NOX1 loss-of-function genetic variants in patients with inflammatory bowel disease.

Genetic defects that affect intestinal epithelial barrier function can present with very early-onset inflammatory bowel disease (VEOIBD). Using whole-genome sequencing, a novel hemizygous defect in NOX1 encoding NAPDH oxidase 1 was identified in a patient with ulcerative colitis-like VEOIBD. Exome screening of 1,878 pediatric patients identified further seven male inflammatory bowel disease (IBD) patients with rare NOX1 mutations. Loss-of-function was validated in p.N122H and p.T497A, and to a lesser degree in p.Y470H, p.R287Q, p.I67M, p.Q293R as well as the previously described p.P330S, and the common NOX1 SNP p.D360N (rs34688635) variant. The missense mutation p.N122H abrogated reactive oxygen species (ROS) production in cell lines, ex vivo colonic explants, and patient-derived colonic organoid cultures. Within colonic crypts, NOX1 constitutively generates a high level of ROS in the crypt lumen. Analysis of 9,513 controls and 11,140 IBD patients of non-Jewish European ancestry did not reveal an association between p.D360N and IBD. Our data suggest that loss-of-function variants in NOX1 do not cause a Mendelian disorder of high penetrance but are a context-specific modifier. Our results implicate that variants in NOX1 change brush border ROS within colonic crypts at the interface between the epithelium and luminal microbes.

Toll-like receptor 3 L412F polymorphism promotes a persistent clinical phenotype in pulmonary sarcoidosis.

BACKGROUND/INTRODUCTION: Sarcoidosis is a multi-systemic disorder of unknown etiology, characterized by the presence of non-caseating granulomas in target organs. In 90% of cases, there is thoracic involvement. Fifty to seventy percent of pulmonary sarcoidosis patients will experience acute, self-limiting disease. For the subgroup of patients who develop persistent disease, no targeted therapy is currently available. AIM: To investigate the potential of the single nucleotide polymorphism (SNP), Toll-like receptor 3 Leu412Phe (TLR3 L412F; rs3775291), as a causative factor in the development of and in disease persistence in pulmonary sarcoidosis. To investigate the functionality of TLR3 L412F in vitro in primary human lung fibroblasts from pulmonary sarcoidosis patients. DESIGN: SNP-genotyping and cellular assays, respectively, were used to investigate the role of TLR3 L412F in the development of persistent pulmonary sarcoidosis. METHODS: Cohorts of Irish sarcoidosis patients (n = 228), healthy Irish controls (n = 263) and a secondary cohort of American sarcoidosis patients (n = 123) were genotyped for TLR3 L412F. Additionally, the effect of TLR3 L412F in primary lung fibroblasts from pulmonary sarcoidosis patients was quantitated following TLR3 activation in the context of cytokine and type I interferon production, TLR3 expression and apoptotic- and fibroproliferative-responses. RESULTS: We report a significant association between TLR3 L412F and persistent clinical disease in two cohorts of Irish and American Caucasians with pulmonary sarcoidosis. Furthermore, activation of TLR3 in primary lung fibroblasts from 412 F-homozygous pulmonary sarcoidosis patients resulted in reduced IFN-ß and TLR3 expression, reduced apoptosis- and dysregulated fibroproliferative-responses compared with TLR3 wild-type patients. DISCUSSION/CONCLUSION: This study identifies defective TLR3 function as a previously unidentified factor in persistent clinical disease in pulmonary sarcoidosis and reveals TLR3 L412F as a candidate biomarker.

ROS in gastrointestinal inflammation: Rescue Or Sabotage?

The intestine is composed of many distinct cell types that respond to commensal microbiota or pathogens with immune tolerance and proinflammatory signals respectively. ROS produced by mucosa-resident cells or by newly recruited innate immune cells are essential for antimicrobial responses and regulation of signalling pathways including processes involved in wound healing. Impaired ROS production due to inactivating patient variants in genes encoding NADPH oxidases as ROS source has been associated with Crohn's disease and pancolitis, whereas overproduction of ROS due to up-regulation of oxidases or altered mitochondrial function was linked to ileitis and ulcerative colitis. Here, we discuss recent advances in our understanding of how maintaining a redox balance is crucial to preserve gut homeostasis. LINKED ARTICLES: This article is part of a themed section on Redox Biology and Oxidative Stress in Health and Disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.12/issuetoc.

