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Jean-vianney Barnier - One of the best experts on this subject based on the ideXlab platform.
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PAK3 controls the tangential to radial migration switch of cortical interneurons by coordinating changes in cell shape and polarity
2020Co-Authors: Lucie Viou, Zhengping Jia, Véronique Rousseau, Pierre Launay, Justine Masson, Clarisse Pace, Robert S. Adelstein, Fujio Murakami, Jean-vianney BarnierAbstract:SUMMARY During the embryonic development, cortical interneurons migrate a long distance tangentially and then re-orient radially to settle in the cortical plate where they contribute to cortical circuits. Migrating interneurons express PAK3, a p21-activated kinase that switches between active and inactive states and controls interneuron migration by unknown mechanism(s). Here we examined the role of the kinase activity of PAK3 to regulate the migration of cortical interneurons. We showed that interneurons expressing a constitutively active PAK3 mutant (PAK3-ca) oriented preferentially radially in the cortex, extended short leading processes and exhibited unstable polarity. On the contrary, interneurons expressing an inactive PAK3 mutant (PAK3-kd for kinase dead) extended branched leading processes, showed directed nuclear movements and remained in the tangential pathways. Results showed that PAK3 kinase activity controls the switch between the tangential and radial modes of migration of cortical interneurons and identified myosin 2B as an effector of this switch.
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The intellectual disability protein PAK3 regulates oligodendrocyte precursor cell differentiation
Neurobiology of Disease, 2017Co-Authors: Majistor Raj Luxman Maglorius Renkilaraj, Lucas Baudouin, Vidjeacoumary Cannaya, Jean-vianney Barnier, Zhengping Jia, C M Wells, Rosine Wehrlé, Mohamed Doulazmi, Corinne Bachelin, Brahim Nait-oumesmarAbstract:Oligodendrocyte and myelin deficits have been reported in mental/psychiatric diseases. The p21-activated kinase 3 (PAK3), a serine/threonine kinase, whose activity is stimulated by the binding of active Rac and Cdc42 GTPases is affected in these pathologies. Indeed, many mutations of PAK3 gene have been described in non-syndromic intellectual disability diseases. PAK3 is expressed mainly in the brain where its role has been investigated in neurons but not in glial cells. Here, we showed that PAK3 is highly expressed in oligodendrocyte precursors (OPCs) and its expression decreases in mature oligodendrocytes. In the developing white matter of the PAK3 knockout mice, we found defects of oligodendrocyte differentiation in the corpus callosum and to a lesser extent in the anterior commissure, which were compensated at the adult stage. In vitro experiments in OPC cultures, derived from PAK3 knockout and wild type brains, support a developmental and cell-autonomous role for PAK3 in regulating OPC differentiation into mature oligodendrocytes. Moreover, we did not detect any obvious alterations of the proliferation or migration of PAK3 null OPCs compared to wild type. Overall, our data highlight PAK3 as a new regulator of OPC differentiation.
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PAK3 promotes cell cycle exit and differentiation of β-cells in the embryonic pancreas and is necessary to maintain glucose homeostasis in adult mice.
Diabetes, 2013Co-Authors: Julie Piccand, Jean-vianney Barnier, Zhengping Jia, Aline Meunier, Carole Merle, Gérard GradwohlAbstract:The transcription factor neurogenin3 (Ngn3) triggers islet cell differentiation in the developing pancreas. However, little is known about the molecular mechanisms coupling cell cycle exit and differentiation in Ngn3(+) islet progenitors. We identified a novel effector of Ngn3 endocrinogenic function, the p21 protein-activated kinase PAK3, known to control neuronal differentiation and implicated in X-linked intellectual disability in humans. We show that PAK3 expression is initiated in Ngn3(+) endocrine progenitor cells and next maintained in maturing hormone-expressing cells during pancreas development as well as in adult islet cells. In PAK3-deficient embryos, the proliferation of Ngn3(+) progenitors and β-cells is transiently increased concomitantly with an upregulation of Ccnd1. β-Cell differentiation is impaired at E15.5 but resumes at later stages. PAK3-deficient mice do not develop overt diabetes but are glucose intolerant under high-fat diet (HFD). In the intestine, PAK3 is expressed in enteroendocrine cells but is not necessary for their differentiation. Our results indicate that PAK3 is a novel regulator of β-cell differentiation and function. PAK3 acts downstream of Ngn3 to promote cell cycle exit and differentiation in the embryo by a mechanism that might involve repression of Ccnd1. In the adult, PAK3 is required for the proper control of glucose homeostasis under challenging HFD.
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The p21-activated kinase PAK3 forms heterodimers with PAK1 in brain implementing trans-regulation of PAK3 activity.
