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Jonathan Chernoff - One of the best experts on this subject based on the ideXlab platform.
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p21 activated kinase 2 regulates hspc cytoskeleton migration and homing via cdc42 activation and interaction with β pix
Blood, 2016Co-Authors: Pavankumar N G Reddy, Jonathan Chernoff, Maria Radu, Jenna Wood, Chad E Harris, David A WilliamsAbstract:Cytoskeletal remodeling of hematopoietic stem and progenitor cells (HSPCs) is essential for homing to the bone marrow (BM). The Ras-related C3 botulinum toxin substrate (Rac)/cell division control protein 42 homolog (CDC42) effector p21-activated kinase (PAK2) has been implicated in HSPC homing and engraftment. However, the molecular pathways mediating PAK2 functions in HSPCs are unknown. Here, we demonstrate that both PAK2 kinase activity and its interaction with the PAK-interacting exchange factor-β (β-Pix) are required to reconstitute defective ITALIC! PAK2 (ITALIC! Δ/Δ)HSPC homing to the BM. PAK2 serine/threonine kinase activity is required for stromal-derived factor-1 (SDF1α) chemokine-induced HSPC directional migration, whereas PAK2 interaction with β-Pix is required to regulate the velocity of HSPC migration and precise F-actin assembly. Lack of SDF1α-induced filopodia and associated abnormal cell protrusions seen in ITALIC! PAK2 (ITALIC! Δ/Δ)HSPCs were rescued by wild-type (WT) PAK2 but not by a PAK2-kinase dead mutant (KD). Expression of a β-Pix interaction-defective mutant of PAK2 rescued filopodia formation but led to abnormal F-actin bundles. Although CDC42 has previously been considered an upstream regulator of PAK2, we found a paradoxical decrease in baseline activation of CDC42 in ITALIC! PAK2 (ITALIC! Δ/Δ)HSPCs, which was rescued by expression of PAK2-WT but not by PAK2-KD; defective homing of ITALIC! PAK2-deleted HSPCs was rescued by constitutive active CDC42. These data demonstrate that both PAK2 kinase activity and its interaction with β-Pix are essential for HSPC filopodia formation, cytoskeletal integrity, and homing via activation of CDC42. Taken together, we provide mechanistic insights into the role of PAK2 in HSPC migration and homing.
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PAK2 Links TCR Signaling Strength to the Development of Regulatory T Cells and Maintains Peripheral Tolerance
Journal of immunology (Baltimore Md. : 1950), 2015Co-Authors: Kyle Leonard O'hagan, Jonathan Chernoff, Olga Pryshchep, Jinyong Choi, Hyewon PheeAbstract:Although significant effort has been devoted to understanding the thymic development of Foxp3(+) regulatory T cells (Tregs), the precise signaling pathways that govern their lineage commitment still remain enigmatic. Our findings show a novel role for the actin cytoskeletal remodeling protein, p21-activated kinase 2 (PAK2), in Treg development and homeostasis. The absence of PAK2 in T cells resulted in a marked reduction in both thymus- and peripherally derived Tregs, accompanied by the development of spontaneous colitis in PAK2-deficient mice. Additionally, PAK2 was required for the proper differentiation of in vitro-induced Tregs as well as maintenance of Tregs. Interestingly, PAK2 was necessary for generating the high-affinity TCR- and IL-2-mediated signals that are required by developing Tregs for their lineage commitment. These findings provide novel insight into how developing thymocytes translate lineage-specific high-affinity TCR signals to adopt the Treg fate, and they further posit PAK2 as an essential regulator for this process.
