P21-activated Serine-threonine Kinase Research Articles

Simultaneous Inhibition of BCR-ABL1 Tyrosine Kinase and PAK1/2 Serine/Threonine Kinases Exerts Synergistic Effect Against Chronic Myeloid Leukemia Cells

Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of BCR-ABL1 tyrosine kinase - positive chronic myeloid leukemia in chronic phase (CML-CP). However, it is unlikely that TKIs will "cure" the disease in majority of patients because CML-CP cells are elusive targets even for the most advanced therapies employing second and third generation of TKIs. Therefore, new treatment modalities are necessary to improve therapeutic outcomes. We showed before that class I p21-activated serine/threonine kinases (PAKs) remained active in TKI-naive and TKI-treated CML-CP leukemia stem and early progenitor cells. The aim of the study was to test whether simultaneous inhibition of signaling pathways activated by BCR-ABL1 and PAK kinases may improve the treatment of CML. Special attention was focused on PAK1 and PAK2, which are expressed in hematopoietic cells and can play an important role in the promotion of CML cells proliferation and survival. PAK kinases were targeted by small molecule inhibitor IPA-3 (inhibitor of PAK1) and shRNA construct for PAK2, BCR-ABL1 kinase was inhibited by imatinib. The studies were carried out using (i) primary CML-CP stem/early progenitor cells and normal hematopoietic counterparts isolated from the bone marrow of newly diagnosed CML-CP patients and healthy donors, respectively, (ii) CML-blast phase cell lines (K562 and KCL-22), and (iii) BCR-ABL1-transformed 32Dcl3 cell line cells. We show here that inhibition of PAK1 or/and PAK2 kinases activity enhanced the effect of IM against CML cells without affecting normal counterparts. We observed that the combined use of IM with IPA-3 increased growth inhibition and apoptosis of leukemia cells. To evaluate the type of drugs interaction median effect analysis method was used. The studies revealed that the type and strength of drug interaction depend on drug concentration. Generally, combination of IM with IPA-3 at the 50% of the cell kill level (EC50) generated synergistic effect. Altogether, we postulate that BCR-ABL1 kinase inhibitor should be combined with PAK1/2 inhibitor to facilitate eradication of CML cells. Disclosures No relevant conflicts of interest to declare.

Simultaneous Inhibition of BCR-ABL1 Tyrosine Kinase and PAK1/2 Serine/Threonine Kinase Exerts Synergistic Effect against Chronic Myeloid Leukemia Cells.

Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of chronic myeloid leukemia in the chronic phase (CML-CP). However, it is unlikely that they can completely “cure” the disease. This might be because some subpopulations of CML-CP cells such as stem and progenitor cells are resistant to chemotherapy, even to the new generation of TKIs. Therefore, it is important to look for new methods of treatment to improve therapeutic outcomes. Previously, we have shown that class I p21-activated serine/threonine kinases (PAKs) remained active in TKI-naive and TKI-treated CML-CP leukemia stem and early progenitor cells. In this study, we aimed to determine if simultaneous inhibition of BCR-ABL1 oncogenic tyrosine kinase and PAK1/2 serine/threonine kinase exert better anti-CML effect than that of individual treatments. PAK1 was inhibited by small-molecule inhibitor IPA-3 (p21-activated kinase inhibitor III), PAK2 was downregulated by specific short hairpin RNA (shRNA), and BCR-ABL1 tyrosine kinase was inhibited by imatinib (IM). The studies were conducted by using (i) primary CML-CP stem/early progenitor cells and normal hematopoietic counterparts isolated from the bone marrow of newly diagnosed patients with CML-CP and from healthy donors, respectively, (ii) CML-blast phase cell lines (K562 and KCL-22), and (iii) from BCR-ABL1-transformed 32Dcl3 cell line. Herein, we show that inhibition of the activity of PAK1 and/or PAK2 enhanced the effect of IM against CML cells without affecting the normal cells. We observed that the combined use of IM with IPA-3 increased the inhibition of growth and apoptosis of leukemia cells. To evaluate the type of interaction between the two drugs, we performed median effect analysis. According to our results, the type and strength of drug interaction depend on the concentration of the drugs tested. Generally, combination of IM with IPA-3 at the 50% of the cell kill level (EC50) generated synergistic effect. Based on our results, we hypothesize that IM, a BCR-ABL1 tyrosine kinase inhibitor, combined with a PAK1/2 inhibitor facilitates eradication of CML-CP cells.

