Induction of apoptosis by p110C requires mitochondrial translocation of the proapoptotic BCL-2 family member BAD

p110C, a 50-kDa isoform of the PITSLRE kinase family, was demonstrated to play an important role in cell apoptosis. However, how p110C exactly promotes apoptosis is unclear. Our previous study showed that p110C interacted with p21-activated kinase 1 (PAK1), an important kinase of the proapoptotic BCL-2 family member BAD, and evidently inhibited its kinase activity. Here, we report that overexpression of p110C leads to decreased phosphorylation of BAD and its subsequent translocation from cytosol to mitochondria, which in turn induces the release of cytochrome c and the onset of apoptosis. Knocking down endogenous BAD expression will inhibit p110C induced apoptosis. Two kinase dead forms of p110C, D149N and K36N, lose the ability to inhibit the kinase activity of PAK1 and fail to induce the translocation of BAD and the BAD and such proapoptotic ability is associated with the kinase activity of p110C.     

Interaction of p58(PITSLRE), a G2/M-specific protein kinase,with cyclin D3

The p58(PITSLRE) is a p34(cdc2)-related protein kinase that plays an important role in normal cell cycle progression. Elevated expression of p58(PITSLRE) in eukaryotic cells prevents them from undergoing normal cytokinesis and appears to delay them in late telophase. To investigate the molecular mechanism of p58(PITSLRE) action, we used the yeast two-hybrid system, screened a human fetal liver cDNA library, and identified cyclin D3 as an interacting partner of p58(PITSLRE). In vitro binding assay, in vivo coimmunoprecipitation, and immunofluorescence cell staining further confirmed the association of p58(PITSLRE) with cyclin D3. This binding was observed only in the G(2)/M phase but not in the G(1)/S phase of the cell cycle; meanwhile, no interaction between p110(PITSLRE) and cyclin D3 was observed in all the cell cycle. The overexpression of cyclin D3 in 7721 cells leads to an exclusively accumulation of p58(PITSLRE) in the nuclear region, affecting its cellular distribution. Histone H1 kinase activity of p58(PITSLRE) was greatly enhanced upon interaction with cyclin D3. Furthermore, kinase activity of p58(PITSLRE) was found to increase greatly in the presence of cyclin D3 using a specific substrate, beta-1,4-galactosyltransferase 1. These data provide a new clue to our understanding of the cellular function of p58(PITSLRE) and cyclin D3.     

Activation of p21-activated kinase 6 by MAP kinase kinase 6 and p38 MAP kinase

The p21-activated kinases (PAKs) contain an N-terminal Cdc42/Rac interactive binding domain, which in the group 1 PAKs (PAK1, 2, and 3) regulates the activity of an adjacent conserved autoinhibitory domain. In contrast, the group 2 PAKs (PAK4, 5, and 6) lack this autoinhibitory domain and are not activated by Cdc42/Rac binding, and the mechanisms that regulate their kinase activity have been unclear. This study found that basal PAK6 kinase activity was repressed by a p38 mitogen-activated protein (MAP) kinase antagonist and could be strongly stimulated by constitutively active MAP kinase kinase 6 (MKK6), an upstream activator of p38 MAP kinases. Mutation of a consensus p38 MAP kinase target site at serine 165 decreased PAK6 kinase activity. Moreover, PAK6 was directly activated by MKK6, and mutation of tyrosine 566 in a consensus MKK6 site (threonine-proline-tyrosine, TPY) in the activation loop of the PAK6 kinase domain prevented activation by MKK6. PAK6 activation by MKK6 was also blocked by mutation of an autophosphorylated serine (serine 560) in the PAK6 activation loop, indicating that phosphorylation of this site is necessary for MKK6-mediated activation. PAK4 and PAK5 were similarly activated by MKK6, consistent with a conserved TPY motif in their activation domains. The activation of PAK6 by both p38 MAP kinase and MKK6 suggests that PAK6 plays a role in the cellular response to stress-related signals.     