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PAK2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation
eLife, 2014Co-Authors: Hyewon Phee, Rachelle Kosoff, Maria Radu, Byron B. Au-yeung, Olga Pryshchep, Kyle Leonard O'hagan, Stephanie Grace Fairbairn, Marianne Mollenauer, Debra A. Cheng, Jonathan ChernoffAbstract:The molecular mechanisms that govern thymocyte development and maturation are incompletely understood. The P21-activated kinase 2 (PAK2) is an effector for the Rho family GTPases Rac and Cdc42 that regulate actin cytoskeletal remodeling, but its role in the immune system remains poorly understood. In this study, we show that T-cell specific deletion of PAK2 gene in mice resulted in severe T cell lymphopenia accompanied by marked defects in development, maturation, and egress of thymocytes. PAK2 was required for pre-TCR β-selection and positive selection. Surprisingly, PAK2 deficiency in CD4 single positive thymocytes prevented functional maturation and reduced expression of S1P1 and KLF2. Mechanistically, PAK2 is required for actin cytoskeletal remodeling triggered by TCR. Failure to induce proper actin cytoskeletal remodeling impaired PLCγ1 and Erk1/2 signaling in the absence of PAK2, uncovering the critical function of PAK2 as an essential regulator that governs the actin cytoskeleton-dependent signaling to ensure normal thymocyte development and maturation.DOI: http://dx.doi.org/10.7554/eLife.02270.001.
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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
Jolinda A. Traugh - One of the best experts on this subject based on the ideXlab platform.
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PAK2 activated by Cdc42 and caspase 3 mediates different cellular responses to oxidative stress-induced apoptosis.
Biochimica et biophysica acta. Molecular cell research, 2020Co-Authors: John Huang, Jolinda A. Traugh, Allen Huang, Amelia Poplawski, Frank Dipino, Jun LingAbstract:p21-activated protein kinase (PAK2) is a unique member of the PAK family kinases that plays important roles in stress signaling. It can be activated by binding to the small GTPase, Cdc42 and Rac1, or by caspase 3 cleavage. Cdc42-activated PAK2 mediates cytostasis, whereas caspase 3-cleaved PAK2 contributes to apoptosis. However, the relationship between these two states of PAK2 activation remains elusive. In this study, through protein biochemical analyses and various cell-based assays, we demonstrated that full-length PAK2 activated by Cdc42 was resistant to the cleavage by caspase 3 in vitro and within cells. When mammalian cells were treated by oxidative stress using hydrogen peroxide, PAK2 was highly activated through caspase 3 cleavage that led to apoptosis. However, when PAK2 was pre-activated by Cdc42 or by mild stress such as serum deprivation, it was no longer able to be cleaved by caspase 3 upon hydrogen peroxide treatment, and the subsequent apoptosis was also largely inhibited. Furthermore, cells expressing active mutants of full-length PAK2 became more resistant to hydrogen peroxide-induced apoptosis than inactive mutants. Taken together, this study identified two states of PAK2 activation, wherein Cdc42- and autophosphorylation-dependent activation inhibited the constitutive activation of PAK2 by caspase cleavage. The regulation between these two states of PAK2 activation provides a new molecular mechanism to support PAK2 as a molecular switch for controlling cytostasis and apoptosis in response to different types and levels of stress with broad physiological and pathological relevance.
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Activation of PAK2 by Serum Starvation Sensitizes its Response to InsulinTreatment in Adipocyte 3T3-L1 Cells
Biochemistry & Analytical Biochemistry, 2016Co-Authors: Jun Ling, Siska Corneillie, Colby Cottell, Jolinda A. TraughAbstract:p21-activated kinase-2 (PAK2) is ubiquitously expressed in all mammalian cells and tissues tested so far. It is a unique member of PAK family kinases that can be activated by various stress conditions to induce apoptosis or cytostasis. Although many conditions have been reported to activate PAK2, serum starvation followed by insulin treatment has not been studied. In this study, pre-adipocyte (3T3-L1) sensitive to insulin signaling and important for energy homeostasis was used as the system to address this topic. It was found that serum starvation transiently activated PAK2 activity by about 3-fold within one hour, then returning to the basal level within three hours. Following the activation of PAK2 by serum starvation, insulin treatment resulted in a rapid deactivation of PAK2 through ubiquitination-proteasome mediated protein degradation. AKT1 and PAK2 activities were reversely related, suggesting that AKT1 activation could be a factor to initiate PAK2 degradation. This dynamic change of PAK2 by serum starvation and insulin was found to be correlated with the fluctuation of protein synthesis, a major biological process to influence cell growth rate. Activation of PAK2 by serum starvation was correlated with about 50% inhibition of protein synthesis; subsequent treatment with insulin reversed this inhibition. Down-regulation of PAK2 by siRNA further proved that PAK2 was a causal factor leading to the inhibition of protein synthesis. In conclusion, this study identifies a new pattern of regulation of PAK2 by serum starvation and insulin, suggests an important role of PAK2 in regulating adipocyte function in response to nutrient status and insulin signaling.