Melanoma-associated mutants within the serine-rich domain of PAK5 direct kinase activity to mitogenic pathways.

The overexpression and hyperactivity of p21-activated serine/threonine kinases (PAKs) is known to facilitate tumorigenesis; however, the contribution of cancer-associated PAK mutations to tumor initiation and progression remains unclear. Here, we identify p21-activated serine/threonine kinase 5 (PAK5) as the most frequently altered PAK family member in human melanoma. More than 60% of melanoma-associated PAK5 gene alterations are missense mutations, and distribution of these variants throughout the protein coding sequence make it difficult to distinguish oncogenic drivers from passengers. To address this issue, we stably introduced the five most common melanoma-associated PAK5 missense mutations into human immortalized primary melanocytes (hMELTs). While expression of these mutants did not promote single-cell migration or induce temozolomide resistance, a subset of variants drove aberrant melanocyte proliferation. These mitogenic mutants, PAK5 S364L and D421N, clustered within an unstructured, serine-rich domain of the protein and inappropriately activated ERK and PKA through kinase-independent and -dependent mechanisms, respectively. Together, our findings establish the ability of mutant PAK5 to enhance PKA and MAPK signaling in melanocytes and localize the engagement of mitogenic pathways to a serine-rich region of PAK5.

Abstract 195: Defining the role of PAK7 variants in melanoma

Abstract Melanoma mortality is directly linked to its profoundly metastatic nature and inherent resistance to conventional chemotherapy. For this reason, increased understanding of the molecular mechanisms driving melanoma progression is of utmost importance. The p21-activated serine/threonine kinases (PAKs) are frequently mutated or overexpressed in cancer and have recently been implicated in tumor metastasis, proliferation, and apoptotic resistance. This observation has inspired drug discovery efforts to limit PAK activity; yet, mechanistic understanding of how PAKs (especially Type II PAKs) contribute to tumor initiation and progression is still evolving. PAK7, a type II PAK, is mutated in 16-18% of all melanomas. Melanoma-associated PAK7 mutations occur throughout the gene and have unknown biochemical and physiological consequences. The objective of this study was to determine how tumor-associated PAK7 variants mechanistically contribute to melanoma initiation and progression. First, we characterized PAK7 protein expression in a panel of 39 melanoma cell lines and primary melanocyte cultures. PAK7 levels were similar in both tumorigenic and non-tumorigenic cells and failed to correlate with the status of common melanoma driver mutations (e.g. NRASQ61, BRAFV600). In an array of 101 human tumor biopsies, PAK7 expression did not significantly change with advancing melanoma stage; however, PAK7 levels were higher in regional lymph node biopsies when compared to primary skin lesions. Since mutation is the predominant mechanism affecting PAK7 in melanoma, we investigated the biochemical and physiological consequences of common PAK7 variants. 11 distinct PAK7 mutations were stably expressed at low levels in VMM39 (melanoma) and NCI-H441 (lung) cells. Using these clones, changes in the phosphorylation of downstream PAK7 targets associated with cellular migration (p120), proliferation (CRAF), and apoptotic resistance (BAD, MDM2) were examined. In addition, we defined phenotypic alterations in cellular morphology, proliferation, migration, and apoptotic resistance associated with the expression of each mutant. These data allowed us to classify PAK7 variants of unknown significance into groups associated with specific cellular outcomes. To our knowledge, this is the first study to functionally characterize a wide variety of melanoma-associated PAK7 mutations and represents a critical first step in understanding how this kinase contributes to tumor initiation and progression. Citation Format: Kyle M. LaPak, Michael A. Gross, Denny C. Vroom, Greg B. Lesinski, William E. Carson. Defining the role of PAK7 variants in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 195.

