Activation Of P21-activated Kinase Research Articles

Rho GTPase-independent regulation of mitotic progression by the RhoGEF Net1.

Neuroepithelial transforming gene 1 (Net1) is a RhoA-subfamily-specific guanine nucleotide exchange factor that is overexpressed in multiple human cancers and is required for proliferation. Molecular mechanisms underlying its role in cell proliferation are unknown. Here we show that overexpression or knockdown of Net1 causes mitotic defects. Net1 is required for chromosome congression during metaphase and generation of stable kinetochore microtubule attachments. Accordingly, inhibition of Net1 expression results in spindle assembly checkpoint activation. The ability of Net1 to control mitosis is independent of RhoA or RhoB activation, as knockdown of either GTPase does not phenocopy effects of Net1 knockdown on nuclear morphology, and effects of Net1 knockdown are effectively rescued by expression of catalytically inactive Net1. We also observe that Net1 expression is required for centrosomal activation of p21-activated kinase and its downstream kinase Aurora A, which are critical regulators of centrosome maturation and spindle assembly. These results identify Net1 as a novel regulator of mitosis and indicate that altered expression of Net1, as occurs in human cancers, may adversely affect genomic stability.

N-acetylglucosaminyltransferase V confers hepatoma cells with resistance to anoikis through EGFR/PAK1 activation

Elevated expression and activity of N-acetylglucosaminyltransferase V (Mgat5) in hepatocellular carcinoma (HCC) is a common early event involved in tumor invasion during hepatocarcinogenesis. A better understanding of the functional role and the molecular mechanism for Mgat5-targeted protein and downstream signaling pathway behind hepatoma invasion and metastasis is urgently needed. Here, we show that Mgat5 overexpression promoted anchorage-independent growth and inhibited anoikis in hepatoma cells. This effect was reversed by glycosyltransferase inactive mutant Mgat5 L188R transfection, α-mannosidase II inhibitor swainsonine treatment and N-acetyl glucosamine (GlcNAc) phosphotransferase (GPT) inhibitor tunicamycin administration. Mgat5 overexpression increased p21-activated kinase 1 (PAK1) expression and shRNA-mediated PAK1 knockdown and kinase inactivation with kinase dead mutant PAK1 K299R coexpression or allosteric inhibitor P21-activated kinase inhibitor III (IPA3) treatment reversed anoikis resistance in Mgat5-overexpressed hepatoma cells. Furthermore, Mgat5 overexpression upregulated β-1-6-GlcNAc branched N-glycosylation and following phosphorylation of epidermal growth factor receptor (EGFR) in hepatoma cells. EGFR tyrosine kinase inhibitors AG1478 and Iressa treatment declined anchorage-independent growth and anoikis resistance, which could be rescued by constitutive active mutant PAK1 T423E coexpression in Mgat5-overexpressed hepatoma cells. Conversely, knockdown of Mgat5 reduced EGFR/PAK1-dependent anoikis resistance, which could be reversed by PAK1 T423E. These results identified Mgat5-mediated β-1-6-GlcNAc branched N-glycosylation and following activation of EGFR as a potential novel upstream molecular event for PAK1-induced anoikis resistance in hepatoma cells, implicating that molecular targeted therapeutics against Mgat5/EGFR/PAK1 might open a new avenue for personalized medicine in advanced-stage HCC patients.

PAK inactivation impairs social recognition in 3xTg-AD Mice without increasing brain deposition of tau and Aβ.

Defects in p21-activated kinase (PAK) are suspected to play a role in cognitive symptoms of Alzheimer's disease (AD). Dysfunction in PAK leads to cofilin activation, drebrin displacement from its actin-binding site, actin depolymerization/severing, and, ultimately, defects in spine dynamics and cognitive impairment in mice. To determine the role of PAK in AD, we first quantified PAK by immunoblotting in homogenates from the parietal neocortex of subjects with a clinical diagnosis of no cognitive impairment (n = 12), mild cognitive impairment (n = 12), or AD (n = 12). A loss of total PAK, detected in the cortex of AD patients (-39% versus controls), was correlated with cognitive impairment (r(2) = 0.148, p = 0.027) and deposition of total and phosphorylated tau (r(2) = 0.235 and r(2) = 0.206, respectively), but not with Aβ42 (r(2) = 0.056). Accordingly, we found a decrease of total PAK in the cortex of 12- and 20-month-old 3xTg-AD mice, an animal model of AD-like Aβ and tau neuropathologies. To determine whether PAK dysfunction aggravates AD phenotype, 3xTg-AD mice were crossed with dominant-negative PAK mice. PAK inactivation led to obliteration of social recognition in old 3xTg-AD mice, which was associated with a decrease in cortical drebrin (-25%), but without enhancement of Aβ/tau pathology or any clear electrophysiological signature. Overall, our data suggest that PAK decrease is a consequence of AD neuropathology and that therapeutic activation of PAK may exert symptomatic benefits on high brain function.

