specific activation of ERK1/2, p38 and JNK MAPKs. Recently, the activation of ERK5 MAPK by Gq-coupled receptors has been described to involve a direct interaction between G αqand two novel effectors, PKCζ and MEK5. However, the possible contribution of β-arrestin towards this pathway has not yet been addressed. In the present work we sought to investigate the role of receptor internalization processes and β-arrestin recruitment in the activation of ERK5 by Gq-coupled GPCRs. Our results show that ERK5 activation is independent of M1 or M3 muscarinic receptor internalization. Furthermore, we demonstrate that phosphorylation-deficient muscarinic M1 and M3 receptors are still able to fully activate the ERK5 pathway, despite their reported inability to recruit β-arrestins. Indeed, the overexpression of Gαq, but not that of β-arrestin1 or β-arrestin2, was found to potently enhance ERK5 activation by GPCRs, whereas silencing of β-arrestin2 expression did not affect the activation of this pathway. Finally, we show that a β-arrestin-biased mutant form of angiotensin II (SII; Sar1-Ile4-Ile8AngII) failed to promote ERK5 phosphorylation in primary cardiac fibroblasts, as compared to the natural ligand. Overall, this study shows that the activation of ERK5 MAPK by model Gq-coupled GPCRs does not depend on receptor internalization, β-arrestin recruitment or receptor phosphorylation but rather is dependent on Gαq-signalling.The study was funded by grants from Ministerio de Educación y Ciencia (SAF2011-23800), Fundación Ramón Areces, The Cardiovascular Diseases Network of Ministerio Sanidad y Consumo-Instituto Carlos III (RD12/0042/0012), Comunidad de Madrid (S-2011/BMD-2332) and Instituto de Salud Carlos III (PI11/00126). Collaboration with Dr. Andrew Tobin was possible thanks to a Wood-Whelan Research Felowship (IUMBM) awarded to GSF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer Reviewe

Gαq signalling: The new and the old

In the last few years the interactome of Gαq has expanded considerably, contributing to improve our understanding of the cellular and physiological events controlled by this G alpha subunit. The availability of high-resolution crystal structures has led the identification of an effector-binding region within the surface of Gαq that is able to recognise a variety of effector proteins. Consequently, it has been possible to ascribe different Gαq functions to specific cellular players and to identify important processes that are triggered independently of the canonical activation of phospholipase Cβ (PLCβ), the first identified Gαq effector. Novel effectors include p63RhoGEF, that provides a link between G protein-coupled receptors and RhoA activation, phosphatidylinositol 3-kinase (PI3K), implicated in the regulation of the Akt pathway, or the cold-activated TRPM8 channel, which is directly inhibited upon Gαq binding. Recently, the activation of ERK5 MAPK by Gq-coupled receptors has also been described as a novel PLCβ-independent signalling axis that relies upon the interaction between this G protein and two novel effectors (PKCφ and MEK5). Additionally, the association of Gαq with different regulatory proteins can modulate its effector coupling ability and, therefore, its signalling potential. Regulators include accessory proteins that facilitate effector activation or, alternatively, inhibitory proteins that downregulate effector binding or promote signal termination. Moreover, Gαq is known to interact with several components of the cytoskeleton as well as with important organisers of membrane microdomains, which suggests that efficient signalling complexes might be confined to specific subcellular environments. Overall, the complex interaction network of Gαq underlies an ever-expanding functional diversity that puts forward this G alpha subunit as a major player in the control of physiological functions and in the development of different pathological situations. © 2014 Elsevier Inc.Our laboratory is funded by grants from Instituto de Salud Carlos III (PI11/00126), Ministerio de Educación y Ciencia (SAF2011-23800), Fundación Ramón Areces, The Cardiovascular Diseases Network of Ministerio Sanidad y Consumo-Instituto Carlos III (RD12/0042/0012), Comunidad de Madrid (S-2011/BMD-2332)Peer Reviewe

The G protein-coupled receptor kinase (GRK) interactome: Role of GRKs in GPCR regulation and signaling