Phosphorylation of tyrosine 285 of PAK1 facilitates βPIX/GIT1 binding and adhesion turnover.

The p21-activated serine-threonine kinase (PAK1) regulates cell motility and adhesion. We have previously shown that the prolactin (PRL)-activated tyrosine kinase JAK2 phosphorylates PAK1 in vivo and in vitro and identified tyrosines 153, 201, and 285 in PAK1 as sites of JAK2 tyrosyl phosphorylation. Here, we further investigate the role of the tyrosyl phosphorylated PAK1 (pTyr-PAK1) in regulation of cell adhesion. We use human breast cancer T47D cell lines that stably overexpress PAK1 wild type or PAK1 Y3F mutant in which these 3 JAK2 phosphorylation sites were mutated to phenylalanine. We demonstrate that PRL/JAK2-dependent phosphorylation of these tyrosines promotes a motile phenotype in the cells upon adhesion, participates in regulation of cell adhesion on collagen IV, and is required for maximal PAK1 kinase activity. Down-regulation of PAK1 abolishes the effect of PAK1 on cell adhesion. We show that the tyrosyl phosphorylation of PAK1 promotes PAK1 binding to β-PAK1-interacting guanine-nucleotide exchange factor (βPIX) and G protein-coupled receptor kinase-interacting target 1 (GIT1), phosphorylation of paxillin on Ser273, and formation and distribution of adhesion complexes. Using phosphospecific antibodies (Abs) directed to single phosphorylated tyrosines on PAK1, we identified Tyr285 as a site of PRL-dependent phosphorylation of PAK1 by JAK2. Furthermore, using PAK1 Y285F mutant, we provide evidence for a role of pTyr285 in cell adhesion, enhanced βPIX/GIT1 binding, and adhesion turnover. Our immunohistochemistry analysis demonstrates that pTyr285- PAK1 may modulate PAK1 signaling during tumor progression.

Abstract 2621: PAK4 mediated mechanisms contributing to chemoresistance of ovarian cancer

Abstract Background: Ovarian cancer is one of the most lethal cancers among women worldwide. Despite of an initial response to chemotherapy, relapse may happen due to the chemoresistant phenotypes. However, underlying mechanism conferring chemoresistance is not well defined. p21-activated serine/threonine kinases (PAKs) are serine/threonine kinases involved in various cellular functions including cell motility, survival and angiogenesis.PAK4 is the prototypic member of Group II PAKs. In this study, we aim to delineate the crucial domains in PAK4 that may contribute to the development of chemoresistance in ovarian cancer cells by analysis of various PAK4 mutants. Materials and Methods: Ovarian cancer cell line SKOV-3 stably expressing various PAK4 mutants was used as an in vitro model. SKOV-3 was transfected with Flag-tagged wild type PAK4, constitutively active PAK4 (445N/474E), kinase-dead PAK4 (M350), or the control vector, p3XFLAG-CMV-10. To illustrate the functional impact of nuclear PAK4, PAK4 constructs with various nucleus localization signals (NLS) mutation were generated. PAK4 NLS mutants were then stably transfected into SKOV-3 and the expression and nuclear exclusion were confirmed by western blotting and immunofluorescence microscope respectively.They were also treated with cisplatin and the discrepancies in response to cisplatin were accessed by MTT assay and through the evaluation of apoptotic activity using flow cytometry and caspase-3 activation. In order to unveil the significance of PAK4 amplification and mutation status, genomic DNA of two immortalized ovarian epithelial cell lines HOSE6-3 and HOSE-11-12, and eleven ovarian cancer cell lines were extracted. Exon 3, which encodes the Cdc42-Rac interaction/binding (CRIB) domain and exons 5 - 10, which encode the kinase domain were sequenced to detect possible mutation in comparison to the wild-type sequence. Results: By MTT assay, overexpression of wild type PAK4 was demonstrated to enhance the survival of SKOV3 cells when compared with vector-transfected control cells when challenged with cisplatin. Flow Cytometry Analysis confirmed the above survival pattern. No significant differences were found between the two PAK4 kinase domain mutants and the wild type one. At genomic level, no amplification or mutation in PAK4 gene could be found in the ovarian cancer cell lines studied. Wild type PAK4 expression was found in both nucleus and cytoplasm. Mutation of NLS3, but not NLS1, could abolish the nuclear-localization of PAK4. Conclusion: In summary, aberrant expression of PAK4 contributes to resistance of ovarian cancer cells towards cisplatin.Amplification or mutation in PAK4 kinase domain cannot be detected and therefore may not be the crucial factor underlying overexpression in ovarian cancers. Further study on PAK4, including its subcellular localization, may further shed light on its potential as a novel therapeutic target of ovarian cancer. Citation Format: Ya Nan Du, Oscar GW Wong, Ka Kui Chan, Esther SY Wong, Annie NY Cheung. PAK4 mediated mechanisms contributing to chemoresistance of ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2621. doi:10.1158/1538-7445.AM2014-2621