Development of p21 Activated Kinase-Targeted Multikinase Inhibitors That Inhibit Thyroid Cancer Cell Migration

The p21 activated kinases (PAKs) are a family of serine/threonine kinases that are downstream effectors of small GTPase Cdc42 and Rac. PAKs regulate cell motility, proliferation, and cytoskeletal rearrangement. PAK isoform expression and activity have been shown to be enhanced in cancer and to function as an oncogene in vivo. PAKs also have been implicated in cancer progression. In thyroid cancer, we have previously determined that PAK overactivation is common in the invasive fronts of aggressive tumors and that it is functionally involved in thyroid cancer cell motility using molecular inhibitors. We report the development of two new PAK-inhibiting compounds that were modified from the structure OSU-03012, a previously identified multikinase inhibitor that competitively blocks ATP binding of both phosphoinositide-dependent kinase 1 (PDK1) and PAK1. Seventeen compounds were created by combinatorial chemistry predicted to inhibit PAK activity with reduced anti-PDK1 effect. Two lead compounds were identified based on the ability to inhibit PAK1 activity in an ATP-competitive manner without discernible in vivo PDK1 inhibitory activity in thyroid cancer cell lines. Both compounds reduced thyroid cancer cell viability. Although they are not PAK-specific on a multikinase screening assay, the antimigration activity effect of the compounds in thyroid cancer cells was rescued by overexpression of a constitutively active PAK1, suggesting this activity is involved in this biological effect. We have developed 2 new multikinase inhibitors with anti-PAK activity that may serve as scaffolds for further compound development targeting this progression-related thyroid cancer target.

Rac1 Signaling Is Required for Insulin-Stimulated Glucose Uptake and Is Dysregulated in Insulin-Resistant Murine and Human Skeletal Muscle

The actin cytoskeleton–regulating GTPase Rac1 is required for insulin-stimulated GLUT4 translocation in cultured muscle cells. However, involvement of Rac1 and its downstream signaling in glucose transport in insulin-sensitive and insulin-resistant mature skeletal muscle has not previously been investigated. We hypothesized that Rac1 and its downstream target, p21-activated kinase (PAK), are regulators of insulin-stimulated glucose uptake in mouse and human skeletal muscle and are dysregulated in insulin-resistant states. Muscle-specific inducible Rac1 knockout (KO) mice and pharmacological inhibition of Rac1 were used to determine whether Rac1 regulates insulin-stimulated glucose transport in mature skeletal muscle. Furthermore, Rac1 and PAK1 expression and signaling were investigated in muscle of insulin-resistant mice and humans. Inhibition and KO of Rac1 decreased insulin-stimulated glucose transport in mouse soleus and extensor digitorum longus muscles ex vivo. Rac1 KO mice showed decreased insulin and glucose tolerance and trended toward higher plasma insulin concentrations after intraperitoneal glucose injection. Rac1 protein expression and insulin-stimulated PAKThr423 phosphorylation were decreased in muscles of high fat–fed mice. In humans, insulin-stimulated PAK activation was decreased in both acute insulin-resistant (intralipid infusion) and chronic insulin-resistant states (obesity and diabetes). These findings show that Rac1 is a regulator of insulin-stimulated glucose uptake and a novel candidate involved in skeletal muscle insulin resistance.