P21-activated kinase is required for mitotic progression and regulates Plk1.

P21-activated kinases (Paks), a family of serine/threonine kinases, are effectors of the Rho GTPases Cdc42 and Rac1. Mammalian Pak1 and Pak homologs in simple eukaryotes are implicated in controlling G(2)/M transition and/or mitosis. Another serine/threonine kinase, polo-like kinase 1 (Plk1), is an important regulator of mitotic events, such as centrosome maturation, mitotic entry, spindle formation, sister chromatid cohesion and cytokinesis. Plk1 phosphorylation is thought to be one of the critical regulatory events leading to these Plk1-mediated functions. We show here that Pak1 is required for cell proliferation, mitotic progression and Plk1 activity in HeLa cells. Gain or loss of Pak function directly impacted phosphorylation and activity of Plk1. Phosphorylation of Plk1 on Ser 49 is important for metaphase-associated events. Inhibition of Pak activity leads to delay in G(2)/M progression and abnormal spindle formation, mirroring some attributes of Plk1 deregulation. Our results reveal a role for Pak in regulating Plk1 activity and mitotic progression, and connect Pak to the complex protein interaction network enabling cell division.

Evidence that 12-lipoxygenase product 12-hydroxyeicosatetraenoic acid activates p21-activated kinase.

The effect of 12-hydroxyeicosatetraenoic acid (12-HETE), an arachidonic acid metabolite of 12-lipoxygenase, to activate p21(Rac/Cdc42)-activated kinase (PAK1) was studied in a Chinese hamster ovary fibroblast cell line overexpressing the rat vascular type-1a angiotensin II receptor (CHO-AT(1a)). 12-HETE (0.1 microM) treatment induced a time-dependent activation of PAK1, with a peak effect at 10 min (335 +/- 16% of control; n=3, P<0.001). The stimulatory effect of 12-HETE on PAK1 activity was dose-dependent, with the maximal activation at 0.01 microM (350+/-15% of control; n=3, P<0.001). A PAK1 fragment encoding the Cdc42/Rac binding domain (amino acid residues 67-150 of hPAK1 termed PBD), was transfected into CHO-AT(1a) cells. PBD transfection markedly reduced 12-HETE-induced PAK1 activation. Furthermore, transfection of dominant negative Cdc42 and Rac1 inhibited 12-HETE-induced PAK1, strongly suggesting that Cdc42 and Rac1 are the upstream activators of 12-HETE-induced PAK1 activation. Low concentrations (1.5 microM) of LY294002, a highly specific inhibitor of phosphoinositide 3-kinase (PI-3K), abolished 12-HETE-induced PAK1 activation, suggesting that PI-3K activation is upstream of 12-HETE-induced PAK1 activation. Transfection of dominant negative PAK1 blocked 12-HETE-induced PAK1, cJun N-terminal kinase (JNK1) and extracellular-signal-regulated kinase (ERK) activity, while transfection of constitutively active PAK1 stimulated PAK1, JNK1 and ERK activity, suggesting that PAK1 is an upstream activator of 12-HETE-induced JNK1 and ERK activation in these cells. We conclude that 12-HETE can activate Cdc42, Rac1 and PI-3K, which then participate as upstream signalling molecules for PAK1 and JNK1 activation.

Rho GTPase signaling in inflammation and transformation.

Intracellular Rho GTPases provide an important regulatory mechanism to connect cell-surface-generated signals with the nucleus. By cycling between the active (guanosine 5'-triphosphate [GTP]) and inactive (guanosine 5'-diphosphate) state, these GTP-binding proteins control cellular functions ranging from dynamic actin remodeling and activation of transcription factors to cell-cycle progression and cellular transformation. Their contribution to these very diverse processes makes them an essential part of cell movement, growth, and apoptosis. Upstream regulatory mechanisms, as well as a variety of downstream effector molecules, enable Rho GTPases to act in a specific, orchestrated manner, dictating cellular responses. In this article, I review my laboratory's work centering on the goal of determining how specificity in intracellular signaling is achieved and identifying molecular mechanisms of Rho GTPase-mediated processes in innate immune and transformed cells.

Endogenous, hyperactive Rac3 controls proliferation of breast cancer cells by a p21-activated kinase-dependent pathway.