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Inhibition of cap-dependent translation via phosphorylation of eIF4G by protein kinase PAK2
The EMBO journal, 2005Co-Authors: Jun Ling, Simon J. Morley, Jolinda A. TraughAbstract:Translation is downregulated in response to a variety of moderate stresses, including serum deprivation, hyperosmolarity and ionizing radiation. The cytostatic p21-activated protein kinase 2 (PAK2)/γ-PAK is activated under the same stress conditions. Expression of wild-type PAK2 in cells and addition of PAK2 to reticulocyte lysate inhibit translation, while kinase-inactive mutants have no effect. PAK2 binds to and phosphorylates initiation factor (eIF)4G, which inhibits association of eIF4E with m7GTP, reducing initiation. The PAK2-binding site maps to the region on eIF4G that contains the eIF4E-binding site; PAK2 and eIF4E compete for binding to this site. Using an eIF4G-depleted reticulocyte lysate, reconstitution with mock-phosphorylated eIF4G fully restores translation, while phosphorylated eIF4G reduces translation to 37%. RNA interference releases PAK2-induced inhibition of translation in contact-inhibited cells by 2.7-fold. eIF4G mutants of the PAK2 site show that S896D inhibits translation, while S896A has no effect. Activation of PAK2 in response to hyperosmotic stress inhibits cap-dependent, but not IRES-driven, initiation. Thus, a novel pathway for mammalian cell stress signaling is identified, wherein activation of PAK2 leads to inhibition of cap-dependent translation through phosphorylation of eIF4G.
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Regulation of the interaction of PAK2 with Cdc42 via autophosphorylation of serine 141.
The Journal of biological chemistry, 2005Co-Authors: Jin-hun Jung, Jolinda A. TraughAbstract:PAK2, a member of the p21-activated protein kinase (Pak) family, is activated in response to a variety of stresses and is directly involved in the induction of cytostasis. At the molecular level PAK2 binds Cdc42(GTP), translocating PAK2 to the endoplasmic reticulum where it is autophosphorylated and activated. PAK2 is autophosphorylated at eight sites; Ser-141 and Ser-165 in the regulatory domain and Thr-402 in the activation loop are identified as key sites in activation of the protein kinase. The function of phosphorylation of Ser-141 and Ser-165 on the activation was analyzed with wild-type (WT) and mutants of PAK2. With S141A, the level of autophosphorylation was reduced to 65% as compared with that of WT and S141D with a concomitant 45% reduction in substrate phosphorylation, indicating that phosphorylation at Ser-141 is required for optimal activity. Autophosphorylation inhibited the interaction between WT PAK2 and Cdc42(GTP). In 293T cells, WT PAK2, S141A, and S141D formed a stable complex with the constitutively active mutant Cdc42 L61, but not with the dominant negative Cdc42 N17. As shown in glutathione S-transferase pull-down assays, S141A bound to Cdc42(GTP) at a 6-fold higher level than that of S141D. In contrast, the S165A and S165D mutants had no effect on autophosphorylation, binding to Cdc42, or activation of PAK2. In summary, autophosphorylation of Ser-141 was required for activation of PAK2 and down-regulated the interaction of PAK2 with Cdc42. A model is proposed suggesting that binding of Cdc42 localizes PAK2 to the endoplasmic reticulum, where autophosphorylation alters association of the two proteins.