P21 Activated Kinase Signaling Coordinates Glycoprotein Receptor VI–Mediated Platelet Aggregation, Lamellipodia Formation, and Aggregate Stability Under Shear

Rho GTPase proteins play a central role in regulating the dynamics of the platelet actin cytoskeleton. Yet, little is known regarding how Rho GTPase activation coordinates platelet activation and function. In this study, we aimed to characterize the role of the Rho GTPase effector, p21 activated kinase (PAK), in platelet activation, lamellipodia formation, and aggregate formation under shear. Stimulation of platelets with the glycoprotein receptor VI agonist, collagen-related peptide, rapidly activated PAK in a time course preceding phosphorylation of PAK substrates, LIM domain kinase LIMK1 and the MAPK/ERK kinase MEK, and the subsequent activation of MAPKs and Akt. Pharmacological inhibitors of PAK blocked signaling events downstream of PAK and prevented platelet secretion as well as platelet aggregation in response to collagen-related peptide. PAK inhibitors also prevented PAK activation and platelet spreading on collagen surfaces. PAK was also required for the formation of platelet aggregates and to maintain aggregate stability under physiological shear flow conditions. These results suggest that PAK serves as an orchestrator of platelet functional responses after activation downstream of the platelet collagen receptor, glycoprotein receptor VI.

P21‐activated kinase inhibits vascular reactivity via inhibition of MLCK

Changes in vascular reactivity contribute to the pathology of a number of different diseases including heart failure, hypertension, severe trauma, and sepsis. P21‐activated kinase (PAK) can alter smooth muscle contractile activity and has been implicated in altering vascular reactivity. The objective of this study was to determine the mechanism by which PAK regulates MLC phosphorylation in vascular smooth muscle to alter vascular reactivity. The effects of PAK activation and inhibition on basal and agonist‐induced aortic contraction in aortic rings and on myosin light chain (MLC) and myosin targeting subunit of MLC phosphatase (MYPT1) phosphorylation and MLC kinase (MLCK) activity in primary aortic smooth muscle cells (ASMC) were determined. Finally, the effects of increased mechanical stimulation on PAK activation were determined in ASMC. PAK activation inhibited both basal tone and agonist induced contraction of rat aortic rings. Conversely, PAK inhibition increased agonist induced contraction and basal tone. In ASMC, PAK activation inhibited MLC phosphorylation and PAK inhibition increased MLC phosphorylation. However, PAK activation increased MYPT1 phosphorylation in ASMC. Increased mechanical stimulation of ASMC increased PAK activation and decreased MLCK activation. These data suggest that PAK inhibits vascular reactivity via decreased inhibition of MLCK.

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.

HGF and IGF-1 synergize with SDF-1α in promoting migration of myeloma cells by cooperative activation of p21-activated kinase

Stromal-derived factor (SDF)-1α, insulin-like growth factor (IGF)-1 and hepatocyte growth factor (HGF) are potent mediators of cell migration. We studied the effect of combinations of these cytokines on the migration of myeloma cells. When SDF-1α was combined with either HGF or IGF-1, we found a striking synergy in the cytokines' ability to guide cells across a transwell membrane. Between HGF and IGF-1 there was no cooperativity. However, the effects of HGF and IGF-1 were not redundant. HGF and SDF-1 caused concentration gradient-directed migration, as opposed to IGF-1, which apparently caused randomly directed cell movement. The SDF-1α-driven migration of JJN-3 cells, a myeloma cell line secreting large amounts of HGF, was reduced when JJN-3 cells were given an inhibitor of the HGF receptor, demonstrating a cooperative activity between autocrine HGF and exogenous SDF-1α. There was a clear positive correlation between the degree of cytokine-induced migration and phosphorylation of p21-activated kinase (PAK) both in primary myeloma cells and in cell lines including INA-6 and IH-1. Downregulation of PAK with small interfering RNA in INA-6 cells resulted in decreased cytokine-driven migration. This study shows synergy between SDF-1α and HGF/IGF-1 in inducing migration of myeloma cells, yet each cytokine has distinct properties in the way it regulates cell migration. These findings are likely to be of clinical relevance because multiple myeloma cells are located in an environment containing HGF and IGF-1 and are exposed to an SDF-1α gradient between the bone marrow and peripheral blood.