Uncontrolled cell proliferation is a major feature of cancer. Experimental cellular models have implicated some members of the Rho GTPase family in this process. However, direct evidence for active Rho GTPases in tumors or cancer cell lines has never been provided. In this paper, we show that endogenous, hyperactive Rac3 is present in highly proliferative human breast cancer-derived cell lines and tumor tissues. Rac3 activity results from both its distinct subcellular localization at the membrane and altered regulatory factors affecting the guanine nucleotide state of Rac3. Associated with active Rac3 was deregulated, persistent kinase activity of two isoforms of the Rac effector p21-activated kinase (Pak) and of c-Jun N-terminal kinase (JNK). Introducing dominant-negative Rac3 and Pak1 fragments into a breast cancer cell line revealed that active Rac3 drives Pak and JNK kinase activities by two separate pathways. Only the Rac3-Pak pathway was critical for DNA synthesis, independently of JNK. These findings identify Rac3 as a consistently active Rho GTPase in human cancer cells and suggest an important role for Rac3 and Pak in tumor growth.

Toll-like receptor 2-mediated NF-kappa B activation requires a Rac1-dependent pathway.

Mammalian Toll-like receptors (TLRs) are expressed on innate immune cells and respond to the membrane components of Gram-positive or Gram-negative bacteria. When activated, they convey signals to transcription factors that orchestrate the inflammatory response. However, the intracellular signaling events following TLR activation are largely unknown. Here we show that TLR2 stimulation by Staphylococcus aureus induces a fast and transient activation of the Rho GTPases Rac1 and Cdc42 in the human monocytic cell line THP-1 and in 293 cells expressing TLR2. Dominant-negative Rac1N17, but not dominant-negative Cdc42N17, block nuclear factor-kappa B (NF-kappa B) transactivation. S. aureus stimulation causes the recruitment of active Rac1 and phosphatidylinositol-3 kinase (PI3K) to the TLR2 cytosolic domain. Tyrosine phosphorylation of TLR2 is required for assembly of a multiprotein complex that is necessary for subsequent NF-kappa B transcriptional activity. A signaling cascade composed of Rac1, PI3K and Akt targets nuclear p65 transactivation independently of I kappa B alpha degradation. Thus Rac1 controls a second, I kappa B-independent, pathway to NF-kappa B activation and is essential in innate immune cell signaling via TLR2.

Enhanced expression of neutrophil NADPH oxidase components in intestine of rats after burn injury.

We have evaluated the accumulation of neutrophils in the gut and their infiltration into the intestinal extravascular spaces in rats subjected to a 25% total body surface area scald burn. The accumulation of neutrophils was assessed via measurements of myeloperoxidase (MPO) activity in the intestinal homogenates, and the immunohistochemical localization of neutrophil NADPH oxidase component proteins (p47phox and p67phox) within the intestinal extravascular spaces determined neutrophil tissue infiltration. MPO measurements demonstrated a 12- and 21-fold increase above the control value in the intestinal tissue at day 1 and day 3 post-burn, respectively, suggesting that a substantial total tissue accumulation of neutrophils occurs in the gut after burn injury. The immunohistochemical staining procedures showed both a definitive presence of the neutrophil in the intestinal extravascular spaces and an enhanced immunoreactivity in neutrophils accumulating in intestine after burn injury. There was no evidence of either the presence of neutrophils in the extravascular regions or any significant neutrophil immunoreactivity to NADPH oxidase component proteins in the intestines of sham control rats. These findings indicate that burn injury causes an enhanced migration of circulating neutrophils into the intestinal interstitial spaces and an upregulation of NADPH oxidase activity in the infiltrating neutrophils.

Activation of the leukocyte NADPH oxidase by protein kinase C in a partially recombinant cell-free system.

The leukocyte NADPH oxidase is an enzyme present in phagocytes and B lymphocytes that when activated catalyzes the production of O-2 from oxygen at the expense of NADPH. A correlation between the activation of the oxidase and the phosphorylation of p47(PHOX), a cytosolic oxidase component, is well recognized in whole cells, and direct evidence for a relationship between the phosphorylation of this oxidase component and the activation of the oxidase has been obtained in a number of cell-free systems containing neutrophil membrane and cytosol. Using superoxide dismutase-inhibitable cytochrome c reduction to quantify O-2 production, we now show that p47(PHOX) phosphorylated by protein kinase C activates the NADPH oxidase not only in a cell-free system containing neutrophil membrane and cytosol, but also in a system in which the cytosol is replaced by the recombinant proteins p67(PHOX), Rac2, and phosphorylated p47(PHOX), suggesting that neutrophil plasma membrane plus those three cytosolic proteins are both necessary and sufficient for oxidase activation. In both the cytosol-containing and recombinant cell-free systems, however, activation by SDS yielded greater rates of O-2 production than activation by protein kinase C-phosphorylated p47(PHOX), indicating that a system that employs protein kinase C-phosphorylated p47(PHOX) as the sole activating agent, although more physiological than the SDS-activated system, is nevertheless incomplete.