Molecular pathways: targeting RAC-p21-activated serine-threonine kinase signaling in RAS-driven cancers.

Cancers driven by oncogenic Ras proteins encompass some of the most deadly human cancer types, and there is a pressing need to develop therapies for these diseases. Although recent studies suggest that mutant Ras proteins may yet be druggable, the most promising and advanced efforts involve inhibitors of Ras effector signaling. Most efforts to target Ras signaling have been aimed at the ERK mitogen-activated protein kinase and the phosphoinositide 3-kinase signaling networks. However, to date, no inhibitors of these Ras effector pathways have been effective against RAS-mutant cancers. This ineffectiveness is due, in part, to the involvement of additional effectors in Ras-dependent cancer growth, such as the Rac small GTPase and the p21-activated serine-threonine kinases (PAK). PAK proteins are involved in many survival, cell motility, and proliferative pathways in the cell and may present a viable new target in Ras-driven cancers. In this review, we address the role and therapeutic potential of Rac and group I PAK proteins in driving mutant Ras cancers.

PAK1 Regulates Breast Cancer Cell Invasion through Secretion of Matrix Metalloproteinases in Response to Prolactin and Three-Dimensional Collagen IV

p21-Activated serine-threonine kinase (PAK1) is implicated in breast cancer. We have shown previously that PAK1 is tyrosyl phosphorylated by prolactin (PRL)-activated Janus tyrosine kinase (JAK2). Although a role for both PRL and PAK1 in breast cancer is widely acknowledged, the mechanism remains poorly understood. In the present study, PRL-activated PAK1 stimulates the invasion of TMX2-28 human breast cancer cells through Matrigel. Three-dimensional (3D) collagen IV stimulates the secretion of the matrix proteases, metalloproteinase (MMP)-1 and -3 that is further enhanced by the PRL-dependent tyrosyl phosphorylation of PAK1. 3D collagen IV also stimulates the expression and secretion of MMP-2, but in contrast to MMP-1 and -3, PRL/PAK1 signaling down-regulates MMP-2 expression and secretion. In contrast, MMP-9 expression and secretion are stimulated by 3D collagen I, not collagen IV, and are not affected by PRL but are down-regulated by PAK1. MMP-1 and -3 are required and MMP-2 contributes to PRL-dependent invasion. ERK1/2 signaling appears to be required for the enhanced expression and secretion of MMP-1 and -3 and enhanced PRL-dependent invasion. p38 MAPK and c-Jun N-terminal kinase 1/2 pathways participate in production of MMP-1 and -3 as well as in PRL/PAK1-dependent cell invasion. Together, these data illustrate the complex interaction between the substratum and PRL/PAK1 signaling in human breast cancer cells and suggest a pivotal role for PRL-dependent PAK1 tyrosyl phosphorylation in MMP secretion.