Journal of Biological Chemistry, 2012Co-Authors: Gaëlle Combeau, Patricia Kreis, Véronique Rousseau, Florence Domenichini, Muriel Amar, Philippe Fossier, Jean-vianney BarnierAbstract:p21-activated kinase 1 (PAK1) and PAK3 belong to group I of the PAK family and control cell movement and division. They also regulate dendritic spine formation and maturation in the brain, and play a role in synaptic transmission and synaptic plasticity. PAK3, in particular, is known for its implication in X-linked intellectual disability. The PAK3 gene is expressed in neurons as a GTPase-regulated PAK3a protein and also as three splice variants which display constitutive kinase activity. PAK1 regulation is based on its homodimerization, forming an inactive complex. Here, we analyze the PAK3 capacity to dimerize and show that although PAK3a is able to homodimerize, it is more likely to form heterodimeric complexes with PAK1. We further show that two intellectual disability mutations impair dimerization with PAK1. The b and c inserts present in the regulatory domain of PAK3 splice variants decrease the dimerization but retain the capacity to form heterodimers with PAK1. PAK1 and PAK3 are co-expressed in neurons, are colocalized within dendritic spines, co-purify with post-synaptic densities, and co-immunoprecipitate in brain lysates. Using kinase assays, we demonstrate that PAK1 inhibits the activity of PAK3a but not of the splice variant PAK3b in a trans-regulatory manner. Altogether, these results show that PAK3 and PAK1 signaling may be coordinated by heterodimerization.
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Alteration of Synaptic Network Dynamics by the Intellectual Disability Protein PAK3
Journal of Neuroscience, 2012Co-Authors: Aline Dubois, Jean-vianney Barnier, Gaëlle Combeau, Olivier Hartley, Bernadette Boda, Hubert Gaertner, Yann Bernardinelli, Dominique MullerAbstract:Several gene mutations linked to intellectual disability in humans code for synaptic molecules implicated in small GTPase signaling. This is the case of the Rac/Cdc42 effector p21-activated kinase 3 (PAK3). The mechanisms responsible for the intellectual defects and the consequences of the mutation on the development and wiring of brain networks remain unknown. Here we show that expression of PAK3 mutants, suppression of PAK3, or inhibition of PAK3 function in rat hippocampal slice cultures interfere with activity-mediated spine dynamics. Inhibition of PAK3 resulted in two main alterations: (1) an increased growth of new, unstable spines, occurring in clusters, and mediated by activity; and (2) an impairment of plasticity-mediated spine stabilization interfering with the formation of persistent spines. Additionally, we find that PAK3 is specifically recruited by activity from dendrites into spines, providing a new mechanism through which PAK3 could participate in the control of both spine stabilization and local spine growth. Together, these data identify a novel function of PAK3 in regulating activity-mediated rearrangement of synaptic connectivity associated with learning and suggest that defects in spine formation and refinement during development could account for intellectual disability.
Dominique Muller - One of the best experts on this subject based on the ideXlab platform.
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Alteration of Synaptic Network Dynamics by the Intellectual Disability Protein PAK3
Journal of Neuroscience, 2012Co-Authors: Aline Dubois, Jean-vianney Barnier, Gaëlle Combeau, Olivier Hartley, Bernadette Boda, Hubert Gaertner, Yann Bernardinelli, Dominique MullerAbstract:Several gene mutations linked to intellectual disability in humans code for synaptic molecules implicated in small GTPase signaling. This is the case of the Rac/Cdc42 effector p21-activated kinase 3 (PAK3). The mechanisms responsible for the intellectual defects and the consequences of the mutation on the development and wiring of brain networks remain unknown. Here we show that expression of PAK3 mutants, suppression of PAK3, or inhibition of PAK3 function in rat hippocampal slice cultures interfere with activity-mediated spine dynamics. Inhibition of PAK3 resulted in two main alterations: (1) an increased growth of new, unstable spines, occurring in clusters, and mediated by activity; and (2) an impairment of plasticity-mediated spine stabilization interfering with the formation of persistent spines. Additionally, we find that PAK3 is specifically recruited by activity from dendrites into spines, providing a new mechanism through which PAK3 could participate in the control of both spine stabilization and local spine growth. Together, these data identify a novel function of PAK3 in regulating activity-mediated rearrangement of synaptic connectivity associated with learning and suggest that defects in spine formation and refinement during development could account for intellectual disability.
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Distinct, but compensatory roles of PAK1 and PAK3 in spine morphogenesis.
Hippocampus, 2008Co-Authors: Bernadett Boda, Lorena Jourdain, Dominique MullerAbstract:PAK1 and PAK3 belong to a family of protein kinases that are effectors of small Rho GTPases. In humans, mutations of PAK3 have been associated with mental retardation and result in in vitro studies in defects of spine morphogenesis. The functional specificities of PAK1 and PAK3 remain, however, unclear. Here, we investigated using loss and gain of function experiments how PAK1 and PAK3 affect spine morphology in hippocampal slice cultures. We find that while knockdown of PAK3 is associated with an increase in thin, elongated, immature-type spines, downregulation of PAK1 does not alter spine morphology. Conversely, expression of a constitutively active form of PAK3 remains without effect, while expression of constitutively active PAK1 results in the formation of spines with smaller head diameters. Interestingly, expression of constitutively active PAK1 can rescue the long spine phenotype induced by suppression of PAK3. We conclude that while PAK1 and PAK3 share distinct roles in the regulation of spine morphogenesis, their activity may overlap allowing the compensation of the PAK3 deficit by PAK1. This result opens interesting perspectives in the context of reversing the spine defects associated with PAK3 mutations.