Identification of dynein light chain 2 as an interaction partner of p21-activated kinase 1

p21-Activated kinase 1 (PAK1), a member of the evolutionarily conserved PAK family of serine/threonine kinases, is essential for a variety of cellular functions. Our previous studies showed that PAK1 participated in the apoptotic pathway mediated by p110C. To further investigate its functions, we used the yeast two-hybrid system to screen a human fetal brain cDNA library and identified dynein light chain 2 (DLC2)/myosin light chain (MLC) as an interacting partner of PAK1. The association of PAK1 with DLC2 was further confirmed by in vitro binding assay. With the stimulation of EGF, PAK1 interacted with HA-DLC2 in vivo and relocalized in cytoplasm near the perinuclear location in confocal microscope analysis. The deletion analysis showed that the interaction of DLC2 with PAK1 occurred within the residues 210-332 of PAK1. For that studies showed that DLC2 was a subunit of myosin complex, so it is possible that PAK1 binds to DLC2 and transports by myosin complex.     

p21-activated kinase 1 phosphorylates the death agonist bad and protects cells from apoptosis

Bad is a critical regulatory component of the intrinsic cell death machinery that exerts its death-promoting effect upon heterodimerization with the antiapoptotic proteins Bcl-2 and Bcl-x(L). Growth factors promote cell survival through phosphorylation of Bad, resulting in its dissociation from Bcl-2 and Bcl-x(L) and its association with 14-3-3tau. Survival of interleukin 3 (IL-3)-dependent FL5.12 lymphoid progenitor cells is attenuated upon treatment with the Rho GTPase-inactivating toxin B from Clostridium difficile. p21-activated kinase 1 (PAK1) is activated by IL-3 in FL5.12 cells, and this activation is reduced by the phosphatidylinositol 3-kinase inhibitor LY294002. Overexpression of a constitutively active PAK mutant (PAK1-T423E) promoted cell survival of FL5.12 and NIH 3T3 cells, while overexpression of the autoinhibitory domain of PAK (amino acids 83 to 149) enhanced apoptosis. PAK phosphorylates Bad in vitro and in vivo on Ser112 and Ser136, resulting in a markedly reduced interaction between Bad and Bcl-2 or Bcl-x(L) and the increased association of Bad with 14-3-3tau. Our findings indicate that PAK inhibits the proapoptotic effects of Bad by direct phosphorylation and that PAK may play an important role in cell survival pathways.     

p85 beta-PIX is required for cell motility through phosphorylations of focal adhesion kinase and p38 MAP kinase

Lysophosphatidic acid (LPA) mediates diverse biological responses, including cell migration, through the activation of G-protein-coupled receptors. Recently, we have shown that LPA stimulates p21-activated kinase (PAK) that is critical for focal adhesion kinase (FAK) phosphorylation and cell motility. Here, we provide the direct evidence that p85 beta-PIX is required for cell motility of NIH-3T3 cells by LPA through FAK and p38 MAP kinase phosphorylations. LPA induced p85 beta-PIX binding to FAK in NIH-3T3 cells that was inhibited by pretreatment of the cells with phosphoinositide 3-kinase inhibitor, LY294002. Furthermore, the similar inhibition of the complex formation was also observed, when the cells were transfected with either p85 beta-PIX mutant that cannot bind GIT or dominant negative mutants of Rac1 (N17Rac1) and PAK (PAK-PID). Transfection of the cells with specific p85 beta-PIX siRNA led to drastic inhibition of LPA-induced FAK phosphorylation, peripheral redistribution of p85 beta-PIX with FAK and GIT1, and cell motility. p85 beta-PIX was also required for p38 MAP kinase phosphorylation induced by LPA. Finally, dominant negative mutant of Rho (N19Rho)-transfected cells did not affect PAK activation, while the cells stably transfected with p85 beta-PIX siRNA or N17Rac1 showed the reduction of LPA-induced PAK activation. Taken together, the present data suggest that p85 beta-PIX, located downstream of Rac1, is a key regulator for the activations of FAK or p38 MAP kinase and plays a pivotal role in focal complex formation and cell motility induced by LPA.     