Tyrosyl Phosphorylated PAK1 Regulates Breast Cancer Cell Motility in Response to Prolactin through Filamin A

The p21-activated serine-threonine kinase (PAK1) is activated by small GTPase-dependent and -independent mechanisms and regulates cell motility. Both PAK1 and the hormone prolactin (PRL) have been implicated in breast cancer by numerous studies. We have previously shown that the PRL-activated tyrosine kinase JAK2 (Janus tyrosine kinase 2) phosphorylates PAK1 in vivo and identified tyrosines (Tyr) 153, 201, and 285 in the PAK1 molecule as sites of JAK2 tyrosyl phosphorylation. Here, we have used human breast cancer T47D cells stably overexpressing PAK1 wild type or PAK1 Y3F mutant in which Tyr(s) 153, 201, and 285 were mutated to phenylalanines to demonstrate that phosphorylation of these three tyrosines are required for maximal PRL-dependent ruffling. In addition, phosphorylation of these three tyrosines is required for increased migration of T47D cells in response to PRL as assessed by two independent motility assays. Finally, we show that PAK1 phosphorylates serine (Ser) 2152 of the actin-binding protein filamin A to a greater extent when PAK1 is tyrosyl phosphorylated by JAK2. Down-regulation of PAK1 or filamin A abolishes the effect of PRL on cell migration. Thus, our data presented here bring some insight into the mechanism of PRL-stimulated motility of breast cancer cells.

PAK1-Nck Regulates Cyclin D1 Promoter Activity in Response to Prolactin

Prolactin (PRL) is critical for alveolar proliferation and differentiation in normal mammary development and is also implicated in breast cancer. PRL influences cell proliferation and growth by altering the expression of cyclin D1. Cyclin D1 expression is directly regulated by PRL through the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5-mediated transcriptional activation of the cyclin D1 promoter. A p21-activated serine-threonine kinase (PAK)1 has also been implicated in the regulation of cyclin D1 gene expression. We have previously demonstrated that JAK2 directly phosphorylates PAK1 and extend these data here to demonstrate that PAK1 activates the cyclin D1 promoter in response to PRL. We show that mutation of PAK1 Tyr 153, 201, and 285 (sites of JAK2 phosphorylation; PAK1 Y3F) decreases both PAK1 nuclear translocation in response to PRL and PRL-induced cyclin D1 promoter activity by 55%. Mutation of the PAK1 nuclear localization signals decreases PRL-induced cyclin D1 promoter activity by 46%. A PAK1 Y3F mutant lacking functional nuclear localization signals decreases PRL-induced cyclin D1 activity by 68%, suggesting that there is another PAK1-dependent mechanism to activate the cyclin D1 promoter. We have found that adapter protein Nck sequesters PAK1 in the cytoplasm and that coexpression of both PAK1 and Nck inhibits the amplifying effect of PRL-induced PAK1 on cyclin D1 promoter activity (95% inhibition). This inhibition is partially abolished by disruption of PAK1-Nck binding. We propose two PAK1-dependent mechanisms to activate cyclin D1 promoter activity in response to PRL: via nuclear translocation of tyrosyl-phosphorylated PAK1 and via formation of a Nck-PAK1 complex that sequesters PAK1 in the cytoplasm.