Synergistic Activation of p21-activated Kinase 1 by Phosphatidylinositol 4,5-Bisphosphate and Rho GTPases

Autoinhibited p21-activated kinase 1 (Pak1) can be activated in vitro by the plasma membrane-bound Rho GTPases Rac1 and Cdc42 as well as by the lipid phosphatidylinositol (4,5)-bisphosphate (PIP2). Activator binding is mediated by a GTPase-binding motif and an adjacent phosphoinositide-binding motif. Whether these two classes of activators play alternative, additive, or synergistic roles in Pak1 activation is unknown, as is their contributions to Pak1 activation in vivo. To address these questions, we developed a system to mimic the membrane anchoring of Rho GTPases by creating liposomes containing both PIP2 and a Ni(2+)-NTA modified lipid capable of binding hexahistidine-tagged Cdc42. We find that among all biologically relevant phosphoinositides, only PIP2 is able to synergistically activate Pak1 in concert with Cdc42. Membrane binding of the kinase was highly sensitive to the spatial density of PIP2 and Pak1 demonstrated dramatically enhanced affinity for Cdc42 anchored in a PIP2 environment. To validate these findings in vivo, we utilized an inducible recruitment system to drive the ectopic synthesis of PIP2 on Golgi membranes, which normally have active Cdc42 but lack significant concentrations of PIP2. Pak1 was recruited to PIP2-containing membranes in a manner dependent on the ability of Pak1 to bind to both PIP2 and Cdc42. These findings provide a mechanistic explanation for the essential role of both phosphoinositides and GTPases in Pak1 recruitment and activation. In contrast, Ack, another Cdc42 effector kinase that lacks an analogous phosphoinositide-binding motif, fails to show the same enhancement of membrane binding and activation by PIP2, thus indicating that regulation by PIP2 and Cdc42 could provide a combinatorial code for activation of different GTPase effectors in different subcellular locations.

Inhibition of formyl peptide-stimulated superoxide anion generation by Fal-002-2 occurs mainly through the blockade of the p21-activated kinase and protein kinase C signaling pathways in ratneutrophils

In formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils, a synthetic compound, 6-chloro-2-(2-chlorophenyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (Fal-002-2), inhibited superoxide anion (O2•−) generation with an IC50 value of about 11μM, which was not mediated by scavenging the generated O2•− or by a cytotoxic effect on neutrophils. Fal-002-2 effectively attenuated the phosphorylation of Ser residues in p47phox and the association between p47phox and p22phox in fMLP-stimulated neutrophils. The interaction of p47phox with protein kinase C (PKC) isoforms (α, βI, βII, δ and ζ) was attenuated by Fal-002-2 with a similar IC50 value to that required for inhibition of O2•− generation, whereas Fal-002-2 had no prominent effect on PKC isoform membrane translocation and did not affect the kinase activity. Moreover, Fal-002-2 had no effect on the phosphorylation of Akt and downstream glycogen synthase kinase-3β, only slightly affected the intracellular free Ca2+ concentration, phosphorylation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase (MAPK), but effectively attenuated the downstream MAPK-activated protein kinase-2 phosphorylation. The interaction of p21-activated kinase (PAK) 1with p47phox, phosphorylation of PAK1 (Thr423/Ser144) and the membrane recruitment of PAK1 were effectively inhibited by Fal-002-2. Fal-002-2 also blocked the activation of Rac1 and Cdc42 in a concentration range that effectively inhibited PAK activation. Taken together, these results suggest that Fal-002-2 inhibits fMLP-stimulated O2•− generation in neutrophils mainly through the blockade of PKC and PAK signaling pathways and partly through p38 MAPK signaling.

P-21-Activated kinase 1 mediates gastrin-stimulated proliferation in the colorectal mucosa via multiple signaling pathways

