Serine/threonine protein kinase that plays a role in a variety of different signaling pathways including cytoskeleton regulation, cell motility, cell cycle progression, apoptosis or proliferation. Acts as downstream effector of the small GTPases CDC42 and RAC1. Activation by the binding of active CDC42 and RAC1 results in a conformational change and a subsequent autophosphorylation on several serine and/or threonine residues. Full-length PAK2 stimulates cell survival and cell growth. Phosphorylates MAPK4 and MAPK6 and activates the downstream target MAPKAPK5, a regulator of F-actin polymerization and cell migration. Phosphorylates JUN and plays an important role in EGF-induced cell proliferation. Phosphorylates many other substrates including histone H4 to promote assembly of H3.3 and H4 into nucleosomes, BAD, ribosomal protein S6, or MBP. Additionally, associates with ARHGEF7 and GIT1 to perform kinase-independent functions such as spindle orientation control during mitosis. On the other hand, apoptotic stimuli such as DNA damage lead to caspase-mediated cleavage of PAK2, generating PAK-2p34, an active p34 fragment that translocates to the nucleus and promotes cellular apoptosis involving the JNK signaling pathway. Caspase-activated PAK2 phosphorylates MKNK1 and reduces cellular translation.
The oncoprotein c-Jun is one of the components of the activator protein-1 (AP-1) transcription factor complex. AP-1 regulates the expression of many genes and is involved in a variety of biological functions such as cell transformation, proliferation, differentiation and apoptosis. AP-1 activates a variety of tumor-related genes and therefore promotes tumorigenesis and malignant transformation. Here, we found that epidermal growth factor (EGF) induces phosphorylation of c-Jun by P21-activated kinase (PAK) 2. Our data showed that PAK2 binds and phosphorylates c-Jun at five threonine sites (Thr2, Thr8, Thr89, Thr93 and Thr286) in vitro and ex vivo. Knockdown of PAK2 in JB6 Cl41 (P+) cells had no effect on c-Jun phosphorylation at Ser63 or Ser73 but resulted in decreases in EGF-induced anchorage-independent cell transformation, proliferation and AP-1 activity. Mutation at all five c-Jun threonine sites phosphorylated by PAK2 decreased the transforming ability of JB6 cells. Knockdown of PAK2 in SK-MEL-5 melanoma cells also decreased colony formation, proliferation and AP-1 activity. These results indicated that PAK2/c-Jun signaling plays an important role in EGF-induced cell proliferation and transformation.
Histone H3 variant H3.3, while differing from canonical H3 (H3.1) by only five amino acids, is assembled into nucleosomes, along with histone H4, at genic regions by the histone chaperone HIRA, whereas H3.1 is assembled into nucleosomes in a CAF-1-dependent reaction. Here, we show that phosphorylation of histone H4 Ser 47 (H4S47ph), catalyzed by the PAK2 kinase, promotes nucleosome assembly of H3.3-H4 and inhibits nucleosome assembly of H3.1-H4 by increasing the binding affinity of HIRA to H3.3-H4 and reducing association of CAF-1 with H3.1-H4. These results reveal a mechanism whereby H4S47ph distinctly regulates nucleosome assembly of H3.1 and H3.3.
The mitogen-activated protein kinase-interacting kinase 1 (Mnk1) is phosphorylated by caspase-cleaved protein kinase Pak2/gamma-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 K(m) of 0.6 microm and a V(max) of 14.9 pmol of (32)P/min/microg 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 Thr(22) and Ser(27). 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 Thr(22) and Ser(27). These results indicate a role for Pak2 in the down-regulation of translation initiation in apoptosis by phosphorylation of Mnk1.
Apoptosis of Jurkat T cells induced the caspase-mediated proteolytic cleavage of p21-activated kinase 2 (PAK2). Cleavage occurred between the amino-terminal regulatory domain and the carboxyl-terminal catalytic domain, which generated a constitutively active PAK2 fragment. Stable Jurkat cell lines that expressed a dominant-negative PAK mutant were resistant to the Fas-induced formation of apoptotic bodies, but had an enhanced externalization of phosphatidylserine at the cell surface. Thus, proteolytic activation of PAK2 represents a guanosine triphosphatase-independent mechanism of PAK regulation that allows PAK2 to regulate morphological changes that are seen in apoptotic cells.
The p21-activated kinase (PAK) 2 is known to be involved in numerous biological functions, including the regulation of actin reorganization and cell motility. To better understand the mechanisms underlying this regulation, we herein used a proteomic approach to identify PAK2-interacting proteins in human epidermoid carcinoma A431 cells. We found that MYO18A, an emerging member of the myosin superfamily, is a novel PAK2 binding partner. Using a siRNA knockdown strategy and in vitro binding assay, we discovered that MYO18A binds to PAK2 through the betaPIX/GIT1 complex. Under normal conditions, MYO18A and PAK2 colocalized in lamellipodia and membrane ruffles. Interestingly, knockdown of MYO18A in cells did not prevent formation of the PAK2/betaPIX/GIT1 complex, but rather apparently changed its localization to focal adhesions. Moreover, MYO18A-depleted cells showed dramatic changes in morphology and actin stress fiber and membrane ruffle formation and displayed increases in the number and size of focal adhesions. Migration assays revealed that MYO18A-depleted cells had decreased cell motility, and reexpression of MYO18A restored their migration ability. Collectively, our findings indicate that MYO18A is a novel binding partner of the PAK2/betaPIX/GIT1 complex and suggest that MYO18A may play an important role in regulating epithelial cell migration via affecting multiple cell machineries.