Adapter Protein SH2B1β Binds Filamin A to Regulate Prolactin-Dependent Cytoskeletal Reorganization and Cell Motility

Prolactin (PRL) regulates cytoskeletal rearrangement and cell motility. PRL-activated Janus tyrosine kinase 2 (JAK2) phosphorylates the p21-activated serine-threonine kinase (PAK)1 and the Src homology 2 (SH2) domain-containing adapter protein SH2B1β. SH2B1β is an actin-binding protein that cross-links actin filaments, whereas PAK1 regulates the actin cytoskeleton by different mechanisms, including direct phosphorylation of the actin-binding protein filamin A (FLNa). Here, we have used a FLNa-deficient human melanoma cell line (M2) and its derivative line (A7) that stably expresses FLNa to demonstrate that SH2B1β and FLNa are required for maximal PRL-dependent cell ruffling. We have found that in addition to two actin-binding domains, SH2B1β has a FLNa-binding domain (amino acids 200-260) that binds directly to repeats 17-23 of FLNa. The SH2B1β-FLNa interaction participates in PRL-dependent actin rearrangement. We also show that phosphorylation of the three tyrosines of PAK1 by JAK2, as well as the presence of FLNa, play a role in PRL-dependent cell ruffling. Finally, we show that the actin- and FLNa-binding-deficient mutant of SH2B1β (SH2B1β 3Δ) abolished PRL-dependent ruffling and PRL-dependent cell migration when expressed along with PAK1 Y3F (JAK2 tyrosyl-phosphorylation-deficient mutant). Together, these data provide insight into a novel mechanism of PRL-stimulated regulation of the actin cytoskeleton and cell motility via JAK2 signaling through FLNa, PAK1, and SH2B1β. We propose a model for PRL-dependent regulation of the actin cytoskeleton that integrates our findings with previous studies.

Signaling Mechanisms Underlying Slit2-Induced Collapse of Xenopus Retinal Growth Cones

Slits mediate multiple axon guidance decisions, but the mechanisms underlying the responses of growth cones to these cues remain poorly defined. We show here that collapse induced by Slit2-conditioned medium (Slit2-CM) in Xenopus retinal growth cones requires local protein synthesis (PS) and endocytosis. Slit2-CM elicits rapid activation of translation regulators and MAP kinases in growth cones, and inhibition of MAPKs or disruption of heparan sulfate blocks Slit2-CM-induced PS and repulsion. Interestingly, Slit2-CM causes a fast PS-dependent decrease in cytoskeletal F-actin concomitant with a PS-dependent increase in the actin-depolymerizing protein cofilin. Our findings reveal an unexpected link between Slit2 and cofilin in growth cones and suggest that local translation of actin regulatory proteins contributes to repulsion.

Thrombin activates the p21-activated kinase in pulmonary artery smooth muscle cells

The p21-activated serine/threonine kinases (PAK) play an important role in a variety of cellular functions. However, their role in the smooth muscle response to thrombin, which is activated upon vascular injury and promotes vascular remodelling processes, is not resolved. Here we investigated the role of PAK in thrombin signalling and regulation of tissue factor (TF), the activator of the extrinsic coagulation cascade, in pulmonary artery smooth muscle cells (PASMC), the main cell type responsible for vascular remodelling in pulmonary hypertension. PAK was rapidly phosphorylated in response to thrombin. Thrombin and active PAKT423E phosphorylated p38 MAP kinase (p38MAPK), ERK1/2, phosphatidylinositol-dependent kinase-1 (PDK1) and protein kinase B/Akt (PKB) whereas kinase-deficient PAK1 prevented activation of these kinases by thrombin. In addition, kinase- deficient MKK3 inhibited activation of PDK1 and PKB by thrombin. Further, thrombin and active PAK1 induced TF expression and promoter activity while kinase-deficient PAK1 diminished thrombin-induced TF upregulation. Moreover, kinase-deficient MKK3, PDK1 and PKB inhibited thrombin- and PAK-dependent TF expression and promoter activity. Together these findings show that PAK is a critical element of thrombin signalling in PASMC which is involved in the regulation of TF expression by sequentially activating MKK3/p38MAPK, PDK1 and PKB. Thus, PAK may play an important role in promoting vascular remodelling processes in pulmonary hypertension.