Gastrins, including amidated (Gamide) and glycine-extended (Ggly) forms, function as growth factors for the gastrointestinal mucosa. The p-21-activated kinase 1 (PAK1) plays important roles in growth factor signaling networks that control cell motility, proliferation, differentiation, and transformation. PAK1, activated by both Gamide and Ggly, mediates gastrin-stimulated proliferation and migration, and activation of β-catenin, in gastric epithelial cells. The aim of this study was to investigate the role of PAK1 in the regulation by gastrin of proliferation in the normal colorectal mucosa in vivo. Mucosal proliferation was measured in PAK1 knockout (PAK1 KO) mice by immunohistochemistry. The expression of phosphorylated and unphosphorylated forms of the signaling molecules PAK1, extracellular signal-regulated kinase (ERK), and protein kinase B (AKT), and the expression of β-catenin and its downstream targets c-Myc and cyclin D1, were measured in gastrin knockout (Gas KO) and PAK1 KO mice by Western blotting. The expression and activation of PAK1 are decreased in Gas KO mice, and these decreases are associated with reduced activation of ERK, AKT, and β-catenin. Proliferation in the colorectal mucosa of PAK1 KO mice is reduced, and the reduction is associated with reduced activation of ERK, AKT, and β-catenin. In compensation, antral gastrin mRNA and serum gastrin concentrations are increased in PAK1 KO mice. These results indicate that PAK1 mediates the stimulation of colorectal proliferation by gastrins via multiple signaling pathways involving activation of ERK, AKT, and β-catenin.

Biphasic Regulation of Myosin Light Chain Phosphorylation by p21-activated Kinase Modulates Intestinal Smooth Muscle Contractility

Supraphysiological mechanical stretching in smooth muscle results in decreased contractile activity. However, the mechanism is unclear. Previous studies indicated that intestinal motility dysfunction after edema development is associated with increased smooth muscle stress and decreased myosin light chain (MLC) phosphorylation in vivo, providing an ideal model for studying mechanical stress-mediated decrease in smooth muscle contraction. Primary human intestinal smooth muscle cells (hISMCs) were subjected to either control cyclical stretch (CCS) or edema (increasing) cyclical stretch (ECS), mimicking the biophysical forces in non-edematous and edematous intestinal smooth muscle in vivo. ECS induced significant decreases in phosphorylation of MLC and MLC phosphatase targeting subunit (MYPT1) and a significant increase in p21-activated kinase (PAK) activity compared with CCS. PAK regulated MLC phosphorylation in an activity-dependent biphasic manner. PAK activation increased MLC and MYPT1 phosphorylation in CCS but decreased MLC and MYPT1 phosphorylation in hISMCs subjected to ECS. PAK inhibition had the opposite results. siRNA studies showed that PAK1 plays a critical role in regulating MLC phosphorylation in hISMCs. PAK1 enhanced MLC phosphorylation via phosphorylating MYPT1 on Thr-696, whereas PAK1 inhibited MLC phosphorylation via decreasing MYPT1 on both Thr-696 and Thr-853. Importantly, in vivo data indicated that PAK activity increased in edematous tissue, and inhibition of PAK in edematous intestine improved intestinal motility. We conclude that PAK1 positively regulates MLC phosphorylation in intestinal smooth muscle through increasing inhibitory phosphorylation of MYPT1 under physiologic conditions, whereas PAK1 negatively regulates MLC phosphorylation via inhibiting MYPT1 phosphorylation when PAK activity is increased under pathologic conditions.

Altered nitric oxide production mediates matrix-specific PAK2 and NF-κB activation by flow

Shear stress generated by distinct blood flow patterns modulates endothelial cell phenotype to spatially restrict atherosclerotic plaque development. Signaling through p21-activated kinase (PAK) mediates several of the deleterious effects of shear stress, including enhanced NF-κB activation and proinflammatory gene expression. Whereas shear stress activates PAK in endothelial cells on a fibronectin matrix, basement membrane proteins limit shear-induced PAK activation and inflammation through a protein kinase A-dependent pathway; however, the mechanisms underlying this regulation were unknown. We show that basement membrane proteins limit membrane recruitment of PAK2, the dominant isoform in endothelial cells, by blocking its interaction with the adaptor protein Nck. This uncoupling response requires protein kinase A-dependent nitric oxide production and subsequent PAK2 phosphorylation on Ser-20 in the Nck-binding domain. Of importance, shear stress does not stimulate nitric oxide production in endothelial cells on fibronectin, resulting in enhanced PAK activation, NF-κB phosphorylation, ICAM-1 expression, and monocyte adhesion. These data demonstrate that differential flow-induced nitric oxide production regulates matrix-specific PAK signaling and describe a novel mechanism of nitric oxide-dependent NF-κB inhibition.