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The p21-activated kinase 3 implicated in mental retardation regulates spine morphogenesis through a Cdc42-dependent pathway.
Journal of Biological Chemistry, 2007Co-Authors: Patricia Kreis, Bernadette Boda, Dominique Muller, Véronique Rousseau, Emmanuel Thévenot, Jean-vianney BarnierAbstract:The p21-activated kinase 3 (PAK3) is one of the recently identified genes for which mutations lead to nonsyndromic mental retardation. PAK3 is implicated in dendritic spine morphogenesis and is a key regulator of synaptic functions. However, the underlying roles of PAK3 in these processes remain poorly understood. We report here that the three mutations R419X, A365E, and R67C, responsible for mental retardation have different effects on the biological functions of PAK3. The R419X and A365E mutations completely abrogate the kinase activity. The R67C mutation drastically decreases the binding of PAK3 to the small GTPase Cdc42 and impairs its subsequent activation by this GTPase. We also report that PAK3 binds significantly more Cdc42 than Rac1 and is selectively activated by endogenous Cdc42, suggesting that PAK3 is a specific effector of Cdc42. Interestingly, the expression of the three mutated proteins in hippocampal neurons affects spinogenesis differentially. Both kinase-dead mutants slightly decrease the number of spines but profoundly alter spine morphology, whereas expression of the R67C mutant drastically decreases spine density. These results demonstrate that the Cdc42/PAK3 is a key module in dendritic spine formation and synaptic plasticity.
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Sequential implication of the mental retardation proteins ARHGEF6 and PAK3 in spine morphogenesis.
Journal of cell science, 2006Co-Authors: Roxanne Nodé-langlois, Dominique Muller, Bernadett BodaAbstract:The biological mechanisms underlying the mental retardation associated with mutation of the ARHGEF6 gene, a Rac1/Cdc42 exchange factor, are still unknown, although defects in the plasticity of synaptic networks have been postulated. We have cloned the rat ARHGEF6 gene and investigated, using a transfection approach, its involvement in spine morphogenesis and its relationship to p21-activated kinase 3 (PAK3). We found that expression of tagged ARHGEF6 in hippocampal slice cultures shows a punctate staining in dendritic spines that colocalizes with PSD95. Over-expression of ARHGEF6, of PAK3 or constitutively active PAK3 did not alter spine morphology. By contrast, knockdown of ARHGEF6 using a siRNA approach resulted in abnormalities in spine morphology similar to those reported with knockdown of PAK3. This phenotype could be rescued through co-expression of a constitutively active PAK3 protein, but not with wild-type PAK3. Together, these results indicate that ARHGEF6 is localized in dendritic spines where it contributes to regulate spine morphogenesis probably by acting through a downstream activation of PAK3. Similar mechanisms are thus likely to underlie the mental retardation induced by mutations of ARHGEF6 and PAK3.
Véronique Rousseau - One of the best experts on this subject based on the ideXlab platform.
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PAK3 controls the tangential to radial migration switch of cortical interneurons by coordinating changes in cell shape and polarity
2020Co-Authors: Lucie Viou, Zhengping Jia, Véronique Rousseau, Pierre Launay, Justine Masson, Clarisse Pace, Robert S. Adelstein, Fujio Murakami, Jean-vianney BarnierAbstract:SUMMARY During the embryonic development, cortical interneurons migrate a long distance tangentially and then re-orient radially to settle in the cortical plate where they contribute to cortical circuits. Migrating interneurons express PAK3, a p21-activated kinase that switches between active and inactive states and controls interneuron migration by unknown mechanism(s). Here we examined the role of the kinase activity of PAK3 to regulate the migration of cortical interneurons. We showed that interneurons expressing a constitutively active PAK3 mutant (PAK3-ca) oriented preferentially radially in the cortex, extended short leading processes and exhibited unstable polarity. On the contrary, interneurons expressing an inactive PAK3 mutant (PAK3-kd for kinase dead) extended branched leading processes, showed directed nuclear movements and remained in the tangential pathways. Results showed that PAK3 kinase activity controls the switch between the tangential and radial modes of migration of cortical interneurons and identified myosin 2B as an effector of this switch.
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The p21-activated kinase PAK3 forms heterodimers with PAK1 in brain implementing trans-regulation of PAK3 activity.