Slit Stimulation Recruits Dock and Pak to the Roundabout Receptor and Increases Rac Activity to Regulate Axon Repulsion at the CNS Midline

Drosophila Roundabout (Robo) is the founding member of a conserved family of repulsive axon guidance receptors that respond to secreted Slit proteins. Here we present evidence that the SH3-SH2 adaptor protein Dreadlocks (Dock), the p21-activated serine-threonine kinase (Pak), and the Rac1/Rac2/Mtl small GTPases can function during Robo repulsion. Loss-of-function and genetic interaction experiments suggest that limiting the function of Dock, Pak, or Rac partially disrupts Robo repulsion. In addition, Dock can directly bind to Robo's cytoplasmic domain, and the association of Dock and Robo is enhanced by stimulation with Slit. Furthermore, Slit stimulation can recruit a complex of Dock and Pak to the Robo receptor and trigger an increase in Rac1 activity. These results provide a direct physical link between the Robo receptor and an important cytoskeletal regulatory protein complex and suggest that Rac can function in both attractive and repulsive axon guidance.

Paxillin-dependent paxillin kinase linker and p21-activated kinase localization to focal adhesions involves a multistep activation pathway.

The precise temporal-spatial regulation of the p21-activated serine-threonine kinase PAK at the plasma membrane is required for proper cytoskeletal reorganization and cell motility. However, the mechanism by which PAK localizes to focal adhesions has not yet been elucidated. Indirect binding of PAK to the focal adhesion protein paxillin via the Arf-GAP protein paxillin kinase linker (PKL) and PIX/Cool suggested a mechanism. In this report, we demonstrate an essential role for a paxillin-PKL interaction in the recruitment of activated PAK to focal adhesions. Similar to PAK, expression of activated Cdc42 and Rac1, but not RhoA, stimulated the translocation of PKL from a generally diffuse localization to focal adhesions. Expression of the PAK regulatory domain (PAK1-329) or the autoinhibitory domain (AID 83-149) induced PKL, PIX, and PAK localization to focal adhesions, indicating a role for PAK scaffold activation. We show PIX, but not NCK, binding to PAK is necessary for efficient focal adhesion localization of PAK and PKL, consistent with a PAK-PIX-PKL linkage. Although PAK activation is required, it is not sufficient for localization. The PKL amino terminus, containing the PIX-binding site, but lacking paxillin-binding subdomain 2 (PBS2), was unable to localize to focal adhesions and also abrogated PAK localization. An identical result was obtained after PKLDeltaPBS2 expression. Finally, neither PAK nor PKL was capable of localizing to focal adhesions in cells overexpressing paxillinDeltaLD4, confirming a requirement for this motif in recruitment of the PAK-PIX-PKL complex to focal adhesions. These results suggest a GTP-Cdc42/GTP-Rac triggered multistep activation cascade leading to the stimulation of the adaptor function of PAK, which through interaction with PIX provokes a functional PKL PBS2-paxillin LD4 association and consequent recruitment to focal adhesions. This mechanism is probably critical for the correct subcellular positioning of PAK, thereby influencing the ability of PAK to coordinate cytoskeletal reorganization associated with changes in cell shape and motility.

Identification of the binding surface on Cdc42Hs for p21-activated kinase.

The Ras superfamily of GTP-binding proteins is involved in a number of cellular signaling events including, but not limited to, tumorigenesis, intracellular trafficking, and cytoskeletal organization. The Rho subfamily, of which Cdc42Hs is a member, is involved in cell morphogenesis through a GTPase cascade which regulates cytoskeletal changes. Cdc42Hs has been shown to stimulate DNA synthesis as well as to initiate a protein kinase cascade that begins with the activation of the p21-activated serine/threonine kinases (PAKs). We have determined previously the solution structure of Cdc42Hs [Feltham et al. (1997) Biochemistry 36, 8755-8766] using NMR spectroscopy. A minimal-binding domain of 46 amino acids of PAK was identified (PBD46), which binds Cdc42Hs with a KD of approximately 20 nM and inhibits GTP hydrolysis. The binding interface was mapped by producing a fully deuterated sample of 15N-Cdc42Hs bound to PBD46. A 1H,15N-NOESY-HSQC spectrum demonstrated that the binding surface on Cdc42Hs consists of the second beta-strand (beta2) and a portion of the loop between the first alpha-helix (alpha1) and beta2 (switch I). A complex of PBD46 bound to 15N-Cdc42Hs.GMPPCP exhibited extensive chemical shift changes in the 1H,15N-HSQC spectrum. Thus, PBD46 likely produces structural changes in Cdc42Hs which are not limited to the binding interface, consistent with its effects on GTP hydrolysis. These results suggest that the kinase-binding domain on Cdc42Hs is similar to, but more extensive than, the c-Raf-binding domain on the Ras antagonist, Rap1 [Nassar et al. (1995) Nature 375, 554-560)].