The p21Rac/Cdc42-activated kinases (PAKs).

The p21-activated kinases (PAKs) are mammalian Rac/Cdc42-associated serine/threonine protein kinases. They contain diverse structural regulatory elements that allow them not only to participate as effectors in signaling processes initiated by activated GTPases but also in signal transduction events mediated by Src3 homology domains (SH3) or caspase-mediated proteolytic cleavage. The biological functions of PAK protein kinases result from the interplay of N- and C-terminal-mediated protein-protein interactions and signals derived from phosphorylation of downstream substrates. The potential regulation of microbial killing, stress responses, apoptosis, and cell motility by PAKs suggest it may be a therapeutically useful target in a number of disease states.

Structural requirements for PAK activation by Rac GTPases.

The Rho family GTPases, Rac1 and Rac2, regulate a variety of cellular functions including cytoskeletal reorganization, the generation of reactive oxygen species, G1 cell cycle progression and, in concert with Ras, oncogenic transformation. Among the many putative protein targets identified for Rac (and/or Cdc42), the Ser/Thr kinase p21-activated kinase (PAK) is a prime candidate for mediating some of Rac's cellular effects. This report shows that Rac1 binds to and stimulates the kinase activity of PAK1 approximately 2- and 4-5-fold, respectively, better than Rac2. Mutational analysis was employed to determine the structural elements on Rac and PAK that are important for optimal binding and activation. The most notable difference between the highly homologous Rac isomers is the composition of their C-terminal polybasic domains. Mutation of these six basic residues in Rac1 to neutral amino acids dramatically decreased the ability of Rac1 to bind PAK1 and almost completely abolished its ability to stimulate PAK activity. Moreover, replacing the highly charged polybasic domain of Rac1 with the less charged domain of Rac2 (and vice versa) completely reversed the PAK binding/activation properties of the two Rac isomers. Thus, polybasic domain differences account for the disparate abilities of Rac1 and Rac2 to activate PAK. PAK proteins also contain a basic region, consisting of three contiguous lysine residues (Lys66-Lys67-Lys68), which lies outside of the previously identified Cdc42/Rac-binding domain. Mutation of these Lys residues to neutral residues decreased PAK binding to activated Rac1 and Rac2 (but not Cdc42) and greatly reduced PAK1 activation by Rac1, Rac2, and Cdc42 proteins in vivo. In contrast, mutation of lysines 66-68 to basic Arg residues did not decrease (and in some cases enhanced) the ability of Rac1, Rac2, and Cdc42 to bind and activate PAK1. Our studies suggest that the polybasic domain of Rac is a novel effector domain that may allow the two Rac isomers to activate different effector proteins. In addition, our results indicate that a basic region in PAK is required for PAK activation and that binding of Rac/Cdc42 to PAK is not sufficient for kinase activation.

Two signaling mechanisms for activation of alphaM beta2 avidity in polymorphonuclear neutrophils.

Circulating polymorphonuclear neutrophils (PMN) are quiescent, nonadherent cells that rapidly activate at sites of inflammation, where they develop the capacity to perform a repertoire of functions that are essential for host defense. Induction of integrin-mediated adhesion, which requires an increase in integrin avidity, is critical for the development of these effector functions. Although a variety of stimuli can activate integrins in PMN, the signaling cascades involved are unclear. Phosphatidylinositol (PI) 3-kinase has been implicated in integrin activation in a variety of cells, including PMN. In this work, we have examined activation of the PMN integrin alphaM beta2, assessing both adhesion and generation of the epitope recognized by the activation-specific antibody CBRM1/5. We have found that PI 3-kinase has a role in activation of alphaM beta2 by immune complexes, but we have found no role for it in alphaM beta2 activation by ligands for trimeric G protein-coupled receptors, including formylmethionylleucylphenylalanine (fMLP), interleukin-8, and C5a. Cytochalasin D inhibition suggests a role for the actin cytoskeleton in immune complex activation of alphaM beta2, but cytochalasin has no effect on fMLP-induced activation. Similarly, immune complex activation of the Rac/Cdc42-dependent serine/threonine kinase Pak1 is blocked by PI 3-kinase inhibitors, but fMLP-induced activation is not. These results demonstrate that two signaling pathways exist in PMN for activation of alphaM beta2. One, induced by FcgammaR ligation, is PI 3-kinase-dependent and requires the actin cytoskeleton. The second, initiated by G protein-linked receptors, is PI 3-kinase-independent and cytochalasin-insensitive. Pak1 may be in a final common pathway leading to activation of alphaM beta2.