p21-activated protein kinases (PAKs) are a family of serine/threonine protein kinases that are activated by binding of the p21 G proteins Cdc42 or Rac. The ubiquitous PAK-2 (gamma-PAK) is unique among the PAK isoforms because it is also activated through proteolytic cleavage by caspases or caspase-like proteases. In response to stress stimulants such as tumor necrosis factor alpha or growth factor withdrawal, PAK-2 is activated as a full-length enzyme and as a proteolytic PAK-2p34 fragment. Activation of full-length PAK-2 stimulates cell survival, whereas proteolytic activation of PAK-2p34 is involved in programmed cell death. Here we provide evidence that the proapoptotic effect of PAK-2p34 is regulated by subcellular targeting and degradation by the proteasome. Full-length PAK-2 is localized in the cytoplasm, whereas the proteolytic PAK-2p34 fragment translocates to the nucleus. Subcellular localization of PAK-2 is regulated by nuclear localization and nuclear export signal motifs. A nuclear export signal motif within the regulatory domain prevents nuclear localization of full-length PAK-2. Proteolytic activation removes most of the regulatory domain and disrupts the nuclear export signal. The activated PAK-2p34 fragment contains a nuclear localization signal and translocates to the nucleus. However, levels of activated PAK-2p34 are tightly regulated through ubiquitination and degradation by the proteasome. Inhibition of degradation by blocking polyubiquitination results in significantly increased levels of PAK-2p34 and as a consequence, in stimulation of programmed cell death. Therefore, nuclear targeting and inhibition of degradation appear to be critical for stimulation of the cell death response by PAK-2p34.
p21-activated protein kinase (PAK) 2 is a small GTPase-activated serine/threonine kinase regulating various cytoskeletal functions and is cleaved by caspase-3 during apoptosis. We demonstrate that the caspase-cleaved PAK2 C-terminal kinase fragment (C-t-PAK2) is posttranslationally myristoylated, although myristoylation is typically a cotranslational process. Myristoylation and an adjacent polybasic domain of C-t-PAK2 are sufficient to redirect EGFP from the cytosol to membrane ruffles and internal membranes. Membrane localization and the ability of C-t-PAK2 to induce cell death are significantly reduced when myristoylation is abolished. In addition, the proper myristoylation-dependent membrane localization of C-t-PAK2 significantly increased signaling through the stress-activated c-Jun N-terminal kinase signaling pathway, which often regulates apoptosis. Interestingly, C-t-PAK2 promoted cell death without compromising mitochondrial integrity. Posttranslational myristoylation of caspase-cleaved proteins involved in cytoskeletal dynamics (e.g., PAK2, actin, and gelsolin) might be part of a unique series of mechanisms involved in the regulation of the later events of apoptosis.
The class I p21-activated kinases (Pak1-3) regulate many essential biological processes, including cytoskeletal rearrangement, cell cycle progression, apoptosis, and cellular transformation. Although many Pak substrates, including elements of MAPK signaling cascades, have been identified, it is likely that additional substrates remain to be discovered. Identification of such substrates, and determination of the consequences of their phosphorylation, is essential for a better understanding of class I Pak activity. To identify novel class I Pak substrates, we used recombinant Pak2 to screen high density protein microarrays. This approach identified the atypical MAPK Erk3 as a potential Pak2 substrate. Solution-based in vitro kinase assays using recombinant Erk3 confirmed the protein microarray results, and phospho-specific antisera identified serine 189, within the Erk3 activation loop, as a site directly phosphorylated by Pak2 in vitro. Erk3 protein is known to shuttle between the cytoplasm and the nucleus, and we showed that selective inhibition of class I Pak kinase activity in cells promoted increased nuclear accumulation of Erk3. Pak inhibition in cells additionally reduced the extent of Ser(189) phosphorylation and inhibited the formation of Erk3-Prak complexes. Collectively, our results identify the Erk3 protein as a novel class I Pak substrate and further suggest a role for Pak kinase activity in atypical MAPK signaling.
The p21-activated kinases (PAKs) are serine/threonine kinases that are involved in a wide variety of cellular functions including cytoskeletal motility, apoptosis, and cell cycle regulation. PAKs are inactivated by blockage of the active site of the kinase domain by an N-terminal regulatory domain. GTP-bound forms of Cdc42 and Rac bind to the regulatory domain and displace it, thereby allowing phosphorylation of the kinase domain and maximal activation. A key step in the activation process is the phosphorylation of the activation loop of one PAK kinase domain by another, but little is known about the underlying recognition events that make this phosphorylation specific. We show that the phosphorylated kinase domain of PAK2 dimerizes in solution and that this association is prevented by addition of a substrate peptide. We have identified a crystallographic dimer in a previously determined crystal structure of activated PAK1 in which two kinase domains are arranged face to face and interact through a surface on the large lobe of the kinase domain that is exposed upon release of the auto-inhibitory domain. The crystallographic dimer is suggestive of an engagement that mediates trans-autophosphorylation. Mutations at the predicted dimerization interface block dimerization and reduce the rate of autophosphorylation, supporting the role of this interface in PAK activation.