Journal of Biological Chemistry, 2012Co-Authors: Gaëlle Combeau, Patricia Kreis, Véronique Rousseau, Florence Domenichini, Muriel Amar, Philippe Fossier, Jean-vianney BarnierAbstract:p21-activated kinase 1 (PAK1) and PAK3 belong to group I of the PAK family and control cell movement and division. They also regulate dendritic spine formation and maturation in the brain, and play a role in synaptic transmission and synaptic plasticity. PAK3, in particular, is known for its implication in X-linked intellectual disability. The PAK3 gene is expressed in neurons as a GTPase-regulated PAK3a protein and also as three splice variants which display constitutive kinase activity. PAK1 regulation is based on its homodimerization, forming an inactive complex. Here, we analyze the PAK3 capacity to dimerize and show that although PAK3a is able to homodimerize, it is more likely to form heterodimeric complexes with PAK1. We further show that two intellectual disability mutations impair dimerization with PAK1. The b and c inserts present in the regulatory domain of PAK3 splice variants decrease the dimerization but retain the capacity to form heterodimers with PAK1. PAK1 and PAK3 are co-expressed in neurons, are colocalized within dendritic spines, co-purify with post-synaptic densities, and co-immunoprecipitate in brain lysates. Using kinase assays, we demonstrate that PAK1 inhibits the activity of PAK3a but not of the splice variant PAK3b in a trans-regulatory manner. Altogether, these results show that PAK3 and PAK1 signaling may be coordinated by heterodimerization.
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p21-Activated kinase 3 (PAK3) protein regulates synaptic transmission through its interaction with the Nck2/Grb4 protein adaptor.
Journal of Biological Chemistry, 2011Co-Authors: Emmanuelle Thévenot, Gaëlle Combeau, Patricia Kreis, Véronique Rousseau, Florence Domenichini, Muriel Amar, Olivier Goupille, Alexandre William Moreau, Claire Jacquet, Philippe FossierAbstract:Mutations in the p21-activated kinase 3 gene (PAK3) are responsible for nonsyndromic forms of mental retardation. Expression of mutated PAK3 proteins in hippocampal neurons induces abnormal dendritic spine morphology and long term potentiation anomalies, whereas PAK3 gene invalidation leads to cognitive impairments. How PAK3 regulates synaptic plasticity is still largely unknown. To better understand how PAK3 affects neuronal synaptic plasticity, we focused on its interaction with the Nck adaptors that play a crucial role in PAK signaling. We report here that PAK3 interacts preferentially with Nck2/Grb4 in brain extracts and in transfected cells. This interaction is independent of PAK3 kinase activity. Selective uncoupling of the Nck2 interactions in acute cortical slices using an interfering peptide leads to a rapid increase in evoked transmission to pyramidal neurons. The P12A mutation in the PAK3 protein strongly decreases the interaction with Nck2 but only slightly with Nck1. In transfected hippocampal cultures, expression of the P12A-mutated protein has no effect on spine morphogenesis or synaptic density. The PAK3-P12A mutant does not affect synaptic transmission, whereas the expression of the wild-type PAK3 protein decreases the amplitude of spontaneous miniature excitatory currents. Altogether, these data show that PAK3 down-regulates synaptic transmission through its interaction with Nck2.
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p21 activated kinase 3 PAK3 protein regulates synaptic transmission through its interaction with the nck2 grb4 protein adaptor
Journal of Biological Chemistry, 2011Co-Authors: Emmanuelle Thévenot, Gaëlle Combeau, Véronique Rousseau, Florence Domenichini, Claire Jacquet, Alexandre MoreauAbstract:Mutations in the p21-activated kinase 3 gene (PAK3) are responsible for nonsyndromic forms of mental retardation. Expression of mutated PAK3 proteins in hippocampal neurons induces abnormal dendritic spine morphology and long term potentiation anomalies, whereas PAK3 gene invalidation leads to cognitive impairments. How PAK3 regulates synaptic plasticity is still largely unknown. To better understand how PAK3 affects neuronal synaptic plasticity, we focused on its interaction with the Nck adaptors that play a crucial role in PAK signaling. We report here that PAK3 interacts preferentially with Nck2/Grb4 in brain extracts and in transfected cells. This interaction is independent of PAK3 kinase activity. Selective uncoupling of the Nck2 interactions in acute cortical slices using an interfering peptide leads to a rapid increase in evoked transmission to pyramidal neurons. The P12A mutation in the PAK3 protein strongly decreases the interaction with Nck2 but only slightly with Nck1. In transfected hippocampal cultures, expression of the P12A-mutated protein has no effect on spine morphogenesis or synaptic density. The PAK3-P12A mutant does not affect synaptic transmission, whereas the expression of the wild-type PAK3 protein decreases the amplitude of spontaneous miniature excitatory currents. Altogether, these data show that PAK3 down-regulates synaptic transmission through its interaction with Nck2.
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The four mammalian splice variants encoded by the p21-activated kinase 3 gene have different biological properties.
Journal of Neurochemistry, 2008Co-Authors: Patricia Kreis, Gaëlle Combeau, Véronique Rousseau, Emmanuel Thévenot, Jean-vianney BarnierAbstract:The p21-activated kinases (PAK1), PAK2, and PAK3 are members of the PAK group I and share high sequence identity and common biochemical properties. PAK3 is specifically implicated in neuronal plasticity and also regulates cell cycle progression, neuronal migration, and apoptosis. Loss of function of PAK3 is responsible for X-linked non-syndromic mental retardation whereas gain of PAK3 function is associated with cancer. To understand the functional specificities of PAK3, we analyzed the structure of PAK3 gene products. We report here the characterization of a new alternatively spliced exon called c located upstream of the previously identified exon b. Exon b is detected in all tetrapods and not in fish, exon c is only present in mammals. Mammalian PAK3 genes encode four splice variants and the corresponding proteins were detected with specific antibodies in brain extracts. All PAK3 transcripts are specifically expressed in brain and in particular in neurons. The presence of the exons b and c renders the kinase constitutively active and decreases interaction with GTPases. The expression of the new splice variants in COS7 cells alters cell morphology and modifies the structure of focal adhesions. We propose that the appearance of new alternatively spliced exons during evolution and the resulting increase of complexity of PAK3 gene products may confer new functions to this kinase and contribute to its specific roles in neuronal signaling.
