PAK1 Kinase Is Required for CXCL1-Induced Chemotaxis<sup><a href="#FN2" rid="FN2" class=" fn">†</a></sup>

Dingzhi Wang

Jiging Sai

Glendora Carter

Aristidis Sachpatzidis

Elias Lolis

Ann Richmond

Department of Veterans Affairs, Nashville, Tennessee 37232, Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, and Department of Pharmacology, Yale University, New Haven, Connecticut 06510

^{Author to whom correspondence should be addressed [telephone (615) 343-7777; fax (615) 343-4539; e-mail}ude.tlibrednav.liamcm@dnomhcir.nna]

^{Vanderbilt University.}

^{Department of Veterans Affairs.}

^{The first two authors contributed equally to this paper.}

^{Yale University.}

Abstract

The CXC subfamily of chemokines plays an important role in diverse processes, including inflammation, wound healing, growth regulation, angiogenesis, and tumorigenesis. The CXC chemokine CXCL1, or MGSA/GROα, is traditionally considered to be responsible for attracting leukocytes into sites of inflammation. To better understand the molecular mechanisms by which CXCL1 induces CXCR2-mediated chemotaxis, the signal transduction components involved in CXCL1-induced chemotaxis were examined. It is shown here that CXCL1 induces cdc42 and PAK1 activation in CXCR2-expressing HEK293 cells. Activation of the cdc42-PAK1 cascade is required for CXCL1-induced chemotaxis but not for CXCL1-induced intracellular Ca^{mobilization. Moreover, CXCL1 activation of PAK1 is independent of ERK1/2 activation, a conclusion based on the observations that the inhibition of MEK-ERK activation by expression of dominant negative ERK or by the MEK inhibitor, PD98059, has no effect on CXCL1-induced PAK1 activation or CXCL1-induced chemotaxis.}

Abstract

CXC chemokines¹ are crucial for the timely recruiting of specific populations of leukocytes to sites of tissue damage during the inflammatory responses. These chemokines are also important in angiogenesis, tumor formation, and tumor metastasis (1–6). In this subfamily, ELR-CXC chemokines with the amino acid sequence glutamic acid–leucine–arginine (the ELR motif) at the N-terminal domain of the ligands, including CXCL1 (melanoma growth stimulatory activity/growth regulated protein, MGSA/GRO), CXCL5 (epithelial-derived neutrophil-activating peptide 78, ENA-78), CXCR6 (granulocyte chemotactic protein-2, GCP-2), and CXCL8 (interleukin-8), are all neutrophil-activating CXC chemokines, which bind to the CXCR1, CXCR2 (CXC chemokine receptor 1 or 2), and/or Kaposi’s sarcoma human herpes virus 8 G protein-coupled receptor (1). CXCL1–3 and 5–8 bind to CXCR2 with high affinity, whereas CXCL6 and CXCL8 also bind CXCR1 with high affinity.

Our earlier studies demonstrated that CXCL1 induces activation of the transcription factor NF-κB through a Ras-MEKK1-MEK4/6-p38 MAP kinase cascade in melanocytes (7). This pathway is involved in CXCL1-induced melanocyte transformation (6). Activation of the phospholipase C–β/PKC/IP3 cascade is required for the CXC chemokine-induced intracellular calcium mobilization in neutrophils (8). Although the chemotactic response to CXCL1 and CXCL8 is well characterized, the signal transduction pathways for the chemotactic responses have not been fully elucidated.

The activated GTPases interact with specific targets that serve as effectors to regulate downstream signaling cascades. The Rho GTPase subfamily, including RhoA, RhoB, RhoC, Rac, and cdc42, has been implicated in the regulation of diverse cellular functions, including actin cytoskeletal dynamics, oxidant generation, transformation, membrane trafficking, apoptosis, transcription, and cell cycle control (9–12). Rac and cdc42 appear to be critical downstream components for the classic chemoattractant fMet-Leu-Phe (13–14). Significant Rac/cdc42 targets are the p21-activated kinases (PAKs).

PAKs play an important role in diverse cellular processes, including cytoskeletal rearrangements (15–19), growth, and apoptosis (20–22). PAKs are Ser/Thr protein kinases, which contain a p21 binding domain (PDB). PAK1 undergoes autophosphorylation and activation upon interacting with the active forms of the small GTPase (p21) Rac or Cdc42 (23). PAK activation is regulated by a variety of external stimuli that act through cell surface receptors, including G protein-coupled receptors (24), growth factor receptor tyrosine kinases (25), proinflammatory cytokine receptors (26), Fc receptors (27), and integrins (28–29). Moreover, a variety of chemoattractants induce rapid activation of PAKs (30). However, the role of PAK1 in chemokine gradient-directed cell movement (chemotaxis) has not been clearly delineated.

Mitogen-activated protein (MAP) kinases represent a point of convergence for cell surface signals regulating cell growth and division. MAP kinases are serine/threonine protein kinases. One member of the MAP kinase family is extra-cellular signal-related protein kinase (ERK). ERK is phosphorylated and activated by MAP kinase kinase (MEK1) (31), which in turn is phosphorylated and activated by the Raf (32). CXCL8 has also been demonstrated to activate the PI3-kinase/Ras/Raf cascade in neutrophils (33). Similarly, CXCL1 induces the activation of ERK through Ras/Raf1 dependent or independent pathways (34). However, it remains controversial whether ERK activation is required for the CXC ligand-induced chemotaxis (33, 35). Van Lint et al. reported that ERK activation is involved in IL-8-induced chemotaxis in neutrophils (35). However, Knall et al. reported that the regulation of cell migration by IL-8 is independent of ERK kinase and ERK activation because the ERK kinase inhibitor PD098059 had no effect on IL-8-induced cell migration of human neutrophils (33).

To determine what signal transducers are involved in CXCL1-induced chemotaxis, we used the HEK293 and RBL systems, which provide cellular models to characterize the signaling mechanisms of CXCR2, as such studies are notoriously difficult to perform in primary neutrophils, which express multiple chemokine receptors. Our findings demonstrate that CXCL1 induces PAK1 activation through cdc42. This cdc42–PAK1 cascade is required for CXCL1-induced chemotaxis. In contrast, we demonstrate that the CXCL1 induction of MEK-ERK1/2 is not involved in the CXCL1-induced chemotaxis. Moreover, cdc42–PAK1 and ERK are not required for the intracellular Ca^{mobilization induced by CXCL1.}

Footnotes

^{We are indebted to the NIH for support through Grants CA34590 (A.R.) and CA56704 (A.R.), to the Vanderbilt Ingram Cancer Center for Grant CA68485, and to the Department of Veterans Affairs for a Merit Award (A.R.), a Career Scientist Award (A.R.), and the GRECC Pilot Project (A.R.).}

^{Abbreviations: CXC, chemokine, chemokine with the first two conserved cysteine residues separated by an intervening amino acid; DMEM, Dulbecco’s modified Eagle’s medium; CXCL1 or MGSA/GRO, melanoma growth-stimulatory activity/growth-regulated protein; PAKs, p21-activated kinases; MBP, myelin basic protein; MAP, mitogen-activated protein; MEK, MAP kinase kinase; PBD, p21 binding domain.}

Footnotes

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