Evidence
2:
Inferred from Physical InteractionIntAct
p21-activated kinase (PAK) 2, a member of the PAK family of serine/threonine protein kinases, plays an important role in physiological processes such as motility, survival, mitosis, and apoptosis. However, the role of PAK2 in resistance to chemotherapy is unclear. Here we report that PAK2 is highly expressed in human breast cancer cell lines and human breast invasive carcinoma tissue compared with a human non-tumorigenic mammary epithelial cell line and adjacent normal breast tissue, respectively. Interestingly, we found that PAK2 can bind with caspase-7 and phosphorylate caspase-7 at the Ser-30, Thr-173, and Ser-239 sites. Functionally, the phosphorylation of caspase-7 decreases its activity, thereby inhibiting cellular apoptosis. Our data indicate that highly expressed PAK2 mediates chemotherapeutic resistance in human breast invasive ductal carcinoma by negatively regulating caspase-7 activity.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionUniProtKB
The Nef protein of primate immunodeficiency viruses plays an important role in the pathogenesis of acquired immunodeficiency syndrome (AIDS) [1] [2]. The interaction of Nef with the Nef-associated kinase (NAK) is one of the most conserved properties of different human and simian immunodeficiency virus (HIV and SIV) Nef alleles. The role of NAK association is currently not known but it has been implicated in enhanced viral infectivity in cell culture and in disease progression in SIV-infected macaques [3]. Previous studies have indicated that NAK shares many features with the p21-activated kinases (PAKs) [3], but the molecular identity of NAK has remained unknown. We have generated specific antisera against PAKs 1-3, and expressed these kinases individually as epitope-tagged proteins. By using these reagents in experiments involving partial proteolytic mapping, and exploiting the unique ability of PAK2 to serve as a caspase substrate, we have positively identified NAK as PAK2. Interestingly, although ectopic PAK2 overexpression efficiently replaced endogenous PAK2 from the complex with Nef, the total Nef-associated PAK2 activity was not increased, indicating the abundance of another cellular factor(s) as the limiting factor in Nef-PAK2 complex formation. Identification of NAK as PAK2 should now facilitate elucidation of its role as a mediator of the pathogenic effects of Nef.
Evidence
2:
Inferred from Physical InteractionUniProtKB
Genetic studies have highlighted the key role of Scrib in the development of Metazoans. Deficiency in Scrib impairs many aspects of cell polarity and cell movement although the mechanisms involved remain unclear. In mammals, Scrib belongs to a protein complex containing betaPIX, an exchange factor for Rac/Cdc42, and GIT1, a GTPase activating protein for ARF6 implicated in receptor recycling and exocytosis. Here we show that the Scrib complex associates with PAK, a serine-threonine kinase family crucial for cell migration. PAK colocalizes with members of the Scrib complex at the leading edge of heregulin-treated T47D breast cancer cells. We demonstrate that the Scrib complex is required for epithelial cells and primary mouse embryonic fibroblasts to efficiently respond to chemoattractant cues. In Scrib-deficient cells, the pool of cortical PAK is decreased, thereby precluding its proper activation by Rac. Loss of Scrib also impairs the polarized distribution of active Rac at the leading edge and compromises the regulated activation of the GTPase in T47D cells and mouse embryonic fibroblasts. These data underscore the role of Scrib in cell migration and show the strong impact of Scrib in the function of PAK and Rac, two key molecules implicated in this process.
Evidence
3:
Inferred from Physical InteractionIntAct
The p21-activated kinases (PAKs) are serine/threonine kinases that are involved in a wide variety of cellular functions including cytoskeletal motility, apoptosis, and cell cycle regulation. PAKs are inactivated by blockage of the active site of the kinase domain by an N-terminal regulatory domain. GTP-bound forms of Cdc42 and Rac bind to the regulatory domain and displace it, thereby allowing phosphorylation of the kinase domain and maximal activation. A key step in the activation process is the phosphorylation of the activation loop of one PAK kinase domain by another, but little is known about the underlying recognition events that make this phosphorylation specific. We show that the phosphorylated kinase domain of PAK2 dimerizes in solution and that this association is prevented by addition of a substrate peptide. We have identified a crystallographic dimer in a previously determined crystal structure of activated PAK1 in which two kinase domains are arranged face to face and interact through a surface on the large lobe of the kinase domain that is exposed upon release of the auto-inhibitory domain. The crystallographic dimer is suggestive of an engagement that mediates trans-autophosphorylation. Mutations at the predicted dimerization interface block dimerization and reduce the rate of autophosphorylation, supporting the role of this interface in PAK activation.
Evidence
4:
Inferred from Physical InteractionIntAct
p21-activated kinase (PAK) 2, a member of the PAK family of serine/threonine protein kinases, plays an important role in physiological processes such as motility, survival, mitosis, and apoptosis. However, the role of PAK2 in resistance to chemotherapy is unclear. Here we report that PAK2 is highly expressed in human breast cancer cell lines and human breast invasive carcinoma tissue compared with a human non-tumorigenic mammary epithelial cell line and adjacent normal breast tissue, respectively. Interestingly, we found that PAK2 can bind with caspase-7 and phosphorylate caspase-7 at the Ser-30, Thr-173, and Ser-239 sites. Functionally, the phosphorylation of caspase-7 decreases its activity, thereby inhibiting cellular apoptosis. Our data indicate that highly expressed PAK2 mediates chemotherapeutic resistance in human breast invasive ductal carcinoma by negatively regulating caspase-7 activity.
