Phosphorylation of the activation loop of gamma p21-activated kinase (gamma-Pak) and related kinases (MSTs) in normal and stressed neutrophils

Neutrophils stimulated with a variety of chemoattractants exhibit a rapid activation of two p21-activated kinases (Paks) with molecular masses of approximately 63 and 69 kDa (gamma- and alpha-Pak). A number of in vitro studies suggest that modification of Thr(402) in the activation loop (AL) of gamma-Pak can play a critical role in the regulation of this kinase under certain circumstances. A phosphospecific Ab was generated to this region of Pak (pPak(AL)Ab). This Ab reacted with activated gamma- and alpha-Pak from fMLP-stimulated neutrophils that contain the sequence KRXT(P)XXGTP in their ALS: The rapid but transient activation of Paks in normal stimulated neutrophils coincided with phosphorylation and dephosphorylation at the ALs of these enzymes. In contrast, stressed cells exhibited a prolonged phosphorylation at Thr(402) in both intact gamma-Pak and a proteolytic fragment of this kinase. The pPak(AL)Ab also reacted with the mammalian sterile twenty-like kinases (MSTs) (members of the Pak family) in osmotically stressed neutrophils and neutrophils treated with certain apoptotic agents (i.e., tumor promoters that inhibit type 1 and 2A protein phosphatases) but not in normal fMLP-stimulated cells. Thus, our results indicate that the AL of gamma-Pak undergoes transient phosphorylation during normal neutrophil stimulation and chronic phosphorylation in stressed cells. In addition, we demonstrate that a number of MSTs are present in neutrophils and also undergo phosphorylation during stressful circumstances.     

Role of group A p21-activated kinases in activation of extracellular-regulated kinase by growth factors

The canonical extracellular-regulated kinase (ERK) signaling cascade, consisting of the Ras-Raf-Mek-ERK module, is critically important to many cellular functions. Although the general mechanism of activation of the ERK cascade is well established, additional noncanonical components greatly influence the activity of this pathway. Here, we focus on the group A p21-activated kinases (Paks), which have previously been implicated in regulating both c-Raf and Mek1 activity, by phosphorylating these proteins at Ser(338) and Ser(298), respectively. In NIH-3T3 cells, expression of an inhibitor of all three group A Paks reduced activation of ERK in response to platelet-derived growth factor (PDGF) but not to epidermal growth factor (EGF). Similar results were obtained in HeLa cells using small interference RNA-mediated simultaneous knockdown of both Pak1 and Pak2 to reduce group A Pak function. Inhibition of Pak kinase activity dramatically decreased phosphorylation of Mek1 at Ser(298) in response to either PDGF or EGF, but this inhibition did not prevent Mek1 activation by EGF, suggesting that although Pak can phosphorylate Mek1 at Ser(298), this event is not required for Mek1 activation by growth factors. Inhibition of Pak reduced the Ser(338) phosphorylation of c-Raf in response to both PDGF and EGF; however, in the case of EGF, the reduction in Ser(338) phosphorylation was not accompanied by a significant decrease in c-Raf activity. These findings suggest that Paks are required for the phosphorylation of c-Raf at Ser(338) in response to either growth factor, but that the mechanisms by which EGF and PDGF activate c-Raf are fundamentally different.     

The role of p21-activated kinases in hepatocellular carcinoma metastasis

The p21-activated kinases (PAKs) are downstream effectors of the Rho family small GTPases as well as a wide variety of mitogenic factors and have been implicated in cancer formation, development and metastasis. PAKs phosphorylate a wide spectrum of substrates to mediate extracellular signals and regulate cytoskeletal remodeling, cell motility and survival. In this review, we aim to summarize the findings regarding the oncogenic role and the underlying mechanisms of PAKs signaling in various cancers, and in particular highlight the prime importance of PAKs in hepatocellular carcinoma (HCC) progression and metastasis. Recent studies exploring the potential therapeutic application of PAK inhibitors will also be discussed.     

Emerging functions of p21-activated kinases in human cancer cells

The p21 activated kinases (Paks), an evolutionarily conserved family of serine/threonine kinases, are important for a variety of cellular functions including cell morphogenesis, motility, survival, mitosis, and angiogenesis. Paks are widely expressed in numerous tissues and are activated by growth factors and extracellular signals through GTPase-dependent and -independent mechanisms. Overexpression of Paks in epithelial cancer cells has been shown to increase migration potential, increase anchorage independent growth, and cause abnormalities in mitosis. Dysregulation of Paks has been reported in several human tumors and neurodegenerative diseases. A growing list of novel Pak interacting proteins has opened up exciting avenues of investigation by which to understand the functions of Paks in tumorigenesis. In this review, we will summarize the current knowledge of the Paks family with respect to emerging cellular functions and possible contributions to cancer.     

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.     