Fas-induced apoptosis in human malignant melanoma cell lines is associated with the activation of the p34(cdc2)-related PITSLRE protein kinases.

The Cdc2L locus encoding the PITSLRE protein kinases maps to chromosome band 1p36 and consists of two duplicated and tandemly linked genes. The purpose of the present study was to determine whether diminution of PITSLRE kinases leads to deregulation of apoptosis. The human melanoma cell lines A375 (Cdc2L wild-type alleles) and UACC 1227 (mutant Cdc2L alleles) were tested with agonist anti-Fas monoclonal antibody. We found that exposure of these cells to anti-Fas for 24, 48, or 72 h resulted in differential sensitivity to Fas-induced apoptosis. In A375, cell death started at 24-48 h post-treatment, and it was maximal by 72 h. Conversely, UACC 1227 cells were resistant to Fas-mediated apoptosis. Induction of PITSLRE histone H1 kinase activity was observed in A375 anti-Fas treated but not in UACC 1227 cells. Also, the PITSLRE protein kinase activity in A375 anti-Fas-treated cells preceded maximal levels of apoptosis. Finally, fluorescence confocal microscopy revealed a nuclear localization of PITSLRE proteins in normal melanocytes and A375 cells but a cytoplasmic localization in UACC 1227 cells. The differences in PITSLRE protein and cellular localization between A375 and UACC 1227 cells appear to account for the differences in sensitivity of the two cells lines to anti-Fas and staurosporine. These observations suggest that alterations in PITSLRE gene expression and protein localization may result in the loss of apoptotic signaling.     

p21-Activated kinase 5: A pleiotropic kinase

The PAKs (p21-activated kinases) are highly conserved serine/threonine protein kinases which comprise six mammalian PAKs. PAK5 (p21-activated kinase 5) is the least understood member of PAKs that regulate many intracellular processes when they are stimulated by activated forms of the small GTPases Cdc42 and Rac. PAK5 takes an important part in multiple signal pathways in mammalian cells and controls a variety of cellular functions including cytoskeleton organization, cell motility and apoptosis. The main goal of this review is to describe the structure, mechanisms underlying its activity regulation, its role in apoptosis and the likely directions of further research.     

Regulation of p21-activated kinase-independent Rac1 signal transduction by nischarin

Nischarin regulates Rac1-dependent cell motility by interaction with and inhibition of the p21-activated kinase (PAK1). In addition to regulating the activation of PAK1, Rac1 controls multiple downstream pathways to regulate cell growth and differentiation, as well as cell motility. Signaling by a constitutively activated Rac1 mutant deficient in PAK binding (Rac1Q61L-40C) was examined to determine whether Nischarin impinges on these other Rac1 effector pathways. Nischarin formed immunoprecipitatable complexes with Rac1Q61L and Rac1Q61L-40C when the proteins were co-expressed. In NIH3T3 cells, Rac1Q61L and Rac1Q61L-40C stimulation of a minimal NF-kappaB response element or the cyclin D1 promoter, a downstream target of NF-kappaB, was inhibited by co-expression of Nischarin. Additionally, suppression of endogenous Nischarin protein with small interfering RNA in PC12 cells enhanced Rac1Q61L and Rac1Q61L-40C activation of NF-kappaB. In further support of Nischarin suppressing PAK independent Rac signaling, foci formation in monolayers of NIH3T3 cells by Rac1Q61L-40C in cooperation with c-Raf/CAAX was inhibited by the presence of Nischarin. Nischarin alters the cellular localization of Rac1Q61L and Rac1Q61L-40C to vesicles and this positively correlates with the repression of the Rac1 signal. Thus, Nischarin, in addition to regulating the PAK strand of Rac1 signaling, can also regulate other links in the web of Rac1 signaling pathways.     