Evidence
5:
Inferred from Physical InteractionIntAct
Huntington's disease is caused by a polyglutamine expansion in the huntingtin protein. Wild-type huntingtin, by contrast, appears to protect cells from pro-apoptotic insults. Here we describe a novel anti-apoptotic function for huntingtin. When cells are exposed to Fas-related signals, the ubiquitously expressed p21-activated kinase 2 (Pak2) can be activated via cleavage by caspases to release a constitutively active C-terminal fragment, which mediates cell death. Our data show that huntingtin interacts with Pak2. Overexpression of huntingtin significantly inhibits caspase-3-mediated and caspase-8-mediated cleavage of Pak2 in cells. Moreover, huntingtin prevents Pak2 cleavage by caspase-3 and caspase-8 in vitro. Although huntingtin is cytoprotective in wild-type cells that are exposed to TNFalpha, it has no significant benefit in TNFalpha-treated cells with Pak2 knockdown. Thus, huntingtin exerts anti-apoptotic effects by binding to Pak2, which reduces the abilities of caspase-3 and caspase-8 to cleave Pak2 and convert it into a mediator of cell death.
Phosphorylation of myosin II regulatory light chains (RLC) by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) is a critical step in the initiation of smooth muscle and non-muscle cell contraction. Post-translational modifications to MLCK down-regulate enzyme activity, suppressing RLC phosphorylation, myosin II activation, and tension development. Here we report that PAK2, a member of the Rho family of GTPase-dependent kinases, regulates isometric tension development and myosin II RLC phosphorylation in saponin permeabilized endothelial monolayers. PAK2 blunts tension development by 75% while inhibiting diphosphorylation of myosin II RLC. Cdc42-activated placenta and recombinant, constitutively active PAK2 phosphorylate MLCK in vitro with a stoichiometry of 1.71 +/- 0. 21 mol of PO(4)/mol of MLCK. This phosphorylation inhibits MLCK phosphorylation of myosin II RLC. PAK2 catalyzes MLCK phosphorylation on serine residues 439 and 991. Binding calmodulin to MLCK blocks phosphorylation of Ser-991 by PAK2. These results demonstrate that PAK2 can directly phosphorylate MLCK, inhibiting its activity and limiting the development of isometric tension.
Interacting selectively and non-covalently with a protein kinase, any enzyme that catalyzes the transfer of a phosphate group, usually from ATP, to a protein substrate.
Evidence
1:
Inferred from Physical InteractionBHF-UCL
A member of the p21-activated protein kinase (PAK) family, gamma-PAK has cytostatic properties and is activated by cellular stresses such as hyperosmolarity or DNA damage. We report herein that gamma-PAK is associated in vivo with the nonreceptor protein tyrosine kinase c-Abl. gamma-PAK phosphorylates c-Abl on sites located in the kinase domain, in a region that is implicated in protein-protein interactions and in subcellular localization. Activation of gamma-PAK in human embryonic kidney 293T cells by cotransfection with constitutively active Cdc42 induces activation of c-Abl, resulting in increased phosphotyrosine levels. Cotransfection of c-Abl and gamma-PAK elicits phosphorylation of gamma-PAK on tyrosine and down-regulation of gamma-PAK activity, promoting accumulation of inactive gamma-PAK. gamma-PAK is also phosphorylated in vitro by c-Abl. gamma-PAK activity is regulated by ubiquitination and proteolysis in vivo, as shown by immunoblotting with an anti-ubiquitin antibody in the presence of proteasome inhibitors. In summary, we describe a functional interaction between gamma-PAK and c-Abl in which gamma-PAK stimulates c-Abl tyrosine kinase activity and c-Abl phosphorylates and down-regulates gamma-PAK, suggesting the existence of a negative feedback loop between c-Abl and gamma-PAK.
Mammalian STE20-like kinase (MST) is a member of the yeast STE20-related kinase family and proteolytically activated by caspase during apoptosis. However, its other cellular functions are not known, including its activation mechanism, substrate(s), and subcellular localization. In this report, using anti-MST monoclonal antibodies, we clearly show that endogenous MST is localized in cytoplasm in a leptomycin B-dependent manner. Analyses with serial deletions and point mutations show that MST has two functional nuclear export signals and, unexpectedly, another localization motif for nuclear import. When cells are treated with leptomycin, monomeric MST is accumulated more rapidly in the nucleus than dimeric MST, indicating that dimerization contributes to the cytoplasmic retention of MST. Okadaic acid, an inhibitor of phosphatase 2A, induces activation of MST and translocation into the nucleus. Using phosphopeptide-specific antibody, we directly show that okadaic acid induces phosphorylation in the activation loop of MST, and, once phosphorylated, MST is rapidly translocated to the nucleus. However, kinase-deficient MST does not enter the nucleus, indicating that phosphorylation and activation is required for okadaic acid-induced nuclear translocation. In apoptotic cells, the activation of MST does not require phosphorylation in the activation loop and occurs through the release of C-terminal regulatory domain by caspase-dependent cleavage. Kinase-deficient MST functions dominant-negatively and represses okadaic acid-induced morphological change indicating that MST plays a role in okadaic acid-induced cellular shrinkage. Our identification of cytoplasmic and nuclear localization motifs and phosphorylation-dependent translocation of MST suggests that regulation of localization is important to the biological function of MST, including its effects on cellular morphology.