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phosphorylation of mnk1 by caspase activated PAK2 γ pak inhibits phosphorylation and interaction of eif4g with mnk
Journal of Biological Chemistry, 2004Co-Authors: Kevin C. Orton, Jun Ling, Andrew J. Waskiewicz, Jonathan A. Cooper, William C. Merrick, Nadejda L. Korneeva, Robert E. Rhoads, Nahum Sonenberg, Jolinda A. TraughAbstract:Abstract The mitogen-activated protein kinase-interacting kinase 1 (Mnk1) is phosphorylated by caspase-cleaved protein kinase PAK2/γ-PAK but not by Cdc42-activated PAK2. Phosphorylation of Mnk1 is rapid, reaching 1 mol/mol within 15 min of incubation with PAK2. A kinetic analysis of the phosphorylation of Mnk1 by PAK2 yields a Km of 0.6 μm and a Vmax of 14.9 pmol of 32P/min/μg of PAK2. Two-dimensional tryptic phosphopeptide mapping of Mnk1 phosphorylated by PAK2 yields two distinct phosphopeptides. Analysis of the phosphopeptides by automated microsequencing and manual Edman degradation identified the sites in Mnk1 as Thr22 and Ser27. Mnk1, activated by phosphorylation with Erk2, phosphorylates the eukaryotic initiation factor (eIF) 4E and the eIF4G components of eIF4F. Phosphorylation of Mnk1 by PAK2 does not activate Mnk1, as measured with either eIF4E or eIF4F as substrate. Phosphorylation of Erk2-activated Mnk1 by PAK2 has no effect on phosphorylation of eIF4E but reduces phosphorylation of eIF4G by Mnk1 by up to 50%. Phosphorylation of Mnk1 by PAK2 inhibits binding of eIF4G peptides containing the Mnk1 binding site by up to 80%. When 293T cells are subjected to apoptotic induction by hydrogen peroxide, Mnk1 is phosphorylated at both Thr22 and Ser27. These results indicate a role for PAK2 in the down-regulation of translation initiation in apoptosis by phosphorylation of Mnk1.
Audrey Minden - One of the best experts on this subject based on the ideXlab platform.
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Pdx1-Cre-driven conditional gene depletion suggests PAK4 as dispensable for mouse pancreas development.
Scientific reports, 2017Co-Authors: Miao Zhao, Parisa Rabieifar, Tânia D. F. Costa, Ting Zhuang, Audrey Minden, Matthias Löhr, Rainer Heuchel, Staffan StrömbladAbstract:Constitutive depletion of p21-activated kinase 4 (PAK4) in the mouse causes embryonic lethality associated with heart and brain defects. Given that conventional gene depletion of PAK1 or PAK3 caused functional deficits in the mouse pancreas, while gene depletion of PAK5 or PAK6 did not, we asked if PAK4 might have a functional role in pancreas development. We therefore introduced conditional, Pdx1-Cre-mediated, pancreatic PAK4 gene depletion in the mouse, verified by loss of PAK4 protein expression in the pancreas. PAK4 knock-out (KO) mice were born at Mendelian ratios in both genders. Further, morphological and immunohistochemical examinations and quantifications indicated that exocrine, endocrine and ductal compartments retained the normal proportions and distributions upon PAK4 gene depletion. In addition, body weight records and a glucose tolerance test revealed no differences between WT and PAK4 KO mice. Together, this suggests that PAK4 is dispensable for mouse pancreas development. This will facilitate future use of our Pdx1-Cre-driven conditional PAK4 KO mouse model for testing in vivo potential functions of PAK4 in pancreatic disease models such as for pancreatitis and different pancreatic cancer forms.
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pak4 6 in cancer and neuronal development
Cellular logistics, 2012Co-Authors: Audrey MindenAbstract:PAKs 4, 5 and 6 are members of the group B family of p21-activated kinases. Among this group, PAK4 has been most extensively studied. While it has essential roles in embryonic development, in adults high levels of PAK4 are frequently associated with cancer. PAK4 is overexpressed in a variety of cancers, and the Pak4 gene is amplified in some cancers. PAK4 overexpression is sufficient to cause oncogenic transformation in cells and in mouse models. The tight connection between PAK4 and cancer make it a promising diagnostic tool as well as a potential drug target. The group B PAKs also have important developmental functions. PAK4 is important for many early developmental processes, while PAK5 and PAK6 play roles in learning and memory in mice. This chapter provides an overview of the roles of the group B PAKs in cancer as well as development, and includes a discussion of PAK mediated signaling pathways and cellular functions.