Jonathan Chernoff - One of the best experts on this subject based on the ideXlab platform.
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Abstract B066: Unique roles of group I PAK isoforms in regulating MPNST cell viability
New Molecular Targets, 2018Co-Authors: Galina Semenova, Dina Stepanova, Sofiia Karchugina, Cara Dubyk, Sergey M. Deyev, Alexander J. Lazar, Jonathan ChernoffAbstract:Group I p21-activated kinases (PAKs) include three members (PAK1, PAK2, and PAK3) that represent key components of several cell signaling networks. By transmitting growth factor-induced signals from Rho family GTPases, PAK1/2/3 regulate cytoskeletal dynamics, cell shape, and motility. Group I PAKs modulate several oncogenic and survival pathways and are thought to play a critical role in initiation and progression of certain tumor types. Although group I PAK members share significant sequence similarity, mechanism of regulation, and substrates, PAK isoforms show different gene expression patterns in tissues and play unique functions in development and immune responses. While the described PAK1/2/3 functions in tumorigenesis are generally assigned to PAK1, there are increasing hints of isoform-specific roles for group I PAKs in cancer. We have recently demonstrated that PAK1/2/3 signaling is upregulated in malignant peripheral nerve sheath tumors (MPNSTs) and that small-molecule PAK inhibitors suppress MPNST growth and dissemination. The selective group I PAK inhibitors (Frax1036 and GST-PID) tested in this study target all three isoforms and exhibit moderate effects on MPNST cell viability as single agents. We have now used an RNAi approach to knock down individual group I PAK isoforms and their combinations to show that PAK1 and PAK3 knockdown inhibits MPNST cell proliferation, while PAK2 knockdown does not impede the growth of most MPNST cell lines, and in some cases, augments it. Interestingly, combined depletion of PAK1/2/3 has less profound cytotoxic effects compared to single knockdowns of PAK1 or PAK3, suggesting the potential need for isoform-selective PAK inhibitors as therapeutic agents for MPNSTs. Citation Format: Galina Semenova, Dina Stepanova, Sofiia Karchugina, Cara Dubyk, Sergey Deyev, Alexander Lazar, Jonathan Chernoff. Unique roles of group I PAK isoforms in regulating MPNST cell viability [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B066.
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kinase activity of pak2 an effector of rac cdc42 and its interaction with β pix is required for murine hematopoietic stem cell shape f actin formation directional migration in vitro and for hspc homing to bone marrow in vivo
Blood, 2013Co-Authors: Pavankumar N G Reddy, Jonathan Chernoff, Rachelle Kosoff, Maria Radu, Jenna Wood, Chad E Harris, Meaghan Mcguinness, David A WilliamsAbstract:Hematopoietic stem and progenitor cell (HSPC) migration, marrow homing and engraftment are key physiological processes regulating hematopoiesis post transplantation. These processes are the result of the orchestrated actions of multiple extracellular stimuli, which regulate actin remodeling, cell polarity, chemotaxis and cell-cell interactions. In HSPC, the Rho GTPases Rac and CDC42 act as molecular switches that integrate extracellular stimuli in a spatially regulated manner to control cell migration and mediate homing to marrow and mobilization as well as cell survival/ proliferation pathways to mediate engraftment (Gu et al., Science 2003; Cancelas et al., Nature Medicine 2005; Wang et al., Blood 2006) . Using an inhibitory peptide against Group A p21 activated kinases (Pak1-3), key effectors of Rac/ CDC42 and individual Pak1 & 2 genetic knock-out mice, we recently demonstrated that Pak kinases, specifically Pak2, are important for HSPC homing and engraftment ( Dorrance et al., Blood 2013 ). Pak2 is a multi-domain protein that contains a C-terminal kinase domain and multiple N-terminal protein-interaction domains. Among these is a non-classical SH3-binding site for the guanine-nucleotide-exchange factor β-PIX, which was shown to be critical for both activation of Rac1 and its localization to and induction of membrane ruffles ( Klooster et al., Journal of Cell Biology 2006 ). In this study we further explored the role of these domains of Pak2 in key HSPC functions, including homing to bone marrow in vivo . We employed a multi-cistronic retrovirus vector that simultaneously deleted floxed endogenous Pak2 gene sequences and rescued with either wild type (WT), a kinase dead (KD) mutant (K278A, defective in auto/ trans phosphorylation) or a Δβ-PIX mutant, (P185/R186A, that cannot bind to β-PIX). As previously demonstrated deletion of Pak2 (Pak2 Δ/Δ) was associated with abnormal SDF-1 stimulated cell protrusions containing F-actin (as demonstrated by confocal