A member of the p21-activated protein kinase (PAK) family, gamma-PAK has cytostatic properties and is activated by cellular stresses such as hyperosmolarity or DNA damage. We report herein that gamma-PAK is associated in vivo with the nonreceptor protein tyrosine kinase c-Abl. gamma-PAK phosphorylates c-Abl on sites located in the kinase domain, in a region that is implicated in protein-protein interactions and in subcellular localization. Activation of gamma-PAK in human embryonic kidney 293T cells by cotransfection with constitutively active Cdc42 induces activation of c-Abl, resulting in increased phosphotyrosine levels. Cotransfection of c-Abl and gamma-PAK elicits phosphorylation of gamma-PAK on tyrosine and down-regulation of gamma-PAK activity, promoting accumulation of inactive gamma-PAK. gamma-PAK is also phosphorylated in vitro by c-Abl. gamma-PAK activity is regulated by ubiquitination and proteolysis in vivo, as shown by immunoblotting with an anti-ubiquitin antibody in the presence of proteasome inhibitors. In summary, we describe a functional interaction between gamma-PAK and c-Abl in which gamma-PAK stimulates c-Abl tyrosine kinase activity and c-Abl phosphorylates and down-regulates gamma-PAK, suggesting the existence of a negative feedback loop between c-Abl and gamma-PAK.
A member of the p21-activated protein kinase (PAK) family, gamma-PAK has cytostatic properties and is activated by cellular stresses such as hyperosmolarity or DNA damage. We report herein that gamma-PAK is associated in vivo with the nonreceptor protein tyrosine kinase c-Abl. gamma-PAK phosphorylates c-Abl on sites located in the kinase domain, in a region that is implicated in protein-protein interactions and in subcellular localization. Activation of gamma-PAK in human embryonic kidney 293T cells by cotransfection with constitutively active Cdc42 induces activation of c-Abl, resulting in increased phosphotyrosine levels. Cotransfection of c-Abl and gamma-PAK elicits phosphorylation of gamma-PAK on tyrosine and down-regulation of gamma-PAK activity, promoting accumulation of inactive gamma-PAK. gamma-PAK is also phosphorylated in vitro by c-Abl. gamma-PAK activity is regulated by ubiquitination and proteolysis in vivo, as shown by immunoblotting with an anti-ubiquitin antibody in the presence of proteasome inhibitors. In summary, we describe a functional interaction between gamma-PAK and c-Abl in which gamma-PAK stimulates c-Abl tyrosine kinase activity and c-Abl phosphorylates and down-regulates gamma-PAK, suggesting the existence of a negative feedback loop between c-Abl and gamma-PAK.
p21-Activated protein kinase 2 (PAK-2) has both anti- and pro-apoptotic functions depending on its mechanism of activation. Activation of full-length PAK-2 by the monomeric GTPases Cdc42 or Rac stimulates cell survival, whereas caspase activation of PAK-2 to the PAK-2p34 fragment is involved in the apoptotic response. In this study we use functional knockout of PAK-2 and gene replacement with the caspase cleavage-deficient PAK-2D212N mutant to differentiate the biological functions of full-length PAK-2 and caspase-activated PAK-2p34. Knockout of PAK-2 results in embryonic lethality at early stages before organ development, whereas replacement with the caspase cleavage-deficient PAK-2D212N results in viable and healthy mice, indicating that early embryonic lethality is caused by deficiency of full-length PAK-2 rather than lack of caspase activation to the PAK-2p34 fragment. However, deficiency of caspase activation of PAK-2 decreased spontaneous cell death of primary mouse embryonic fibroblasts and increased cell growth at high cell density. In contrast, stress-induced cell death by treatment with the anti-cancer drug cisplatin was not reduced by deficiency of caspase activation of PAK-2, but switched from an apoptotic to a nonapoptotic, caspase-independent mechanism. Homozygous PAK-2D212N primary mouse embryonic fibroblasts that lack the ability to generate the proapoptotic PAK-2p34 show less activation of the effector caspase 3, 6, and 7, indicating that caspase activation of PAK-2 amplifies the apoptotic response through a positive feedback loop resulting in more activation of effector caspases.
Negative regulation of cysteine-type endopeptidase activity involved in execution phase of apoptosisdefinition[GO:2001271]
Any process that stops, prevents or reduces the frequency, rate or extent of cysteine-type endopeptidase activity involved in execution phase of apoptosis.