AbstractG protein-coupled receptor kinases (GRKs) and arrestins are key participants in the canonical pathways leading to phosphorylation-dependent GPCR desensitization, endocytosis, intracellular trafficking and resensitization as well as in the modulation of important intracellular signaling cascades by GPCR. Novel studies have revealed a phosphorylation-independent desensitization mechanism operating through their RGS-homology (RH) domain and the recent determination of the crystal structures of GRK2 and GRK6 has uncovered interesting details on the structure–function relationships of these kinases. Emerging evidence indicates that the activity of GRKs is tightly modulated by mechanisms including phosphorylation by different kinases and interaction with several cellular proteins such as calmodulin, caveolin or RKIP. In addition, GRKs are involved in multiple interactions with non-receptor proteins (PI3K, Akt, GIT or MEK) that point to novel GRK cellular roles. In this article, our purpose is to describe the ever increasing map of functional interactions for GRK proteins as a basis to better understand its contribution to cellular processes

Protein kinase C (PKC)ζ-mediated Gα q stimulation of ERK5 protein pathway in cardiomyocytes and cardiac fibroblasts

Gq-coupled G protein-coupled receptors (GPCRs) mediate the actions of a variety of messengers that are key regulators of cardiovascular function. Enhanced Gα q-mediated signaling plays an important role in cardiac hypertrophy and in the transition to heart failure. We have recently described that Gα q acts as an adaptor protein that facilitates PKCζ-mediated activation of ERK5 in epithelial cells. Because the ERK5 cascade is known to be involved in cardiac hypertrophy, we have investigated the potential relevance of this pathway in cardiovascular Gq-dependent signaling using both cultured cardiac cell types and chronic administration of angiotensin II in mice. We find that PKCζ is required for the activation of the ERK5 pathway by Gq-coupled GPCRin neonatal and adult murine cardiomyocyte cultures and in cardiac fibroblasts. Stimulation of ERK5 by angiotensin II is blocked upon pharmacological inhibition or siRNA-mediated silencing of PKCζ in primary cultures of cardiac cells and in neonatal cardiomyocytes isolated from PKCζ-deficient mice. Moreover, upon chronic challenge with angiotensin II, these mice fail to promote the changes in the ERK5 pathway, in gene expression patterns, and in hypertrophic markers observed in wild-type animals. Taken together, our results show that PKCζ is essential for Gq-dependent ERK5 activation in cardiomyocytes and cardiac fibroblasts and indicate a key cardiac physiological role for the Gα q/PKCζ/ERK5 signaling axis. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.This work was supported by grants from the Ministerio de Ciencia e Innovación (SAF2008-00552), Fundación Ramón Areces, The Cardiovascular Network (RECAVA) of Instituto de Salud Carlos III (RD06-0014/0037), Comunidad de Madrid (S-SAL-0159-2006 to F. M.), and Instituto de Salud Carlos III (PI080461 to C. R.).Peer Reviewe

β-arrestins are not involved in ERK5 activation by Gq-coupled muscarinic receptors.

<p>(A) CHO cells stably expressing <i>wild-type</i> muscarinic M3 receptor (CHO-M3 cells) were transfected with cDNAs encoding for HA-ERK5 and either pcDNA3, Gαq, β-arrestin1-Flag or β-arrestin2-GFP. Twenty-four hours after transfection, cells were serum-starved for 2h and stimulated with carbachol (10µM) for the indicated times. ERK5 phosphorylation was assessed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084174#pone-0084174-g002" target="_blank">Figure 2</a>. Data (mean +/- SEM of 3 independent experiments) were normalised using HA-ERK5 as loading control and expressed as fold-induction over basal conditions (*p<0.05, **p<0.005, two-tailed T-test). Gαq, β-arrestin1-Flag and β-arrestin2-GFP, and α-tubulin expression levels were assessed with specific antibodies. (B) NIH-3T3-M1 cells were transfected with siRNA oligonucleotides targeting β-arrestin2 or non-targeting scrambled oligonucleotides (100nM) as detailed in the Materials and Methods section. After 72h of incubation, cells were serum-starved for 5h and challenged with carbachol (10µM) for the indicated times. Endogenous ERK5 phosphorylation was assessed in cell lysates with a phospho-ERK5 specific antibody. Total ERK5 appears as a double band corresponding to basal (lower) and hyper-phosphorylated (upper) kinase. A representative blot for 3 independent experiments with similar results is shown. (C) β-arrestin2 expression levels were also determined and quantified to estimate the overall efficiency of protein silencing. Data (mean +/- SEM of 3 independent experiments) were normalised using α-tubulin as loading control and expressed as the relative difference (%) to β-arrestin2 protein levels in scrambled siRNA-treated cells.</p

Gq-coupled muscarinic receptor-induced activation of the ERK5 pathway does not require receptor phosphorylation.