and electron microscopy) and these HSPC displayed decreased directional migration (Euclidean distance in Pak2Δ/Δ vs. Pak2WT/WT: 39.6µm ±9.6 vs. 96.6µm ±21.6; P<0.05). This phenotype of abnormal cell protrusions and decreased directional migration was rescued by expressing Pak2-WT (Pak2WT/WT vs. Pak2-WT: 96.6µm ±21.6 vs. 74.0µm ±18.7; P: not significant) but not by expressing Pak2-KD (Pak2WT/WT vs. Pak2-KD: 96.6µm ±21.6 vs. 33.6µm ±6.3; P<0.05) demonstrating the requirement of Pak2 kinase activity in SDF1-induced cell polarization and directed cell migration. Interestingly, we found abnormal F-actin clustering associated with defective polarization (by confocal microscopy) and decreased velocity of cell migration in time-lapsed video microscopy when Pak2-deletion was rescued with Pak2-Δβ-PIX (velocity of migration Pak2WT/WT vs. Pak2-Δβ-PIX, 0.32µm/minute ±0.02 vs. 0.13µm/minute ±0.02; P<0.001), indicating the requirement of β-PIX exchange factor interaction with Pak2 in directed migration. To test whether these in vitro phenotypes were associated with changes in homing efficiency to bone marrow, we performed in vivo homing assays of rescued HSPC. Transduced, GFP-sorted Lin-Sca1+Kit+ cells of each genotype were injected into lethally-irradiated C57BL/6 recipient mice (N= 12-29 /genotype). Twelve hours post-transplantation the number of EGFP+ cells in the bone marrow was determined and percent homing is calculated. Compared to Pak2 WT/WT, Pak2Δ/Δ HSPC displayed reduced homing (99.26%± 4.9 vs. 53.4% ± 4.2; P< 0.0001). The homing defect was rescued by Pak2-WT (Pak2WT/WT vs. Pak2-WT rescue: 99.26%± 4.9 vs. 86% ± 8.5; P: not significant). However neither Pak2-KD nor Pak2-Δβ-PIX rescued in vivo homing: 99.26% ±4.9 vs. 38.9% ±3.7 vs. 33.0%± 6.0; P< 0.0001 each mutant vs.Pak2WT/WT) proving the necessity of kinase activity and interaction with β-PIX for bone marrow homing. Taken together we show that both Pak2-kinase activity and its interaction with β-PIX exchange factor are required for coordinated HSPC F-actin formation and cell polarization, directed cell migration in vitro and homing to bone marrow in vivo . These data directly link the in vitro effects of Pak2 kinase with in vivo bone marrow homing. Note All p values are calculated by Mann Whitney test. Disclosures: No relevant conflicts of interest to declare.
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Kinase Activity Of Pak2, An Effector Of Rac/CDC42 and Its Interaction With β-PIX Is Required For Murine Hematopoietic Stem Cell Shape, F-Actin Formation, Directional Migration In Vitro and For HSPC Homing To Bone Marrow In Vivo
Blood, 2013Co-Authors: Pavankumar N G Reddy, Jonathan Chernoff, Rachelle Kosoff, Maria Radu, Jenna Wood, Chad E Harris, Meaghan Mcguinness, David A WilliamsAbstract:Hematopoietic stem and progenitor cell (HSPC) migration, marrow homing and engraftment are key physiological processes regulating hematopoiesis post transplantation. These processes are the result of the orchestrated actions of multiple extracellular stimuli, which regulate actin remodeling, cell polarity, chemotaxis and cell-cell interactions. In HSPC, the Rho GTPases Rac and CDC42 act as molecular switches that integrate extracellular stimuli in a spatially regulated manner to control cell migration and mediate homing to marrow and mobilization as well as cell survival/ proliferation pathways to mediate engraftment (Gu et al., Science 2003; Cancelas et al., Nature Medicine 2005; Wang et al., Blood 2006) . Using an inhibitory peptide against Group A p21 activated kinases (Pak1-3), key effectors of Rac/ CDC42 and individual Pak1 & 2 genetic knock-out mice, we recently demonstrated that Pak kinases, specifically Pak2, are important for HSPC homing and engraftment ( Dorrance et al., Blood 2013 ). Pak2 is a multi-domain protein that contains a C-terminal kinase domain and multiple N-terminal protein-interaction domains. Among these is a non-classical SH3-binding site for the guanine-nucleotide-exchange factor β-PIX, which was shown to be critical for both activation of Rac1 and its localization to and induction of membrane ruffles ( Klooster et al., Journal of Cell Biology 2006 ). In this study we further explored the role of these domains of Pak2 in key HSPC functions, including homing to bone marrow in vivo . We employed a multi-cistronic retrovirus vector that simultaneously deleted floxed endogenous Pak2 gene sequences and rescued with either wild type (WT), a kinase dead (KD) mutant (K278A, defective in auto/ trans phosphorylation) or a Δβ-PIX mutant, (P185/R186A, that cannot bind to β-PIX). As previously demonstrated deletion of Pak2 (Pak2 Δ/Δ) was associated with abnormal SDF-1 stimulated cell protrusions containing F-actin (as demonstrated by confocal and electron microscopy) and these HSPC displayed decreased directional migration (Euclidean distance in Pak2Δ/Δ vs. Pak2WT/WT: 39.6µm ±9.6 vs. 96.6µm ±21.6; P