A GTPase-independent mechanism of p21-activated kinase activation. Regulation by sphingosine and other biologically active lipids.

p21-activated kinases (PAKs) are serine/threonine kinases that have been identified as targets for the small GTPases Rac and Cdc42. PAKs have been implicated in cytoskeletal regulation, stimulation of mitogen-activated protein kinase cascades, and in control of the phagocyte NADPH oxidase. Membrane targeting of PAK1 induced increased kinase activity in a GTPase-independent manner, suggesting that other mechanisms for PAK regulation exist. We observed concentration- and time-dependent activation of PAK1 by sphingosine and several related long chain sphingoid bases but not by ceramides or a variety of other lipids. Although phospholipids were generally ineffective, phosphatidic acid and phosphatidylinositol also had stimulatory effects on PAK1. Lipid stimulation induced a similar level of PAK1 activity as did stimulation by GTPases, and the patterns of PAK1 autophosphorylation determined after partial tryptic digestion and two-dimensional peptide analysis were similar with each class of activator. Lipid stimulation of PAK1 activity was dependent upon intact PAK kinase activity, as indicated by studies with a kinase-dead PAK1 mutant. Treatment of COS-7 cells expressing wild type PAK1 with sphingosine, fumonisin B, or sphingomyelinase, all of which are able to elevate the levels of free sphingosine, induced increased activity of PAK1 as determined using a p47(phox) peptide substrate. Studies using PAK1 mutants suggest that lipids act at a site overlapping or identical to the GTPase-binding domain on PAK. The inactive sphingosine derivative N,N-dimethylsphingosine was an effective inhibitor of PAK1 activation in response to either sphingosine or Cdc42. Our results demonstrate a novel GTPase-independent mechanism of PAK activation and, additionally, suggest that PAK(s) may be important mediators of the biological effects of sphingolipids.

Human p21-activated kinase (Pak1) regulates actin organization in mammalian cells.

BACKGROUND: The Rho family GTPases Cdc42, Rac1 and RhoA regulate the reorganization of the actin cytoskeleton induced by extracellular signals such as growth factors. In mammalian cells, Cdc42 regulates the formation of filopodia, whereas Rac regulates lamellipodia formation and membrane ruffling, and RhoA regulates the formation of stress fibers. Recently, the serine/threonine protein kinase p65(pak) autophosphorylates, thereby increasing its catalytic activity towards exogenous substrates. This kinase is therefore a candidate effector for the changes in cell shape induced by growth factors. RESULTS: Here, we report that the microinjection of activated Pak1 protein into quiescent Swiss 3T3 cells induces the rapid formation of polarized filopodia and membrane ruffles. The prolonged overexpression of Pak1 amino-terminal mutants that are unable to bind Cdc42 or Rac1 results in the accumulation of filamentous actin in large, polarized membrane ruffles and the formation of vinculin-containing focal complexes within these structures. This phenotype resembles that seen in motile fibroblasts. The amino-terminal Pak1 mutant displays enhanced binding to the adaptor protein Nck, which contains three Src-homology 3 (SH3) domains. Mutation of a proline residue within a conserved SH3-binding region at the amino terminus of Pak1 interferes with SH3-protein binding and alters the effects of Pak1 on the cytoskeleton. CONCLUSIONS: These results indicate that Pak1, acting through a protein that contains an SH3 domain, regulates the structure of the actin cytoskeleton in mammalian cells, and may serve as an effector for Cdc42 and/or Rac1 in promoting cell motility.

p21-activated kinase has substrate specificity similar to Acanthamoeba myosin I heavy chain kinase and activates Acanthamoeba myosin I.