The p21-activated kinases in neural cytoskeletal remodeling and related neurological disorders

The serine/threonine p21-activated kinases(PAKs),as main effectors of the Rho GTPases Cdc42 and Rac,represent a group of important molecular switches linking the complex cytoskeletal networks to broad neural activity.PAKs show wide expression in the brain,but they differ in specific cell types,brain regions,and developmental stages.PAKs play an essential and differential role in controlling neural cytoskeletal remodeling and are related to the development and fate of neurons as well as the structural and functional plasticity of dendritic spines.PAK-mediated actin signaling and interacting functional networks represent a common pathway frequently affected in multiple neurodevelopmental and neurodegenerative disorders.Considering specific small-molecule agonists and inhibitors for PAKs have been developed in cancer treatment,comprehensive knowledge about the role of PAKs in neural cytoskeletal remodeling will promote our understanding of the complex mechanisms underlying neurological diseases,which may also represent potential therapeutic targets of these diseases.     

Substrates enhance autophosphorylation and activation of p21-activated protein kinase gamma-PAK in the absence of activation loop phosphorylation.

The p21-activated protein kinase gamma-PAK from rabbit, expressed in insect cells, is activated following binding of Cdc42(GTPgammaS). The rate of autophosphorylation is increased fivefold and the protein kinase activity 13-fold, as measured with the synthetic heptapeptide (AKRESAA). The mutant K278R, where the invariant lysine in the catalytic site is replaced by arginine, shows neither autophosphorylation nor activity. Replacement of the conserved threonine in the catalytic domain with alanine (T402A) reduces autophosphorylation and protein kinase activity to 1% that of the wild-type gamma-PAK, indicating autophosphorylation of Thr402 in the activation loop is essential for protein kinase activity. In contrast, certain protein substrates such as histone 2B, histone 4 and myelin basic protein, stimulate both autophosphorylation and protein kinase activity to levels similar to those observed with Cdc42(GTPgammaS). This substrate-level activation does not require autophosphorylation of Thr402 in the activation loop. As shown with T402A, the protein kinase activity with histone 4 is similar to that observed with recombinant wild-type gamma-PAK. Basic proteins or peptides which are not substrates of gamma-PAK, such as histone 1 and polylysine, do not stimulate autophosphorylation or activity. Other substrates such as the Rous sarcoma virus protein NC are phosphorylated by gamma-PAK following activation by Cdc42(GTPgammaS), but are not phosphorylated by T402A. The data suggest that some substrates can override the requirement for Cdc42(GTPgammaS), by activating gamma-PAK directly.     

Multisite autophosphorylation of p21-activated protein kinase gamma-PAK as a function of activation

p21-activated protein kinase (PAK) is a family of serine/threonine kinases whose activity is stimulated by binding to small G-proteins such as Cdc42 and subsequent autophosphorylation. Focusing on the ubiquitous gamma-isoform of PAK in this study, baculovirus-infected insect cells were used to obtain recombinant gamma-PAK, while native gamma-PAK was isolated from rabbit reticulocytes. Two-dimensional gel electrophoresis of gamma-PAK followed by immunoblot analysis revealed a similar profile for native and recombinant gamma-PAK, both consisting of multiple protein spots. Following Cdc42-stimulated autophosphorylation, the two-dimensional profiles of native and recombinant gamma-PAK were characterized by a similar acidic shift, suggesting a common response to Cdc42. To understand the effect of differential phosphorylation on its activation status, gamma-PAK autophosphorylation was conducted in the presence or absence of activators such as Cdc42 and histone II-AS, followed by tryptic digestion and comparative two-dimensional phosphopeptide mapping. The major phosphopeptides were subjected to a combination of manual and automated amino acid sequencing. Overall, eight autophosphorylation sites were identified in Cdc42-activated gamma-PAK, six of which are in common with those previously reported in alpha-PAK, while Ser-19 and Ser-165 appear to be uniquely phosphorylated in the gamma-form. Further, the phosphorylation of Ser-141, Ser-165, and Thr-402 was found to correlate with gamma-PAK activation.     

p21-activated kinase-aberrant activation and translocation in Alzheimer disease pathogenesis

Defects in dendritic spines and synapses contribute to cognitive deficits in mental retardation syndromes and, potentially, Alzheimer disease. p21-activated kinases (PAKs) regulate actin filaments and morphogenesis of dendritic spines regulated by the Rho family GTPases Rac and Cdc42. We previously reported that active PAK was markedly reduced in Alzheimer disease cytosol, accompanied by downstream loss of the spine actin-regulatory protein Drebrin. beta-Amyloid (Abeta) oligomer was implicated in PAK defects. Here we demonstrate that PAK is aberrantly activated and translocated from cytosol to membrane in Alzheimer disease brain and in 22-month-old Tg2576 transgenic mice with Alzheimer disease. This active PAK coimmunoprecipitated with the small GTPase Rac and both translocated to granules. Abeta42 oligomer treatment of cultured hippocampal neurons induced similar effects, accompanied by reduction of dendrites that were protected by kinase-active but not kinase-dead PAK. Abeta42 oligomer treatment also significantly reduced N-methyl-d-aspartic acid receptor subunit NR2B phosphotyrosine labeling. The Src family tyrosine kinase inhibitor PP2 significantly blocked the PAK/Rac translocation but not the loss of p-NR2B in Abeta42 oligomer-treated neurons. Src family kinases are known to phosphorylate the Rac activator Tiam1, which has recently been shown to be Abeta-responsive. In addition, anti-oligomer curcumin comparatively suppressed PAK translocation in aged Tg2576 transgenic mice with Alzheimer amyloid pathology and in Abeta42 oligomer-treated cultured hippocampal neurons. Our results implicate aberrant PAK in Abeta oligomer-induced signaling and synaptic deficits in Alzheimer disease.     

Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members.

The mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that are regulated by distinct extracellular stimuli. The currently known members include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP kinases. We find that overexpression of the Ste20-related enzymes p21-activated kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a lesser extent p38 MAP kinase but not ERK2. Rat MAP/ERK kinase kinase 1 can stimulate the activity of each of these MAP kinases. Although neither activated Rac nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative Rac2 or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway. Furthermore, constitutively active Rac2, Cdc42hs, and RhoA synergize with an activated form of Raf to increase ERK2 activity. These findings reveal a previously unrecognized connection between Rho family small G proteins and the ERK pathway.     

Role of the p21-activated kinases (PAKs) in influenza A virus replication

Influenza A virus infection stimulates a wide range of virus-supportive or antiviral mechanisms in host cells. p21-Activated kinase 1 (PAK1) is a serine/threonine kinase that regulates a number of fundamental cellular processes and has been implicated in the modulation of virus replication. Here, we investigated the role of PAK1 activation during influenza A virus infection and found that virus propagation corresponded to stimulated PAK1 phosphorylation. Moreover, transfection of the active form of PAK1 (PAK1-T423E) in A549 cells induced higher viral titers (∼10-fold differences) compared to that in the control vector or inactive PAK1 (PAK1-K299R)-transfected cells. PAK1-specific siRNA knockdown also resulted in 10–100-fold reductions in virus yields compared to that in the control siRNA-treatment (p < 0.05). We further showed that treatment with PAK18, a PAK1 peptide inhibitor, resulted in marked suppression of both ERK 1/2 phosphorylation and infectious virus production, which was comparable to that by U0126, a specific MEK/ERK inhibitor. These results provide evidence for the importance of PAK1 activation during influenza virus infection and its association with ERK in regulating virus replication. The present study also implicates PAK1 as a potential therapeutic target for managing influenza virus infections.     

Matrix-specific p21-activated kinase activation regulates vascular permeability in atherogenesis

Elevated permeability of the endothelium is thought to be crucial in atherogenesis because it allows circulating lipoproteins to access subendothelial monocytes. Both local hemodynamics and cytokines may govern endothelial permeability in atherosclerotic plaque. We recently found that p21-activated kinase (PAK) regulates endothelial permeability. We now report that onset of fluid flow, atherogenic flow profiles, oxidized LDL, and proatherosclerotic cytokines all stimulate PAK phosphorylation and recruitment to cell-cell junctions. Activation of PAK is higher in cells plated on fibronectin (FN) compared to basement membrane proteins in all cases. In vivo, PAK is activated in atherosclerosis-prone regions of arteries and correlates with FN in the subendothelium. Inhibiting PAK in vivo reduces permeability in atherosclerosis-prone regions. Matrix-specific PAK activation therefore mediates elevated vascular permeability in atherogenesis.     

Cdc42-independent activation and translocation of the cytostatic p21-activated protein kinase gamma-PAK by sphingosine.

Autophosphorylation of p21-activated protein kinase gamma-PAK is stimulated at 10 microM sphingosine in vitro and is maximal at 100 microM. Sites autophosphorylated on gamma-PAK in response to sphingosine are identical to those obtained with Cdc42(GTP). Autophosphorylation is paralleled by stimulation of gamma-PAK activity as measured with peptide and protein substrates. In 3T3-L1 cells, sphingosine stimulates the autophosphorylation and activity of gamma-PAK associated with the membrane-containing particulate fraction by 2.8-fold, but does not stimulate the activity of the soluble enzyme. Thus, gamma-PAK is activatable via a Cdc42-independent mechanism, suggesting sphingosine has a role in gamma-PAK activation under conditions of cell stress.     

Group I p21-activated kinases: Emerging roles in immune function and viral pathogenesis

Group I p21-activated kinases are a highly conserved three-member family of serine/threonine kinases that act as key effectors for the small GTPases Cdc42 and Rac. In man, these enzymes have been implicated in a wide range of biological processes and are beginning to draw the attention of the pharmaceutical industry as potential therapeutic targets in cancer and in inflammatory processes. In this review, we summarize basic properties of group I Paks and discuss recently uncovered roles for these kinases in immune function and in viral infection.