p67/MetAP2 suppresses K-RasV12-mediated transformation of NIH3T3 mouse fibroblasts in culture and in athymic mice

In many tumor cells, the activation and activity of extracellular signal-regulated kinases (ERK1/2) are very high because of the constitutive activation of the Ras-mediated signaling pathway. Here, we ectopically expressed the human homologue of rat eukaryotic initiation factor 2-associated glycoprotein, p67/MetAP2, in EGF-treated mouse embryonic NIH3T3 fibroblasts and C2C12 myoblasts and NIH3T3 cell lines expressing the constitutively active form of MAP kinase kinase (MEK) to inhibit the activation and activity of ERK1/2 MAP kinases. In addition, we also ectopically expressed rat p67/MetAP2 in oncogenic Ras-induced transformed NIH3T3 fibroblasts and inhibited their transformed phenotype both in culture and in athymic nude mice possibly by inhibiting angiogenesis. This inhibition of ERK1/2 MAP kinases is due to the direct binding with rat p67/MetAP2, and this leads to the inhibition of activity of ERK1/2 MAP kinases both in vitro and in vivo. Furthermore, expression of p67/MetAP2 siRNA in both NIH3T3 fibroblasts and C2C12 myoblasts causes activation and activity of ERK1/2 MAP kinases. Our results thus suggest that ectopic expression of rat p67/MetAP2 in transformed cells can inhibit the tumorigenic phenotype by inhibiting the activation and activity of ERK1/2 MAP kinases and, thus, that p67/MetAP2 has tumor suppression activity.     

Hsp72 functions as a natural inhibitory protein of c-Jun N-terminal kinase

Hsp72, a major inducible member of the heat shock protein family, can protect cells against many cellular stresses including heat shock. In our present study, we observed that pretreatment of NIH 3T3 cells with mild heat shock (43 degrees C for 20 min) suppressed UV-stimulated c-Jun N-terminal kinase 1 (JNK1) activity. Constitutively overexpressed Hsp72 also inhibited JNK1 activation in NIH 3T3 cells, whereas it did not affect either SEK1 or MEKK1 activity. Both in vitro binding and kinase studies indicated that Hsp72 bound to JNK1 and that the peptide binding domain of Hsp72 was important to the binding and inhibition of JNK1. In vivo binding between endogenous Hsp72 and JNK1 in NIH 3T3 cells was confirmed by co-immunoprecipitation. Hsp72 also inhibited JNK-dependent apoptosis. Hsp72 antisense oligonucleotides blocked Hsp72 production in NIH 3T3 cells in response to mild heat shock and concomitantly abolished the suppressive effect of mild heat shock on UV-induced JNK activation and apoptosis. Collectively, our data suggest strongly that Hsp72 can modulate stress-activated signaling by directly inhibiting JNK.     

Merosin-integrin promotion of skeletal myofiber cell survival: Differentiation state-distinct involvement of p60Fyn tyrosine kinase and p38alpha stress-activated MAP kinase