p21-Activated protein kinase 2 (PAK-2) has both anti- and pro-apoptotic functions depending on its mechanism of activation. Activation of full-length PAK-2 by the monomeric GTPases Cdc42 or Rac stimulates cell survival, whereas caspase activation of PAK-2 to the PAK-2p34 fragment is involved in the apoptotic response. In this study we use functional knockout of PAK-2 and gene replacement with the caspase cleavage-deficient PAK-2D212N mutant to differentiate the biological functions of full-length PAK-2 and caspase-activated PAK-2p34. Knockout of PAK-2 results in embryonic lethality at early stages before organ development, whereas replacement with the caspase cleavage-deficient PAK-2D212N results in viable and healthy mice, indicating that early embryonic lethality is caused by deficiency of full-length PAK-2 rather than lack of caspase activation to the PAK-2p34 fragment. However, deficiency of caspase activation of PAK-2 decreased spontaneous cell death of primary mouse embryonic fibroblasts and increased cell growth at high cell density. In contrast, stress-induced cell death by treatment with the anti-cancer drug cisplatin was not reduced by deficiency of caspase activation of PAK-2, but switched from an apoptotic to a nonapoptotic, caspase-independent mechanism. Homozygous PAK-2D212N primary mouse embryonic fibroblasts that lack the ability to generate the proapoptotic PAK-2p34 show less activation of the effector caspase 3, 6, and 7, indicating that caspase activation of PAK-2 amplifies the apoptotic response through a positive feedback loop resulting in more activation of effector caspases.
Phosphorylation of myosin II regulatory light chains (RLC) by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) is a critical step in the initiation of smooth muscle and non-muscle cell contraction. Post-translational modifications to MLCK down-regulate enzyme activity, suppressing RLC phosphorylation, myosin II activation, and tension development. Here we report that PAK2, a member of the Rho family of GTPase-dependent kinases, regulates isometric tension development and myosin II RLC phosphorylation in saponin permeabilized endothelial monolayers. PAK2 blunts tension development by 75% while inhibiting diphosphorylation of myosin II RLC. Cdc42-activated placenta and recombinant, constitutively active PAK2 phosphorylate MLCK in vitro with a stoichiometry of 1.71 +/- 0. 21 mol of PO(4)/mol of MLCK. This phosphorylation inhibits MLCK phosphorylation of myosin II RLC. PAK2 catalyzes MLCK phosphorylation on serine residues 439 and 991. Binding calmodulin to MLCK blocks phosphorylation of Ser-991 by PAK2. These results demonstrate that PAK2 can directly phosphorylate MLCK, inhibiting its activity and limiting the development of isometric tension.
A member of the p21-activated protein kinase (PAK) family, gamma-PAK has cytostatic properties and is activated by cellular stresses such as hyperosmolarity or DNA damage. We report herein that gamma-PAK is associated in vivo with the nonreceptor protein tyrosine kinase c-Abl. gamma-PAK phosphorylates c-Abl on sites located in the kinase domain, in a region that is implicated in protein-protein interactions and in subcellular localization. Activation of gamma-PAK in human embryonic kidney 293T cells by cotransfection with constitutively active Cdc42 induces activation of c-Abl, resulting in increased phosphotyrosine levels. Cotransfection of c-Abl and gamma-PAK elicits phosphorylation of gamma-PAK on tyrosine and down-regulation of gamma-PAK activity, promoting accumulation of inactive gamma-PAK. gamma-PAK is also phosphorylated in vitro by c-Abl. gamma-PAK activity is regulated by ubiquitination and proteolysis in vivo, as shown by immunoblotting with an anti-ubiquitin antibody in the presence of proteasome inhibitors. In summary, we describe a functional interaction between gamma-PAK and c-Abl in which gamma-PAK stimulates c-Abl tyrosine kinase activity and c-Abl phosphorylates and down-regulates gamma-PAK, suggesting the existence of a negative feedback loop between c-Abl and gamma-PAK.
The process of introducing a phosphate group into a molecule, usually with the formation of a phosphoric ester, a phosphoric anhydride or a phosphoric amide.
p21-Activated protein kinase 2 (PAK-2) has both anti- and pro-apoptotic functions depending on its mechanism of activation. Activation of full-length PAK-2 by the monomeric GTPases Cdc42 or Rac stimulates cell survival, whereas caspase activation of PAK-2 to the PAK-2p34 fragment is involved in the apoptotic response. In this study we use functional knockout of PAK-2 and gene replacement with the caspase cleavage-deficient PAK-2D212N mutant to differentiate the biological functions of full-length PAK-2 and caspase-activated PAK-2p34. Knockout of PAK-2 results in embryonic lethality at early stages before organ development, whereas replacement with the caspase cleavage-deficient PAK-2D212N results in viable and healthy mice, indicating that early embryonic lethality is caused by deficiency of full-length PAK-2 rather than lack of caspase activation to the PAK-2p34 fragment. However, deficiency of caspase activation of PAK-2 decreased spontaneous cell death of primary mouse embryonic fibroblasts and increased cell growth at high cell density. In contrast, stress-induced cell death by treatment with the anti-cancer drug cisplatin was not reduced by deficiency of caspase activation of PAK-2, but switched from an apoptotic to a nonapoptotic, caspase-independent mechanism. Homozygous PAK-2D212N primary mouse embryonic fibroblasts that lack the ability to generate the proapoptotic PAK-2p34 show less activation of the effector caspase 3, 6, and 7, indicating that caspase activation of PAK-2 amplifies the apoptotic response through a positive feedback loop resulting in more activation of effector caspases.