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pak1 kinase links erbb2 to β catenin in transformation of breast epithelial cells
Cancer Research, 2013Co-Authors: Luis E Ariasromero, Olga Villamarcruz, Min Huang, Klaus P Hoeflich, Jonathan ChernoffAbstract:p21-Activated kinase-1 (Pak1) is frequently upregulated in human breast cancer and is required for transformation of mammary epithelial cells by ErbB2. Here, we show that loss of Pak1, but not the closely related Pak2, leads to diminished expression of β-catenin and its target genes. In MMTV-ErbB2 transgenic mice, loss of Pak1 prolonged survival, and mammary tissues of such mice showed loss of β-catenin. Expression of a β-catenin mutant bearing a phospho-mimetic mutation at Ser 675, a specific Pak1 phosphorylation site, restored transformation to ErbB2-positive, Pak1-deficient mammary epithelial cells. Mice bearing xenografts of ErbB2-positive breast cancer cells showed tumor regression when treated with small-molecule inhibitors of Pak or β-catenin, and combined inhibition by both agents was synergistic. These data delineate a signaling pathway from ErbB2 to Pak to β-catenin that is required for efficient transformation of mammary epithelial cells, and suggest new therapeutic strategies in ErbB2-positive breast cancer.
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pak2 kinase restrains mast cell fcϵri receptor signaling through modulation of rho protein guanine nucleotide exchange factor gef activity
Journal of Biological Chemistry, 2013Co-Authors: Rachelle Kosoff, Hoi Yee Chow, Maria Radu, Jonathan ChernoffAbstract:Abstract p21-activated kinase-1 (Pak1) is a serine/threonine kinase that plays a key role in mediating antigen-stimulated extracellular calcium influx and degranulation in mast cells. Another isoform in this kinase family, Pak2, is expressed at very high levels in mast cells, but its function is unknown. Here we show that Pak2 loss in murine bone-marrow-derived mast cells (BMMCs), unlike loss of Pak1, induces increased antigen-mediated adhesion, degranulation, and cytokine secretion without changes to extracellular calcium influx. This phenotype is associated with an increase in RhoA-GTPase signaling activity to downstream effectors, including myosin light chain and p38MAPK, and is reversed upon treatment with a Rho-specific inhibitor. Pak2, but not Pak1, negatively regulates RhoA via phosphorylation of the guanine-nucleotide exchange factor GEF-H1 at an inhibitory site, leading to increased GEF-H1 microtubule binding and loss of RhoA stimulation. These data suggest that Pak2 plays a unique inhibitory role in mast cell degranulation by downregulating RhoA via GEF-H1.
Patricia Kreis - One of the best experts on this subject based on the ideXlab platform.
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The p21-activated kinase PAK3 forms heterodimers with PAK1 in brain implementing trans-regulation of PAK3 activity.
Journal of Biological Chemistry, 2012Co-Authors: Gaëlle Combeau, Patricia Kreis, Véronique Rousseau, Florence Domenichini, Muriel Amar, Philippe Fossier, Jean-vianney BarnierAbstract:p21-activated kinase 1 (PAK1) and PAK3 belong to group I of the PAK family and control cell movement and division. They also regulate dendritic spine formation and maturation in the brain, and play a role in synaptic transmission and synaptic plasticity. PAK3, in particular, is known for its implication in X-linked intellectual disability. The PAK3 gene is expressed in neurons as a GTPase-regulated PAK3a protein and also as three splice variants which display constitutive kinase activity. PAK1 regulation is based on its homodimerization, forming an inactive complex. Here, we analyze the PAK3 capacity to dimerize and show that although PAK3a is able to homodimerize, it is more likely to form heterodimeric complexes with PAK1. We further show that two intellectual disability mutations impair dimerization with PAK1. The b and c inserts present in the regulatory domain of PAK3 splice variants decrease the dimerization but retain the capacity to form heterodimers with PAK1. PAK1 and PAK3 are co-expressed in neurons, are colocalized within dendritic spines, co-purify with post-synaptic densities, and co-immunoprecipitate in brain lysates. Using kinase assays, we demonstrate that PAK1 inhibits the activity of PAK3a but not of the splice variant PAK3b in a trans-regulatory manner. Altogether, these results show that PAK3 and PAK1 signaling may be coordinated by heterodimerization.
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p21-Activated kinase 3 (PAK3) protein regulates synaptic transmission through its interaction with the Nck2/Grb4 protein adaptor.