Regulation of phosphorylation pathways by p21 GTPases. The p21 Ras-related Rho subfamily and its role in phosphorylation signalling pathways.

The oncogenic Ras p21 GTPases regulate phosphorylation pathways that underlie a wealth of activities, including growth and differentiation, in organisms ranging from yeast to human. In metazoa, growth factors trigger conversion of Ras from an inactive GDP-bound form to an active GTP-bound form. This activation of Ras leads to activation of Raf. Raf is one of the initial kinases in the cytoplasmic mitogen-activated protein kinase (MAPK) cascade, involving extracellular-signal-regulated kinases (ERK), which culminates in nuclear transcription. The Ras-related subfamily of Rho p21s, including Rho, Rac and Cdc42 are similarly active in their GTP-bound forms. These p21s mediate growth-factor-induced morphological changes involving actin-based cellular structures. For example, in mammalian fibroblasts, Rho mediates the formation of cytoskeletal stress fibres induced by lysophosphatidic acid, while Rac mediates the formation of membrane ruffles induced by platelet-derived growth factor, and Cdc42 mediates the formation of peripheral filopodia by bradykinin. In some cases, factor-induced Rac activation results in Rho activation, and factor-induced Cdc42 activation leads to Rac activation, as determined by specific morphological changes. Although separate Cdc42/Rac and Rac/Rho hierarchies exist, these might not extend into a linear form (i.e. Cdc42-->Rac-->Rho) since Cdc42 and Rho activities may be competitive or even antagonistic. Thus Cdc42-mediated formation of filopodia is accompanied by loss of stress fibres (whose formation is mediated by Rho). Recently, mammalian kinases that bind to the GTP-bound forms of Rho p21s have been isolated. These kinases include the p21-activated serine/threonine kinase (PAK), which is stimulated by binding to Cdc42 and Rac, and the Rho-binding serine/threonine kinase (ROK), which is not as strongly stimulated by binding. These kinases act as effectors for their p21 partners since they can directly affect the reorganization of the relevant actin-containing structures. ROK promotes the formation of Rho-induced actin-containing stress fibres and focal-adhesion complexes, to which the ends of the stress fibres attach. PAK stimulates the disassembly of stress fibres, which has been shown to accompany formation of Cdc42-induced peripheral-actin-containing structures, including filopodia, which with Rac-induced membrane ruffles play a role in cell movement. PAK also fosters loss of focal-adhesion complexes. Thus, there is cooperation between different Rho p21s as well as antagonism, with their associated kinases having a role in the integration of the reorganization of the actin cytoskeleton. The similarity of PAK to the Saccharomyces cerevisiae kinase Ste20p, which initiates the yeast mating/pheromone MAPK cascade, led to experiments showing that Cdc42 regulates Ste20p in this MAPK pathway. This similarity has also led to the demonstration that mammalian Cdc42 and Rac can signal to the nucleus through MAPK pathways. However, c-Jun N-terminal kinase (JNK, stress-activated protein kinase) rather than ERK, is involved. PAK have been implicated in the JNK pathway, but their exact roles are uncertain. Thus members of the Rho subfamily, and kinases that bind to these p21s are intimately involved in immediate morphological processes as well as long-term transcriptional events.