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PAK4–6 in cancer and neuronal development
Cellular logistics, 2012Co-Authors: Audrey MindenAbstract:PAKs 4, 5 and 6 are members of the group B family of p21-activated kinases. Among this group, PAK4 has been most extensively studied. While it has essential roles in embryonic development, in adults high levels of PAK4 are frequently associated with cancer. PAK4 is overexpressed in a variety of cancers, and the Pak4 gene is amplified in some cancers. PAK4 overexpression is sufficient to cause oncogenic transformation in cells and in mouse models. The tight connection between PAK4 and cancer make it a promising diagnostic tool as well as a potential drug target. The group B PAKs also have important developmental functions. PAK4 is important for many early developmental processes, while PAK5 and PAK6 play roles in learning and memory in mice. This chapter provides an overview of the roles of the group B PAKs in cancer as well as development, and includes a discussion of PAK mediated signaling pathways and cellular functions.
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cytoskeletal changes regulated by the pak4 serine threonine kinase are mediated by lim kinase 1 and cofilin
Journal of Biological Chemistry, 2001Co-Authors: Chuntao Dan, April P. Kelly, Ora Bernard, Audrey MindenAbstract:Abstract PAK4 is the most recently identified member of the PAK family of serine/threonine kinases. PAK4 differs from other members of the PAK family in sequence and in many of its functions. Previously, we have shown that an important function of this kinase is to mediate the induction of filopodia in response to the Rho GTPase Cdc42. Here we show that PAK4 also regulates the activity of the protein kinase LIM kinase 1 (LIMK1). PAK4 was shown to interact specifically with LIMK1 in binding assays. Immune complex kinase assays revealed that both wild-type and constitutively active PAK4 phosphorylated LIMK1 even more strongly than PAK1, and activated PAK4 stimulated LIMK1's ability to phosphorylate cofilin. Immunofluorescence experiments revealed that PAK4 and LIMK1 cooperate to induce cytoskeletal changes in C2C12 cells. Furthermore, dominant negative LIMK1 and a mutant cofilin inhibited the specific cytoskeletal and cell shape changes that were induced in response to a recently characterized constitutively activated PAK4 mutant.
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Cytoskeletal changes regulated by the PAK4 serine/threonine kinase are mediated by LIM kinase 1 and cofilin.
The Journal of biological chemistry, 2001Co-Authors: Chuntao Dan, April P. Kelly, Ora Bernard, Audrey MindenAbstract:Abstract PAK4 is the most recently identified member of the PAK family of serine/threonine kinases. PAK4 differs from other members of the PAK family in sequence and in many of its functions. Previously, we have shown that an important function of this kinase is to mediate the induction of filopodia in response to the Rho GTPase Cdc42. Here we show that PAK4 also regulates the activity of the protein kinase LIM kinase 1 (LIMK1). PAK4 was shown to interact specifically with LIMK1 in binding assays. Immune complex kinase assays revealed that both wild-type and constitutively active PAK4 phosphorylated LIMK1 even more strongly than PAK1, and activated PAK4 stimulated LIMK1's ability to phosphorylate cofilin. Immunofluorescence experiments revealed that PAK4 and LIMK1 cooperate to induce cytoskeletal changes in C2C12 cells. Furthermore, dominant negative LIMK1 and a mutant cofilin inhibited the specific cytoskeletal and cell shape changes that were induced in response to a recently characterized constitutively activated PAK4 mutant.
Dana Gabuzda - One of the best experts on this subject based on the ideXlab platform.
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Nef-mediated enhancement of cellular activation and human immunodeficiency virus type 1 replication in primary T cells is dependent on association with p21-activated kinase 2
Retrovirology, 2011Co-Authors: Kevin C. Olivieri, Joya Mukerji, Dana GabuzdaAbstract:The HIV-1 accessory protein Nef is an important determinant of lentiviral pathogenicity that contributes to disease progression by enhancing viral replication and other poorly understood mechanisms. Nef mediates diverse functions including downmodulation of cell surface CD4 and MHC Class I, enhancement of viral infectivity, and enhancement of T cell activation. Nef interacts with a multiprotein signaling complex that includes Src family kinases, Vav1, CDC42, and activated PAK2 (p21-activated kinase 2). Although previous studies have attempted to identify a biological role for the Nef-PAK2 signaling complex, the importance of this complex and its constituent proteins in Nef function remains unclear. Here, we show that Nef mutants defective for PAK2-association, but functional for CD4 and MHC Class I downmodulation and infectivity enhancement, are also defective for the ability to enhance viral replication in primary T cells that are infected and subsequently activated by sub-maximal stimuli (1 μg/ml PHA-P). In contrast, these Nef mutants had little or no effect on HIV-1 replication in T cells activated by stronger stimuli (2 μg/ml PHA-P or anti-CD3/CD28-coated beads). Viruses bearing wild-type Nefs, but not Nef mutants defective for PAK2 association, enhanced NFAT and IL2 receptor promoter activity in Jurkat cells. Moreover, expression of wild-type Nefs, but not mutant Nefs defective for PAK2 association, was sufficient to enhance responsiveness of primary CD4 and CD8 T cells to activating stimuli in Nef-expressing and bystander cells. siRNA knockdown of PAK2 in Jurkat cells reduced NFAT activation induced by anti-CD3/CD28 stimulation both in the presence and absence of Nef, and expression of a PAK2 dominant mutant inhibited Nef-mediated enhancement of CD25 expression. Nef-mediated enhancement of cellular activation and viral replication in primary T cells is dependent on PAK2 and on the strength of the activating stimuli, and correlates with the ability of Nef to associate with PAK2. PAK2 is likely to play a role in Nef-mediated enhancement of viral replication and immune activation in vivo.