Huntington's disease is caused by a polyglutamine expansion in the huntingtin protein. Wild-type huntingtin, by contrast, appears to protect cells from pro-apoptotic insults. Here we describe a novel anti-apoptotic function for huntingtin. When cells are exposed to Fas-related signals, the ubiquitously expressed p21-activated kinase 2 (Pak2) can be activated via cleavage by caspases to release a constitutively active C-terminal fragment, which mediates cell death. Our data show that huntingtin interacts with Pak2. Overexpression of huntingtin significantly inhibits caspase-3-mediated and caspase-8-mediated cleavage of Pak2 in cells. Moreover, huntingtin prevents Pak2 cleavage by caspase-3 and caspase-8 in vitro. Although huntingtin is cytoprotective in wild-type cells that are exposed to TNFalpha, it has no significant benefit in TNFalpha-treated cells with Pak2 knockdown. Thus, huntingtin exerts anti-apoptotic effects by binding to Pak2, which reduces the abilities of caspase-3 and caspase-8 to cleave Pak2 and convert it into a mediator of cell death.
A member of the p21-activated protein kinase (PAK) family, gamma-PAK has cytostatic properties and is activated by cellular stresses such as hyperosmolarity or DNA damage. We report herein that gamma-PAK is associated in vivo with the nonreceptor protein tyrosine kinase c-Abl. gamma-PAK phosphorylates c-Abl on sites located in the kinase domain, in a region that is implicated in protein-protein interactions and in subcellular localization. Activation of gamma-PAK in human embryonic kidney 293T cells by cotransfection with constitutively active Cdc42 induces activation of c-Abl, resulting in increased phosphotyrosine levels. Cotransfection of c-Abl and gamma-PAK elicits phosphorylation of gamma-PAK on tyrosine and down-regulation of gamma-PAK activity, promoting accumulation of inactive gamma-PAK. gamma-PAK is also phosphorylated in vitro by c-Abl. gamma-PAK activity is regulated by ubiquitination and proteolysis in vivo, as shown by immunoblotting with an anti-ubiquitin antibody in the presence of proteasome inhibitors. In summary, we describe a functional interaction between gamma-PAK and c-Abl in which gamma-PAK stimulates c-Abl tyrosine kinase activity and c-Abl phosphorylates and down-regulates gamma-PAK, suggesting the existence of a negative feedback loop between c-Abl and gamma-PAK.
A member of the p21-activated protein kinase (PAK) family, gamma-PAK has cytostatic properties and is activated by cellular stresses such as hyperosmolarity or DNA damage. We report herein that gamma-PAK is associated in vivo with the nonreceptor protein tyrosine kinase c-Abl. gamma-PAK phosphorylates c-Abl on sites located in the kinase domain, in a region that is implicated in protein-protein interactions and in subcellular localization. Activation of gamma-PAK in human embryonic kidney 293T cells by cotransfection with constitutively active Cdc42 induces activation of c-Abl, resulting in increased phosphotyrosine levels. Cotransfection of c-Abl and gamma-PAK elicits phosphorylation of gamma-PAK on tyrosine and down-regulation of gamma-PAK activity, promoting accumulation of inactive gamma-PAK. gamma-PAK is also phosphorylated in vitro by c-Abl. gamma-PAK activity is regulated by ubiquitination and proteolysis in vivo, as shown by immunoblotting with an anti-ubiquitin antibody in the presence of proteasome inhibitors. In summary, we describe a functional interaction between gamma-PAK and c-Abl in which gamma-PAK stimulates c-Abl tyrosine kinase activity and c-Abl phosphorylates and down-regulates gamma-PAK, suggesting the existence of a negative feedback loop between c-Abl and gamma-PAK.
Mammalian STE20-like kinase (MST) is a member of the yeast STE20-related kinase family and proteolytically activated by caspase during apoptosis. However, its other cellular functions are not known, including its activation mechanism, substrate(s), and subcellular localization. In this report, using anti-MST monoclonal antibodies, we clearly show that endogenous MST is localized in cytoplasm in a leptomycin B-dependent manner. Analyses with serial deletions and point mutations show that MST has two functional nuclear export signals and, unexpectedly, another localization motif for nuclear import. When cells are treated with leptomycin, monomeric MST is accumulated more rapidly in the nucleus than dimeric MST, indicating that dimerization contributes to the cytoplasmic retention of MST. Okadaic acid, an inhibitor of phosphatase 2A, induces activation of MST and translocation into the nucleus. Using phosphopeptide-specific antibody, we directly show that okadaic acid induces phosphorylation in the activation loop of MST, and, once phosphorylated, MST is rapidly translocated to the nucleus. However, kinase-deficient MST does not enter the nucleus, indicating that phosphorylation and activation is required for okadaic acid-induced nuclear translocation. In apoptotic cells, the activation of MST does not require phosphorylation in the activation loop and occurs through the release of C-terminal regulatory domain by caspase-dependent cleavage. Kinase-deficient MST functions dominant-negatively and represses okadaic acid-induced morphological change indicating that MST plays a role in okadaic acid-induced cellular shrinkage. Our identification of cytoplasmic and nuclear localization motifs and phosphorylation-dependent translocation of MST suggests that regulation of localization is important to the biological function of MST, including its effects on cellular morphology.