Group I p21-Activated Kinases (PAKs) Promote Tumor Cell Proliferation and Survival through the AKT1 and Raf–MAPK Pathways

Group I p21-activated kinases (PAK) are important effectors of the small GTPases Rac and Cdc42, which regulate cell motility/migration, survival, proliferation, and gene transcription. Hyperactivation of these kinases have been reported in many tumor types, making PAKs attractive targets for therapeutic intervention. PAKs are activated by growth factor-mediated signaling and are negatively regulated by the tumor suppressor neurofibromatosis type 2 (NF2)/Merlin. Thus, tumors characterized by NF2 inactivation would be expected to show hyperactivated PAK signaling. On the basis of this rationale, we evaluated the status of PAK signaling in malignant mesothelioma, an aggressive neoplasm that is resistant to current therapies and shows frequent inactivation of NF2. We show that group I PAKs are activated in most mesotheliomas and mesothelioma cell lines and that genetic or pharmacologic inhibition of PAKs is sufficient to inhibit mesothelioma cell proliferation and survival. We also identify downstream effectors and signaling pathways that may contribute mechanistically to PAK-related tumorigenesis. Specifically, we show that inhibition of PAK results in attenuation of AKT and Raf-MAPK signaling and decreased tumor cell viability. Collectively, these data suggest that pharmacologic inhibition of group I PAKs may have therapeutic efficacy in tumors characterized by PAK activation.

Molecular Pathways: Targeting P21-Activated Kinase 1 Signaling in Cancer—Opportunities, Challenges, and Limitations

The evolution of cancer cells involves deregulation of highly regulated fundamental pathways that are central to normal cellular architecture and functions. p21-activated kinase 1 (PAK1) was initially identified as a downstream effector of the GTPases Rac and Cdc42. Subsequent studies uncovered a variety of new functions for this kinase in growth factor and steroid receptor signaling, cytoskeleton remodeling, cell survival, oncogenic transformation, and gene transcription, largely through systematic discovery of its direct, physiologically relevant substrates. PAK1 is widely upregulated in several human cancers, such as hormone-dependent cancer, and is intimately linked to tumor progression and therapeutic resistance. These exciting developments combined with the kinase-independent role of PAK1-centered phenotypic signaling in cancer cells elevated PAK1 as an attractive drug target. Structural and biochemical studies revealed the precise mechanism of PAK1 activation, offering the possibility to develop PAK1-targeted cancer therapeutic approaches. In addition, emerging reports suggest the potential of PAK1 and its specific phosphorylated substrates as cancer prognostic markers. Here, we summarize recent findings about the PAK1 molecular pathways in human cancer and discuss the current status of PAK1-targeted anticancer therapies.

Synapses of Amphids Defective (SAD-A) Kinase Promotes Glucose-stimulated Insulin Secretion through Activation of p21-activated Kinase (PAK1) in Pancreatic β-Cells

The p21-activated kinase-1 (PAK1) is implicated in regulation of insulin exocytosis as an effector of Rho GTPases. PAK1 is activated by the onset of glucose-stimulated insulin secretion (GSIS) through phosphorylation of Thr-423, a major activation site by Cdc42 and Rac1. However, the kinase(s) that phosphorylates PAK1 at Thr-423 in islet β-cells remains elusive. The present studies identified SAD-A (synapses of amphids defective), a member of AMP-activated protein kinase-related kinases exclusively expressed in brain and pancreas, as a key regulator of GSIS through activation of PAK1. We show that SAD-A directly binds to PAK1 through its kinase domain. The interaction is mediated by the p21-binding domain (PBD) of PAK1 and requires both kinases in an active conformation. The binding leads to direct phosphorylation of PAK1 at Thr-423 by SAD-A, triggering the onset of GSIS from islet β-cells. Consequently, ablation of PAK1 kinase activity or depletion of PAK1 expression completely abolishes the potentiating effect of SAD-A on GSIS. Consistent with its role in regulating GSIS, overexpression of SAD-A in MIN6 islet β-cells significantly stimulated cytoskeletal remodeling, which is required for insulin exocytosis. Together, the present studies identified a critical role of SAD-A in the activation of PAK1 during the onset of insulin exocytosis.