Myofiber survival and suppression of anoikis depend in large part on the merosin (laminin-2/-4)-integrin alpha7beta1D cell adhesion system; however, the question remains as to the nature of the signaling molecules/pathways involved. In the present study, we investigated this question using the C2C12 cell model of myogenic differentiation and its merosin- and laminin-deficient derivatives. Herein, we report that: 1) of four members of the Src family of tyrosine kinases studied (p60Src, p53/56Lyn, p59Yes, or p60Fyn), the expression and activity of p60Fyn are found in myotubes exclusively; 2) a severe decrease of p60Fyn activity correlates with myotube apoptosis/anoikis induced by pharmocological compounds (herbimycin A or PP2) which inhibit tyrosine kinases of the Src family, by merosin deficiency and by beta1 integrin inhibition; 3) myoblast survival depends on Fak and the MEK/Erk pathway, in contrast to myotubes; 4) the PI3-K pathway is not involved in either myoblast or myotube survival; and 5) p38alpha SAPK stimulation and activity (but not that of p38beta) are required in the progression of myotube apoptosis/anoikis induced by p60Fyn inhibition, merosin deficiency or beta1 integrin-inhibition; however, p38 is not involved in myoblast apoptosis. Taken together, these results suggest that the promotion of myotube survival by the merosin-alpha7beta1D adhesion system involves p60Fyn, and that disruptions in this cell adhesion system induce myotube apoptosis/anoikis through a p38alpha SAPK-dependent pathway.     

A novel role for p21-activated protein kinase 2 in T cell activation

To identify novel components of the TCR signaling pathway, a large-scale retroviral-based functional screen was performed using CD69 expression as a marker for T cell activation. In addition to known regulators, two truncated forms of p21-activated kinase 2 (PAK2), PAK2DeltaL(1-224) and PAK2DeltaS(1-113), both lacking the kinase domain, were isolated in the T cell screen. The PAK2 truncation, PAK2DeltaL, blocked Ag receptor-induced NFAT activation and TCR-mediated calcium flux in Jurkat T cells. However, it had minimal effect on PMA/ionomycin-induced CD69 up-regulation in Jurkat cells, on anti-IgM-mediated CD69 up-regulation in B cells, or on the migratory responses of resting T cells to chemoattractants. We show that PAK2 kinase activity is increased in response to TCR stimulation. Furthermore, a full-length kinase-inactive form of PAK2 blocked both TCR-induced CD69 up-regulation and NFAT activity in Jurkat cells, demonstrating that kinase activity is required for PAK2 function downstream of the TCR. We also generated a GFP-fused PAK2 truncation lacking the Cdc42/Rac interactive binding region domain, GFP-PAK2(83-149). We show that this construct binds directly to the kinase domain of PAK2 and inhibits anti-TCR-stimulated T cell activation. Finally, we demonstrate that, in primary T cells, dominant-negative PAK2 prevented anti-CD3/CD28-induced IL-2 production, and TCR-induced CD40 ligand expression, both key functions of activated T cells. Taken together, these results suggest a novel role for PAK2 as a positive regulator of T cell activation.     

Klotho Endows Hepatoma Cells with Resistance to Anoikis via VEGFR2/PAK1 Activation in Hepatocellular Carcinoma

Klotho was originally characterized as an aging suppressor gene that predisposed Klotho-deficient mice to premature aging-like syndrome. Although Klotho was recently reported to exhibit tumor suppressive properties during various malignant transformations, the functional role and molecular mechanism of Klotho in hepatocarcinogenesis remains poorly understood. In our present study, immunohistochemical Klotho staining levels in a clinical follow-up of 52 hepatoma patients were significantly associated with liver cirrhosis, tumor multiplicity and venous invasion. The overall survival rate of hepatoma patients with high Klotho expression was significantly lower than those patients with low Klotho expression. Moreover, Klotho overexpression increased cellular migration, anchorage-independent growth, and anoikis resistance in hepatoma cells. Klotho overexpression elevated p21-activated kinase 1 (PAK1) expression and shRNA-mediated PAK1 knockdown and kinase activity inhibition with kinase dead mutant PAK1 K299R coexpression or allosteric inhibitor IPA3 treatment reversed anoikis resistance in Klotho-overexpressed hepatoma cells. More importantly, the pivotal significance of upregulated VEGFR2 protein levels mediated by Klotho expression was confirmed by VEGFR2 inhibitor Axitinib and blocking antibody treatment in hepatoma cells. Axitinib treatment sensitized anoikis was reversed by constitutive active mutant PAK1 T423E coexpression in Klotho-overexpressed hepatoma cells. Conversely, knockdown of Klotho reduced VEGFR2/PAK1 dependent anoikis resistance, which could be reversed by PAK1 T423E. These results revealed a novel oncogenic function of Klotho in promoting anoikis resistance via activating VEGFR2/PAK1 signaling, thus facilitating tumor migration and invasion during hepatoma progression, which could provide a putative molecular mechanism for tumor metastasis.     