<p>Different stable lines of CHO cells (previously characterised in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084174#B11" target="_blank">11</a>]) that stably overexpress <i>wild-type</i> or phosphorylation-deficient muscarinic M3 receptor (A) and <i>wild-type</i> or phosphorylation-deficient muscarinic M1 receptor (B) were utilised. All cell lines were transfected with HA-ERK5. Twenty-four hours after transfection, cells were serum-starved for 2h and stimulated with acetylcholine (100µM) for the indicated times (A) or incubated for 15min with acetylcholine at various concentrations (B). ERK5 phosphorylation was assessed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084174#pone-0084174-g002" target="_blank">Figure 2</a>. Data (mean +/- SD of 2-3 independent experiments) were normalised using HA-ERK5 as loading control and expressed as fold-induction over basal conditions or over maximum activation (*p<0.05, **p<0.005; two-tailed T-test). </p

Protein kinase C (PKC)ζ-mediated Gαq stimulation of ERK5 protein pathway in cardiomyocytes and cardiac fibroblasts

Gq-coupled G protein-coupled receptors (GPCRs) mediate the actions of a variety of messengers that are key regulators of cardiovascular function. Enhanced Gα(q)-mediated signaling plays an important role in cardiac hypertrophy and in the transition to heart failure. We have recently described that Gα(q) acts as an adaptor protein that facilitates PKCζ-mediated activation of ERK5 in epithelial cells. Because the ERK5 cascade is known to be involved in cardiac hypertrophy, we have investigated the potential relevance of this pathway in cardiovascular Gq-dependent signaling using both cultured cardiac cell types and chronic administration of angiotensin II in mice. We find that PKCζ is required for the activation of the ERK5 pathway by Gq-coupled GPCR in neonatal and adult murine cardiomyocyte cultures and in cardiac fibroblasts. Stimulation of ERK5 by angiotensin II is blocked upon pharmacological inhibition or siRNA-mediated silencing of PKCζ in primary cultures of cardiac cells and in neonatal cardiomyocytes isolated from PKCζ-deficient mice. Moreover, upon chronic challenge with angiotensin II, these mice fail to promote the changes in the ERK5 pathway, in gene expression patterns, and in hypertrophic markers observed in wild-type animals. Taken together, our results show that PKCζ is essential for Gq-dependent ERK5 activation in cardiomyocytes and cardiac fibroblasts and indicate a key cardiac physiological role for the Gα(q)/PKCζ/ERK5 signaling axis

Gq-coupled muscarinic receptor-induced activation of the ERK5 pathway does not require receptor internalization.

<p>(A) CHO cells stably overexpressing <i>wild-type</i> muscarinic M3 receptor or internalization-deficient M3 receptor (SASS motif mutant, characterised in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084174#B10" target="_blank">10</a>]) were transfected with HA-ERK5. Twenty-four hours after transfection, cells were serum-starved for 2h and stimulated with carbachol (10µM). HA-ERK5 was immunoprecipitated with an anti-HA agarose-conjugated antibody as detailed in the Materials and Methods section. ERK5 phosphorylation was assessed in the immunoprecipitate using a phosphospecific antibody. Data (mean +/- SEM of 3 independent experiments) were normalised using HA-ERK5 as loading control and expressed as fold-induction over basal conditions (*p<0.05, two-tailed T-test). (B) Quantification of cell surface receptor density was performed through [³H]-NMS binding at 4°C. The two cell types utilised in (A) were serum-starved for 2h and stimulated with carbachol (100µM) for 30 minutes. Data were normalised to unspecific binding (atropine treatment) and binding percentage was expressed as the mean +/- SEM of 3 independent experiments.</p