Characterization of EHop-016, Novel Small Molecule Inhibitor of Rac GTPase

The Rho GTPase Rac regulates actin cytoskeleton reorganization to form cell surface extensions (lamellipodia) required for cell migration/invasion during cancer metastasis. Rac hyperactivation and overexpression are associated with aggressive cancers; thus, interference of the interaction of Rac with its direct upstream activators, guanine nucleotide exchange factors (GEFs), is a viable strategy for inhibiting Rac activity. We synthesized EHop-016, a novel inhibitor of Rac activity, based on the structure of the established Rac/Rac GEF inhibitor NSC23766. Herein, we demonstrate that EHop-016 inhibits Rac activity in the MDA-MB-435 metastatic cancer cells that overexpress Rac and exhibits high endogenous Rac activity. The IC(50) of 1.1 μM for Rac inhibition by EHop-016 is ∼100-fold lower than for NSC23766. EHop-016 is specific for Rac1 and Rac3 at concentrations of ≤5 μM. At higher concentrations, EHop-016 inhibits the close homolog Cdc42. In MDA-MB-435 cells that demonstrate high active levels of the Rac GEF Vav2, EHop-016 inhibits the association of Vav2 with a nucleotide-free Rac1(G15A), which has a high affinity for activated GEFs. EHop-016 also inhibits the Rac activity of MDA-MB-231 metastatic breast cancer cells and reduces Rac-directed lamellipodia formation in both cell lines. EHop-016 decreases Rac downstream effects of PAK1 (p21-activated kinase 1) activity and directed migration of metastatic cancer cells. Moreover, at effective concentrations (<5 μM), EHop-016 does not affect the viability of transformed mammary epithelial cells (MCF-10A) and reduces viability of MDA-MB-435 cells by only 20%. Therefore, EHop-016 holds promise as a targeted therapeutic agent for the treatment of metastatic cancers with high Rac activity.

The PAKs come of age

Protein kinases are versatile signaling molecules that are involved in the regulation most physiological responses. The p21-activated kinases (PAKs) can be activated directly by the small GTPases Rac and Cdc42 and are among the best characterized downstream effectors of these Rho proteins. The structure, substrate specificity and functional role of PAKS are evolutionarily conserved from protozoa to mammals. Vertebrate PAKs are particularly important for cytoskeletal remodeling and focal adhesion assembly, thereby contributing to dynamic processes such as cell migration and synaptic plasticity. This issue of Cellular Logistics focuses on the PAK family of kinases, with ten reviews written by researchers currently working in the field. Here in this introductory overview we highlight some of the most interesting recent discoveries regarding PAK biochemistry and biology. The reviews in this issue cover a range of topics including the atomic structures of PAK1 and PAK4, their role in animals as assessed by knockout studies, and how PAKs are likely to contribute to cancer and neurodegenerative diseases. The promise remains that PAK inhibitors will emerge that validate current pre-clinical studies suggesting that blocking PAK activity will positively contribute to human health.

Phosphorylation of β-catenin at serine 663 regulates its transcriptional activity

β-Catenin, a component of Wnt signaling, plays a key role in colorectal carcinogenesis. The phosphorylation status of β-catenin determines its fate and affects its cellular function, and serine 675 (S675) was previously identified as a common target of p21-activated kinase 1 (PAK1) and protein kinase A. In the present study, we explored the PAK1-specific phosphorylation site(s) in β-catenin. Active PAK1 T423E but not inactive PAK1 K299R interacted with and phosphorylated β-catenin. Mutagenesis followed by a kinase assay revealed that PAK1 phosphorylated S663 in addition to S675, and an anti-phospho-β-cateninS663 antibody detected the phosphorylation of S663 downstream of PAK1 in various human colon cancer cells. Furthermore, the Wnt3a-stimulated S663 phosphorylation was inhibited by the PAK1-specific inhibitor, IPA-3, but not by H-89 or LY294002. The non-phosphorylatable mutant forms of β-catenin, S663A, S675A and S663/675A, showed similar defects in their PAK1-induced TCF/LEF transactivation, whereas the phosphomimetic form of β-catenin, S663D, demonstrated a transcriptional activity that was comparable to that of β-catenin S675D and β-catenin S663D/S675D. Taken together, these results provide evidence that PAK1 specifically phosphorylates β-catenin at S663 and that this phosphorylation is essential for the PAK1-mediated transcriptional activation of β-catenin.