Mammalian STE20-like kinase (MST) is a member of the yeast STE20-related kinase family and proteolytically activated by caspase during apoptosis. However, its other cellular functions are not known, including its activation mechanism, substrate(s), and subcellular localization. In this report, using anti-MST monoclonal antibodies, we clearly show that endogenous MST is localized in cytoplasm in a leptomycin B-dependent manner. Analyses with serial deletions and point mutations show that MST has two functional nuclear export signals and, unexpectedly, another localization motif for nuclear import. When cells are treated with leptomycin, monomeric MST is accumulated more rapidly in the nucleus than dimeric MST, indicating that dimerization contributes to the cytoplasmic retention of MST. Okadaic acid, an inhibitor of phosphatase 2A, induces activation of MST and translocation into the nucleus. Using phosphopeptide-specific antibody, we directly show that okadaic acid induces phosphorylation in the activation loop of MST, and, once phosphorylated, MST is rapidly translocated to the nucleus. However, kinase-deficient MST does not enter the nucleus, indicating that phosphorylation and activation is required for okadaic acid-induced nuclear translocation. In apoptotic cells, the activation of MST does not require phosphorylation in the activation loop and occurs through the release of C-terminal regulatory domain by caspase-dependent cleavage. Kinase-deficient MST functions dominant-negatively and represses okadaic acid-induced morphological change indicating that MST plays a role in okadaic acid-induced cellular shrinkage. Our identification of cytoplasmic and nuclear localization motifs and phosphorylation-dependent translocation of MST suggests that regulation of localization is important to the biological function of MST, including its effects on cellular morphology.
Any process that modulates the frequency, rate or extent of the growth of all or part of an organism so that it occurs at its proper speed, either globally or in a specific part of the organism's development.
The cellular process in which a signal is conveyed to trigger a change in the activity or state of a cell. Signal transduction begins with reception of a signal (e.g. a ligand binding to a receptor or receptor activation by a stimulus such as light), or for signal transduction in the absence of ligand, signal-withdrawal or the activity of a constitutively active receptor. Signal transduction ends with regulation of a downstream cellular process, e.g. regulation of transcription or regulation of a metabolic process. Signal transduction covers signaling from receptors located on the surface of the cell and signaling via molecules located within the cell. For signaling between cells, signal transduction is restricted to events at and within the receiving cell.
We identified three proteins in neutrophil cytosol of molecular size 65, 62 and 68 kDa which interact in a GTP-dependent manner with rac1 and CDC42Hs, but not with rho. Purification of p65 and subsequent peptide sequencing revealed identity to rat brain PAK65 and to yeast STE20 kinase domains. Based on these sequences we screened a human placenta library and cloned the full-length cDNA. The complete amino acid sequence of the human cDNA shares approximately identity with rat brain PAK65; within the kinase domain the human protein shares > 95% and approximately 63% identity with rat PAK65 and yeast STE20 respectively. The new human (h)PAK65 mRNA is ubiquitously expressed and hPAK65 protein is distinct from either human or rat brain PAK65. Recombinant hPAK65 exhibits identical specificity to the endogenous p65; both can bind rac1 and CDC42Hs in a GTP-dependent manner. The GTP-bound forms of rac1 and CDC42Hs induce autophosphorylation of hPAK65 on serine residues only. hPAK65 activated by either rac1 or CDC42Hs is phosphorylated on the same sites. Induction of hPAK65 autophosphorylation by rac1 or CDC42Hs stimulates hPAK65 kinase activity towards myelin basic protein and once hPAK65 is activated, rac1 or CDC42Hs are no longer required to keep it active. The affinities of rac/CDC42Hs for the non-phosphorylated and phosphorylated hPAK65 were similar. hPAK65 had only a marginal effect on the intrinsic GTPase activity of CDC42Hs, but significantly affected the binding and GAP activity of p190. These data are consistent with a model in which hPAK65 functions as an effector molecule for rac1 and CDC42Hs.
Interactions, directly with the host cell macromolecular machinery, to allow virus replication.
IEAUniProtKB KW
Enzymatic activity
This protein acts as an enzyme. It is known to catalyze the following reaction
EC 2.7.11.1: ATP + a protein ⇄ ADP + a phosphoprotein.
CuratedUniProtKB
It is regulated in the following manner
Activated by binding small G proteins. Binding of GTP-bound CDC42 or RAC1 to the autoregulatory region releases monomers from the autoinhibited dimer, enables phosphorylation of Thr-402 and allows the kinase domain to adopt an active structure (By similarity). Following caspase cleavage, autophosphorylted PAK-2p34 is constitutively active.
Protein involved in apoptotic programmed cell death. Apoptosis is characterized by cell morphological changes, including blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation and chromosomal DNA fragmentation, and eventually death. Unlike necrosis, apoptosis produces cell fragments, called apoptotic bodies, that phagocytic cells are able to engulf and quickly remove before the contents of the cell can spill out onto surrounding cells and cause damage. In general, apoptosis confers advantages during an organism's life cycle.
Viral protein involved in a direct and specific interaction with a host macromolecule. Viruses interact with many cellular pathways to achieve their replication cycle. Entry into the host cell, transport to the viral replication sites or viral budding are all steps that require interaction between the host and the virus. Additionally, the evasion from the host immune response requires a lot of viral proteins to associate with and inhibit cellular proteins with antiviral functions.
Protein which catalyzes the phosphorylation of serine or threonine residues on target proteins by using ATP as phosphate donor. Such phosphorylation may cause changes in the function of the target protein. Protein kinases share a conserved catalytic core common to both serine/ threonine and tyrosine protein kinases.
Enzyme whose activity is modified by the noncovalent binding of an allosteric effector at a site other than the active site. This binding mediates conformational changes, altering its catalytic or binding properties.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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