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Nef-mediated enhancement of cellular activation and human immunodeficiency virus type 1 replication in primary T cells is dependent on association with p21-activated kinase 2
Retrovirology, 2011Co-Authors: Kevin C. Olivieri, Joya Mukerji, Dana GabuzdaAbstract:Background The HIV-1 accessory protein Nef is an important determinant of lentiviral pathogenicity that contributes to disease progression by enhancing viral replication and other poorly understood mechanisms. Nef mediates diverse functions including downmodulation of cell surface CD4 and MHC Class I, enhancement of viral infectivity, and enhancement of T cell activation. Nef interacts with a multiprotein signaling complex that includes Src family kinases, Vav1, CDC42, and activated PAK2 (p21-activated kinase 2). Although previous studies have attempted to identify a biological role for the Nef-PAK2 signaling complex, the importance of this complex and its constituent proteins in Nef function remains unclear. Results Here, we show that Nef mutants defective for PAK2-association, but functional for CD4 and MHC Class I downmodulation and infectivity enhancement, are also defective for the ability to enhance viral replication in primary T cells that are infected and subsequently activated by sub-maximal stimuli (1 μg/ml PHA-P). In contrast, these Nef mutants had little or no effect on HIV-1 replication in T cells activated by stronger stimuli (2 μg/ml PHA-P or anti-CD3/CD28-coated beads). Viruses bearing wild-type Nefs, but not Nef mutants defective for PAK2 association, enhanced NFAT and IL2 receptor promoter activity in Jurkat cells. Moreover, expression of wild-type Nefs, but not mutant Nefs defective for PAK2 association, was sufficient to enhance responsiveness of primary CD4 and CD8 T cells to activating stimuli in Nef-expressing and bystander cells. siRNA knockdown of PAK2 in Jurkat cells reduced NFAT activation induced by anti-CD3/CD28 stimulation both in the presence and absence of Nef, and expression of a PAK2 dominant mutant inhibited Nef-mediated enhancement of CD25 expression. Conclusion Nef-mediated enhancement of cellular activation and viral replication in primary T cells is dependent on PAK2 and on the strength of the activating stimuli, and correlates with the ability of Nef to associate with PAK2. PAK2 is likely to play a role in Nef-mediated enhancement of viral replication and immune activation in vivo .
Jun Ling - One of the best experts on this subject based on the ideXlab platform.
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PAK2 activated by Cdc42 and caspase 3 mediates different cellular responses to oxidative stress-induced apoptosis.