Journal of Biological Chemistry, 2011Co-Authors: Emmanuelle Thévenot, Gaëlle Combeau, Patricia Kreis, Véronique Rousseau, Florence Domenichini, Muriel Amar, Olivier Goupille, Alexandre William Moreau, Claire Jacquet, Philippe FossierAbstract:Mutations in the p21-activated kinase 3 gene (PAK3) are responsible for nonsyndromic forms of mental retardation. Expression of mutated PAK3 proteins in hippocampal neurons induces abnormal dendritic spine morphology and long term potentiation anomalies, whereas PAK3 gene invalidation leads to cognitive impairments. How PAK3 regulates synaptic plasticity is still largely unknown. To better understand how PAK3 affects neuronal synaptic plasticity, we focused on its interaction with the Nck adaptors that play a crucial role in PAK signaling. We report here that PAK3 interacts preferentially with Nck2/Grb4 in brain extracts and in transfected cells. This interaction is independent of PAK3 kinase activity. Selective uncoupling of the Nck2 interactions in acute cortical slices using an interfering peptide leads to a rapid increase in evoked transmission to pyramidal neurons. The P12A mutation in the PAK3 protein strongly decreases the interaction with Nck2 but only slightly with Nck1. In transfected hippocampal cultures, expression of the P12A-mutated protein has no effect on spine morphogenesis or synaptic density. The PAK3-P12A mutant does not affect synaptic transmission, whereas the expression of the wild-type PAK3 protein decreases the amplitude of spontaneous miniature excitatory currents. Altogether, these data show that PAK3 down-regulates synaptic transmission through its interaction with Nck2.
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PAK signalling in neuronal physiology.
Cellular Signalling, 2009Co-Authors: Patricia Kreis, Jean-vianney BarnierAbstract:Group I p21-activated kinases are a family of key effectors of Rac1 and Cdc42 and they regulate many aspects of cellular function, such as cytoskeleton dynamics, cell movement and cell migration, cell proliferation and differentiation, and gene expression. The three genes PAK1/2/3 are expressed in brain and recent evidence indicates their crucial roles in neuronal cell fate, in axonal guidance and neuronal polarisation, and in neuronal migration. Moreover they are implicated in neurodegenerative diseases and play an important role in synaptic plasticity, with PAK3 being specifically involved in mental retardation. The main goal of this review is to describe the molecular mechanisms that govern the different functions of group I PAK in neuronal signalling and to discuss the specific functions of each isoform.
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The four mammalian splice variants encoded by the p21-activated kinase 3 gene have different biological properties.
Journal of Neurochemistry, 2008Co-Authors: Patricia Kreis, Gaëlle Combeau, Véronique Rousseau, Emmanuel Thévenot, Jean-vianney BarnierAbstract:The p21-activated kinases (PAK1), PAK2, and PAK3 are members of the PAK group I and share high sequence identity and common biochemical properties. PAK3 is specifically implicated in neuronal plasticity and also regulates cell cycle progression, neuronal migration, and apoptosis. Loss of function of PAK3 is responsible for X-linked non-syndromic mental retardation whereas gain of PAK3 function is associated with cancer. To understand the functional specificities of PAK3, we analyzed the structure of PAK3 gene products. We report here the characterization of a new alternatively spliced exon called c located upstream of the previously identified exon b. Exon b is detected in all tetrapods and not in fish, exon c is only present in mammals. Mammalian PAK3 genes encode four splice variants and the corresponding proteins were detected with specific antibodies in brain extracts. All PAK3 transcripts are specifically expressed in brain and in particular in neurons. The presence of the exons b and c renders the kinase constitutively active and decreases interaction with GTPases. The expression of the new splice variants in COS7 cells alters cell morphology and modifies the structure of focal adhesions. We propose that the appearance of new alternatively spliced exons during evolution and the resulting increase of complexity of PAK3 gene products may confer new functions to this kinase and contribute to its specific roles in neuronal signaling.
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The p21-activated kinase 3 implicated in mental retardation regulates spine morphogenesis through a Cdc42-dependent pathway.
Journal of Biological Chemistry, 2007Co-Authors: Patricia Kreis, Bernadette Boda, Dominique Muller, Véronique Rousseau, Emmanuel Thévenot, Jean-vianney BarnierAbstract:The p21-activated kinase 3 (PAK3) is one of the recently identified genes for which mutations lead to nonsyndromic mental retardation. PAK3 is implicated in dendritic spine morphogenesis and is a key regulator of synaptic functions. However, the underlying roles of PAK3 in these processes remain poorly understood. We report here that the three mutations R419X, A365E, and R67C, responsible for mental retardation have different effects on the biological functions of PAK3. The R419X and A365E mutations completely abrogate the kinase activity. The R67C mutation drastically decreases the binding of PAK3 to the small GTPase Cdc42 and impairs its subsequent activation by this GTPase. We also report that PAK3 binds significantly more Cdc42 than Rac1 and is selectively activated by endogenous Cdc42, suggesting that PAK3 is a specific effector of Cdc42. Interestingly, the expression of the three mutated proteins in hippocampal neurons affects spinogenesis differentially. Both kinase-dead mutants slightly decrease the number of spines but profoundly alter spine morphology, whereas expression of the R67C mutant drastically decreases spine density. These results demonstrate that the Cdc42/PAK3 is a key module in dendritic spine formation and synaptic plasticity.