Tumor Suppressive Function of p21-activated Kinase 6 in Hepatocellular Carcinoma

Our previous studies identified the oncogenic role of p21-activated kinase 1 (PAK1) in hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC). Contrarily, PAK6 was found to predict a favorable prognosis in RCC patients. Nevertheless, the ambiguous tumor suppressive function of PAK6 in hepatocarcinogenesis remains obscure. Herein, decreased PAK6 expression was found to be associated with tumor node metastasis stage progression and unfavorable overall survival in HCC patients. Additionally, overexpression and silence of PAK6 experiments showed that PAK6 inhibited xenografted tumor growth in vivo, and restricted cell proliferation, colony formation, migration, and invasion and promoted cell apoptosis and anoikis in vitro. Moreover, overexpression of kinase dead and nuclear localization signal deletion mutants of PAK6 experiments indicated the tumor suppressive function of PAK6 was partially dependent on its kinase activity and nuclear translocation. Furthermore, gain or loss of function in polycomb repressive complex 2 (PRC2) components, including EZH2, SUZ12, and EED, elucidated epigenetic control of H3K27me3-arbitrated PAK6 down-regulation in hepatoma cells. More importantly, negative correlation between PAK6 and EZH2 expression was observed in hepatoma tissues from HCC patients. These data identified the tumor suppressive role and potential underlying mechanism of PAK6 in hepatocarcinogenesis.     

Regulation of macropinocytosis by p21-activated kinase-1

The process of macropinocytosis is an essential aspect of normal cell function, contributing to both growth and motile processes of cells. p21-activated kinases (PAKs) are targets for activated Rac and Cdc42 guanosine 5'-triphosphatases and have been shown to regulate the actin-myosin cytoskeleton. In fibroblasts PAK1 localizes to areas of membrane ruffling, as well as to amiloride-sensitive pinocytic vesicles. Expression of a PAK1 kinase autoinhibitory domain blocked both platelet-derived growth factor- and RacQ61L-stimulated uptake of 70-kDa dextran particles, whereas an inactive version of this domain did not, indicating that PAK kinase activity is required for normal growth factor-induced macropinocytosis. The mechanisms by which PAK modulate macropinocytosis were examined in NIH3T3 cell lines expressing various PAK1 constructs under the control of a tetracycline-responsive transactivator. Cells expressing PAK1 (H83,86L), a mutant that dramatically stimulates formation of dorsal membrane ruffles, exhibited increased macropinocytic uptake of 70-kDa dextran particles in the absence of additional stimulation. This effect was not antagonized by coexpression of dominant-negative Rac1-T17N. In the presence of platelet-derived growth factor, both PAK1 (H83,86L) and a highly kinase active PAK1 (T423E) mutant dramatically enhanced the uptake of 70-kDa dextran. Neither wild-type PAK1 nor vector controls exhibited enhanced macropinocytosis, nor did PAK1 (H83,86L) affect clathrin-dependent endocytic mechanisms. Active versions of PAK1 enhanced both growth factor-stimulated 70-kDa dextran uptake and efflux, suggesting that PAK1 activity modulated pinocytic vesicle cycling. These data indicate that PAK1 plays an important regulatory role in the process of macropinocytosis, perhaps related to the requirement for PAK in directed cell motility.     