Biochimica et biophysica acta. Molecular cell research, 2020Co-Authors: John Huang, Jolinda A. Traugh, Allen Huang, Amelia Poplawski, Frank Dipino, Jun LingAbstract:p21-activated protein kinase (PAK2) is a unique member of the PAK family kinases that plays important roles in stress signaling. It can be activated by binding to the small GTPase, Cdc42 and Rac1, or by caspase 3 cleavage. Cdc42-activated PAK2 mediates cytostasis, whereas caspase 3-cleaved PAK2 contributes to apoptosis. However, the relationship between these two states of PAK2 activation remains elusive. In this study, through protein biochemical analyses and various cell-based assays, we demonstrated that full-length PAK2 activated by Cdc42 was resistant to the cleavage by caspase 3 in vitro and within cells. When mammalian cells were treated by oxidative stress using hydrogen peroxide, PAK2 was highly activated through caspase 3 cleavage that led to apoptosis. However, when PAK2 was pre-activated by Cdc42 or by mild stress such as serum deprivation, it was no longer able to be cleaved by caspase 3 upon hydrogen peroxide treatment, and the subsequent apoptosis was also largely inhibited. Furthermore, cells expressing active mutants of full-length PAK2 became more resistant to hydrogen peroxide-induced apoptosis than inactive mutants. Taken together, this study identified two states of PAK2 activation, wherein Cdc42- and autophosphorylation-dependent activation inhibited the constitutive activation of PAK2 by caspase cleavage. The regulation between these two states of PAK2 activation provides a new molecular mechanism to support PAK2 as a molecular switch for controlling cytostasis and apoptosis in response to different types and levels of stress with broad physiological and pathological relevance.
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Activation of PAK2 by Serum Starvation Sensitizes its Response to InsulinTreatment in Adipocyte 3T3-L1 Cells
Biochemistry & Analytical Biochemistry, 2016Co-Authors: Jun Ling, Siska Corneillie, Colby Cottell, Jolinda A. TraughAbstract:p21-activated kinase-2 (PAK2) is ubiquitously expressed in all mammalian cells and tissues tested so far. It is a unique member of PAK family kinases that can be activated by various stress conditions to induce apoptosis or cytostasis. Although many conditions have been reported to activate PAK2, serum starvation followed by insulin treatment has not been studied. In this study, pre-adipocyte (3T3-L1) sensitive to insulin signaling and important for energy homeostasis was used as the system to address this topic. It was found that serum starvation transiently activated PAK2 activity by about 3-fold within one hour, then returning to the basal level within three hours. Following the activation of PAK2 by serum starvation, insulin treatment resulted in a rapid deactivation of PAK2 through ubiquitination-proteasome mediated protein degradation. AKT1 and PAK2 activities were reversely related, suggesting that AKT1 activation could be a factor to initiate PAK2 degradation. This dynamic change of PAK2 by serum starvation and insulin was found to be correlated with the fluctuation of protein synthesis, a major biological process to influence cell growth rate. Activation of PAK2 by serum starvation was correlated with about 50% inhibition of protein synthesis; subsequent treatment with insulin reversed this inhibition. Down-regulation of PAK2 by siRNA further proved that PAK2 was a causal factor leading to the inhibition of protein synthesis. In conclusion, this study identifies a new pattern of regulation of PAK2 by serum starvation and insulin, suggests an important role of PAK2 in regulating adipocyte function in response to nutrient status and insulin signaling.
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Inhibition of cap-dependent translation via phosphorylation of eIF4G by protein kinase PAK2
The EMBO journal, 2005Co-Authors: Jun Ling, Simon J. Morley, Jolinda A. TraughAbstract:Translation is downregulated in response to a variety of moderate stresses, including serum deprivation, hyperosmolarity and ionizing radiation. The cytostatic p21-activated protein kinase 2 (PAK2)/γ-PAK is activated under the same stress conditions. Expression of wild-type PAK2 in cells and addition of PAK2 to reticulocyte lysate inhibit translation, while kinase-inactive mutants have no effect. PAK2 binds to and phosphorylates initiation factor (eIF)4G, which inhibits association of eIF4E with m7GTP, reducing initiation. The PAK2-binding site maps to the region on eIF4G that contains the eIF4E-binding site; PAK2 and eIF4E compete for binding to this site. Using an eIF4G-depleted reticulocyte lysate, reconstitution with mock-phosphorylated eIF4G fully restores translation, while phosphorylated eIF4G reduces translation to 37%. RNA interference releases PAK2-induced inhibition of translation in contact-inhibited cells by 2.7-fold. eIF4G mutants of the PAK2 site show that S896D inhibits translation, while S896A has no effect. Activation of PAK2 in response to hyperosmotic stress inhibits cap-dependent, but not IRES-driven, initiation. Thus, a novel pathway for mammalian cell stress signaling is identified, wherein activation of PAK2 leads to inhibition of cap-dependent translation through phosphorylation of eIF4G.