Acanthamoeba class I myosins are unconventional, single-headed myosins that express actin-activated Mg2+-ATPase and in vitro motility activities only when a single serine or threonine in the heavy chain is phosphorylated by myosin I heavy chain kinase (MIHCK). Some other, but not most, class I myosins have the same consensus phosphorylation site sequence, and the two known class VI myosins have a phosphorylatable residue in the homologous position, where most myosins have an aspartate or glutamate residue. Recently, we found that the catalytic domain of Acanthamoeba MIHCK has extensive sequence similarity to the p21-activated kinase (PAK)/STE20 family of kinases from mammals and yeast, which are activated by small GTP-binding proteins. The physiological substrates of the PAK/STE20 kinases are not well characterized. In this paper we show that PAK1 has similar substrate specificity as MIHCK when assayed against synthetic substrates and that PAK1 phosphorylates the heavy chain (1 mol of P(i) per mol) and activates Acanthamoeba myosin I as MIHCK does. These results, together with the known involvement of Acanthamoeba myosin I, yeast myosin I, STE20, PAK, and small GTP-binding proteins in membrane- and cytoskeleton-associated morphogenetic transformations and activities, suggest that myosins may be physiological substrates for the PAK/STE20 family and thus mediators of these events.

The renaturable 69- and 63-kDa protein kinases that undergo rapid activation in chemoattractant-stimulated guinea pig neutrophils are p21-activated kinases.

Neutrophils stimulated with the chemoattractant fMet-Leu-Phe (fMLP) are known to exhibit rapid activation of four protein kinases with molecular masses of approximately 69, approximately 63, approximately 49, and approximately 40-kDa. Activation of these kinases is blocked by antagonists of phosphatidylinositol 3-kinase and type 1 and/or type 2A protein phosphatases. These enzymes can be detected by their ability to undergo renaturation and catalyze the phosphorylation of a peptide substrate that corresponds to amino acid residues 297-331 of the 47-kDa subunit of the NADPH-oxidase complex fixed within a gel. In this report, we demonstrate that an antibody generated to a fusion protein containing amino acid residues 175-306 of p21-activated protein kinase 1 (Pak1) reacts with three proteins in guinea pig neutrophils with molecular masses in the 60-70-kDa range during Western blotting. This antibody immunoprecipitates both the 69- and 63-kDa renaturable kinases from lysates of stimulated cells along with a minor 60-kDa kinase. No activities were observed for any of these enzymes in immunoprecipitates from unstimulated neutrophils. However, addition of ATP and activated Rac 1 or Cdc42 to immunoprecipitates from unstimulated cells resulted in the stimulation of two renaturable kinases with molecular masses in the 69- and 63-kDa range. These immunoprecipitates also contained two novel protein kinases with masses of approximately49 and 40 kDa that were selectively activated by Cdc42. In contrast, the 69- and 63-kDa kinases were not immunoprecipitated from lysates of stimulated neutrophils with an antibody to Pak2 or with nonimmune serum. These data indicate that the renaturable 69- and 63-kDa kinases are Paks and reveal some of the upstream events that are necessary for the rapid activation of this family of protein kinases in neutrophils.

Interaction of the Nck adapter protein with p21-activated kinase (PAK1).

The p21-activated kinases (PAKs) link G protein-coupled receptors and growth factor receptors (S. Dharmawardhane, R. H. Daniels, and G. M. Bokoch, submitted for publication) to activation of MAP kinase cascades and to cytoskeletal reorganization (M. A. Sells, U. G. Knaus, D. Ambrose, S. Bagrodia, G. M. Bokoch, and J. Chernoff, submitted for publication). The proteins that interact with PAK to mediate its cellular effects and to couple it to upstream receptors are unknown. We describe here a specific interaction of the Nck adapter molecule with PAK1 both in vitro and in vivo. PAK1 and Nck associate in COS-7 and Swiss 3T3 cells constitutively, but this interaction is strengthened upon platelet-derived growth factor receptor stimulation. We show that Nck binds to PAK1 through its second Src homology 3 (SH3) domain, while PAK1 interacts with Nck via the first proline-rich SH3 binding motif at its amino terminus. The interaction of active PAK1 with Nck leads to the phosphorylation of Nck at multiple sites. Association of Nck with PAK1 may serve to link this important regulatory kinase to cell activation by growth factor receptors.