Enhanced ROS production in oncogenically transformed cells potentiates c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activation and sensitization to genotoxic stress

Many primary tumors as well as transformed cell lines display high sensitivity to chemotherapeutic drugs and radiation. The molecular mechanisms that underlie this sensitivity are largely unknown. Here we show that the sensitization of transformed cells to stress stimuli is due to the potentiation of the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase pathways. Activation of these pathways by the antitumor drug cis-platin (CDDP) and by other stress agents is markedly enhanced and is induced by lower stress doses in NIH 3T3 cells overexpressing epidermal growth factor receptor, HER1-2 kinase, or oncogenic Ras than in nontransformed NIH 3T3 cells. Inhibition of stress kinase activity by specific inhibitors reduces CDDP-mediated cell death in transformed cells, whereas overactivation of stress kinase pathways augments cells death. Potentiation of stress kinases is a common feature of cells transformed by different oncogenes, including cells derived from human tumors, and is shown here to be independent of the activity of the particular transforming oncoprotein. We further show that the mechanism that underlies potentiation of stress kinases in transformed cells involves reactive oxygen species (ROS), whose production is elevated in these cells. JNK/p38 activation is inhibited by antioxidants and in particular by inhibitors of the mitochondrial respiratory chain and NADPH oxidase. Conversely, by artificially elevating ROS levels in nontransformed NIH 3T3 cells we were able to induce potentiation of JNK/p38 activation. Taken together, our findings suggest that ROS-dependent potentiation of stress kinase pathways accounts for the sensitization of transformed cells to stress and anticancer drugs.     

p21-activated kinase 1 participates in tracheal smooth muscle cell migration by signaling to p38 Mapk

Cell migration contributesto many physiological processes and requires dynamic changes in thecytoskeleton. These migration-dependent cytoskeletal changes are partlymediated by p21-activated protein kinases (PAKs). At least four closelyrelated isoforms, PAK1, PAK2, PAK3, and PAK4, exist in mammalian cells.In smooth muscle cells, little is known about the expression,activation, or ability of PAKs to regulate migration. Our studyrevealed the existence of three PAK isoforms in cultured trachealsmooth muscle cells (TSMCs). Additionally, we constructed adenoviralvectors encoding wild type and a catalytically inactive PAK1 mutant toinvestigate PAK activation and its role in TSMC migration. Stimulationof TSMCs with platelet-derived growth factor (PDGF) increased the activity of PAK1 over time. Overexpression of mutant PAK1 blocked PDGF-induced chemotactic cell migration. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) in cells overexpressing wild-type PAK1 was similar to vector controls; however, p38 MAPK phosphorylation was severely reduced by overexpression of the PAK1mutant. Collectively, these results suggest a role for PAK1 inchemotactic TSMC migration that involves catalytic activity and mayrequire signaling to p38 MAPK among other pathways.

     

Heat shock protein hsp72 is a negative regulator of apoptosis signal-regulating kinase 1

Heat shock protein 72 (Hsp72) is thought to protect cells against cellular stress. The protective role of Hsp72 was investigated by determining the effect of this protein on the stress-activated protein kinase signaling pathways. Prior exposure of NIH 3T3 cells to mild heat shock (43 degrees C for 20 min) resulted in inhibition of H(2)O(2)-induced activation of apoptosis signal-regulating kinase 1 (ASK1). Overexpression of Hsp72 also inhibited H(2)O(2)-induced activation of ASK1 as well as that of downstream kinases in the p38 mitogen-activated protein kinase (MAPK) signaling cascade. Recombinant Hsp72 bound directly to ASK1 and inhibited ASK1 activity in vitro. Furthermore, coimmunoprecipitation analysis revealed a physical interaction between endogenous Hsp72 and ASK1 in NIH 3T3 cells exposed to mild heat shock. Hsp72 blocked both the homo-oligomerization of ASK1 and ASK1-dependent apoptosis. Hsp72 antisense oligonucleotides prevented the inhibitory effects of mild heat shock on H(2)O(2)-induced ASK1 activation and apoptosis. These observations suggest that Hsp72 functions as an endogenous inhibitor of ASK1.