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phosphorylation of mnk1 by caspase activated PAK2 γ pak inhibits phosphorylation and interaction of eif4g with mnk
Journal of Biological Chemistry, 2004Co-Authors: Kevin C. Orton, Jun Ling, Andrew J. Waskiewicz, Jonathan A. Cooper, William C. Merrick, Nadejda L. Korneeva, Robert E. Rhoads, Nahum Sonenberg, Jolinda A. TraughAbstract:Abstract The mitogen-activated protein kinase-interacting kinase 1 (Mnk1) is phosphorylated by caspase-cleaved protein kinase PAK2/γ-PAK but not by Cdc42-activated PAK2. Phosphorylation of Mnk1 is rapid, reaching 1 mol/mol within 15 min of incubation with PAK2. A kinetic analysis of the phosphorylation of Mnk1 by PAK2 yields a Km of 0.6 μm and a Vmax of 14.9 pmol of 32P/min/μg of PAK2. Two-dimensional tryptic phosphopeptide mapping of Mnk1 phosphorylated by PAK2 yields two distinct phosphopeptides. Analysis of the phosphopeptides by automated microsequencing and manual Edman degradation identified the sites in Mnk1 as Thr22 and Ser27. Mnk1, activated by phosphorylation with Erk2, phosphorylates the eukaryotic initiation factor (eIF) 4E and the eIF4G components of eIF4F. Phosphorylation of Mnk1 by PAK2 does not activate Mnk1, as measured with either eIF4E or eIF4F as substrate. Phosphorylation of Erk2-activated Mnk1 by PAK2 has no effect on phosphorylation of eIF4E but reduces phosphorylation of eIF4G by Mnk1 by up to 50%. Phosphorylation of Mnk1 by PAK2 inhibits binding of eIF4G peptides containing the Mnk1 binding site by up to 80%. When 293T cells are subjected to apoptotic induction by hydrogen peroxide, Mnk1 is phosphorylated at both Thr22 and Ser27. These results indicate a role for PAK2 in the down-regulation of translation initiation in apoptosis by phosphorylation of Mnk1.
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Phosphorylation of Mnk1 by Caspase-activated PAK2/γ-PAK Inhibits Phosphorylation and Interaction of eIF4G with Mnk
The Journal of biological chemistry, 2004Co-Authors: Kevin C. Orton, Jun Ling, Andrew J. Waskiewicz, Jonathan A. Cooper, William C. Merrick, Nadejda L. Korneeva, Robert E. Rhoads, Nahum Sonenberg, Jolinda A. TraughAbstract:Abstract The mitogen-activated protein kinase-interacting kinase 1 (Mnk1) is phosphorylated by caspase-cleaved protein kinase PAK2/γ-PAK but not by Cdc42-activated PAK2. Phosphorylation of Mnk1 is rapid, reaching 1 mol/mol within 15 min of incubation with PAK2. A kinetic analysis of the phosphorylation of Mnk1 by PAK2 yields a Km of 0.6 μm and a Vmax of 14.9 pmol of 32P/min/μg of PAK2. Two-dimensional tryptic phosphopeptide mapping of Mnk1 phosphorylated by PAK2 yields two distinct phosphopeptides. Analysis of the phosphopeptides by automated microsequencing and manual Edman degradation identified the sites in Mnk1 as Thr22 and Ser27. Mnk1, activated by phosphorylation with Erk2, phosphorylates the eukaryotic initiation factor (eIF) 4E and the eIF4G components of eIF4F. Phosphorylation of Mnk1 by PAK2 does not activate Mnk1, as measured with either eIF4E or eIF4F as substrate. Phosphorylation of Erk2-activated Mnk1 by PAK2 has no effect on phosphorylation of eIF4E but reduces phosphorylation of eIF4G by Mnk1 by up to 50%. Phosphorylation of Mnk1 by PAK2 inhibits binding of eIF4G peptides containing the Mnk1 binding site by up to 80%. When 293T cells are subjected to apoptotic induction by hydrogen peroxide, Mnk1 is phosphorylated at both Thr22 and Ser27. These results indicate a role for PAK2 in the down-regulation of translation initiation in apoptosis by phosphorylation of Mnk1.
