Phosphorylation of RhoGDI1 by p21-activated kinase 2 mediates basic fibroblast growth factor-stimulated neurite outgrowth in PC12 cells

We previously showed that p21-activated kinase 2 (PAK2), a major PAK isoform expressed in PC12 cells, mediates neurite outgrowth via Rac1 GTPase. RhoGDI1 forms a complex with Rac1, resulting in its inhibition. Rac1 activation requires dissociation from RhoGDI1. Here, we show that PAK2 mediates basic fibroblast growth factor (bFGF)-stimulated neurite outgrowth via phosphorylation of RhoGDI1. RhoGDI1 was shown to be associated with PAK2, with phosphorylation of Ser34 and Ser101 by active PAK2 evident in vitro and in vivo. A RhoGDI1 phosphomimetic mutant (S34E/S101E) was dissociated from Rac1/Cdc42, whereas the wild-type or a nonphosphorylatable mutant (S34A/S101A) formed a tight complex. Consistent with this, PC12 cells expressing the phosphomimetic mutant displayed Rac1/Cdc42 activation in response to bFGF stimulation. Neurite outgrowth was also enhanced in PC12 cells expressing the phosphomimetic mutant. These results suggest that PAK2-mediated RhoGDI1 phosphorylation stimulates dissociation of RhoGDI1-Rac1/Cdc42 complex accompanied by relief of inhibitory effect on Rac1/Cdc42, which promotes neuronal differentiation.     

LKB1 Suppresses p21-activated Kinase-1 (PAK1) by Phosphorylation of Thr109 in the p21-binding Domain

The serine/threonine protein kinase LKB1 is a tumor suppressor gene mutated in Peutz-Jeghers syndrome patients. The mutations are found also in several types of sporadic cancer. Although LKB1 is implicated in suppression of cell growth and metastasis, the detailed mechanisms have not yet been elucidated. In this study, we investigated the effect of LKB1 on cell motility, whose acquisition occurs in early metastasis. The knockdown of LKB1 enhanced cell migration and PAK1 activity in human colon cancer HCT116 cells, whereas forced expression of LKB1 in Lkb1-null mouse embryonic fibroblasts suppressed PAK1 activity and PAK1-mediated cell migration simultaneously. Notably, LKB1 directly phosphorylated PAK1 at Thr¹⁰⁹ in the p21-binding domain in vitro. The phosphomimetic T109E mutant showed significantly lower protein kinase activity than wild-type PAK1, suggesting that the phosphorylation at Thr¹⁰⁹ by LKB1 was responsible for suppression of PAK1. Consistently, the nonphosphorylatable T109A mutant was resistant to suppression by LKB1. Furthermore, we found that PAK1 was activated in the hepatocellular carcinomas and the precancerous liver lesions of Lkb1(+/−) mice. Taken together, these results suggest that PAK1 is a direct downstream target of LKB1 and plays an essential role in LKB1-induced suppression of cell migration.     

NCK and PAK participate in the signaling pathway by which vascular endothelial growth factor stimulates the assembly of focal adhesions

Vascular endothelial growth factor (VEGF)-induced endothelial cell migration is a key step in the angiogenic response and is mediated, in part, by an accelerated rate of focal adhesion complex assembly and disassembly. We investigated the signaling pathway by which VEGF regulates focal adhesion complex assembly by examining the signaling proteins involved. VEGF stimulated the tyrosine phosphorylation of the SH2 domain-containing signaling proteins NCK and CRK in human umbilical vein endothelial cells. The signaling pathways that couple the kinase insert domain-containing receptor to NCK and CRK is most likely mediated by another cellular protein, as NCK and CRK were tyrosine-phosphorylated in response to VEGF in cells expressing receptors mutated at each of several candidate SH2 domain-interacting cytosolic tyrosines. In the absence of VEGF treatment, NCK (but not CRK) associated with the p21 GTPase-activated kinase PAK. PAK catalytic activity was augmented after VEGF treatment; an association of PAK with 60- and 90-kDa tyrosine-phosphorylated proteins accompanied this. VEGF stimulated the recruitment of PAK to focal adhesions, and FAK immunoprecipitated with both NCK and PAK in VEGF-treated (but not untreated) human umbilical vein endothelial cells. Inhibition of NCK protein expression using antisense oligonucleotides led to the inhibition of both VEGF-induced focal adhesion assembly and VEGF-induced cell migration, demonstrating a necessary role of NCK in these cellular responses.     

PAK1 regulates myosin II-B phosphorylation, filament assembly, localization and cell chemotaxis

Serine/threonine p21-activated kinase is an effector of Rac with a key role in the regulation of cytoskeletal organization. Non-muscle myosin II is a molecular motor, which is an important component of the cytoskeleton. Non-muscle myosin II-B plays a major role in cell motility and chemotaxis. We investigated the role of Rac and p21-activated kinase 1 (PAK1) in the regulation of myosin II-B in prostate cancer cells in response to epidermal growth factor (EGF) stimulation. We found that both Rac and PAK1 affect EGF-dependent non-muscle heavy chain II-B localization and cell morphology. We further found that a dominant negative mutant of PAK1 significantly inhibits EGF-dependent myosin II-B heavy chains phosphorylation and filament disassembly. Furthermore, cells expressing the dominant negative mutant exhibited an increase in EGF-dependent myosin light chain phosphorylation and diminished chemotaxis towards EGF. To our knowledge this is the first report exploring the role of PAK1 in the regulation of both non-muscle myosin II-B heavy chains and light chains. Furthermore, the data presented here suggest that PAK1 plays a crucial role in the regulation of cell morphology and chemotaxis by regulating the phosphorylation and cellular localization of myosin II-B.     

Phosphorylation of VE-cadherin controls endothelial phenotypes via p120-catenin coupling and Rac1 activation

To establish the role of vascular endothelial (VE)-cadherin in the regulation of endothelial cell functions, we investigated the effect of phosphorylation of a VE-cadherin site sought to be involved in p120-catenin binding on vascular permeability and endothelial cell migration. To this end, we introduced either wild-type VE-cadherin or Y658 phosphomimetic (Y658E) or dephosphomimetic (Y658F) VE-cadherin mutant constructs into an endothelial cell line (rat fat pad endothelial cells) lacking endogenous VE-cadherin. Remarkably, neither wild-type- nor Y658E VE-cadherin was retained at cell-cell contacts because of p120-catenin preferential binding to N-cadherin, resulting in the targeting of N-cadherin to cell-cell junctions and the exclusion of VE-cadherin. However, Y658F VE-cadherin was able to bind p120-catenin and to localize at adherence junctions displacing N-cadherin. This resulted in an enhanced barrier function and a complete abrogation of Rac1 activation and lamellipodia formation, thereby inhibiting cell migration. These findings demonstrate that VE-cadherin, through the regulation of Y658 phosphorylation, competes for junctional localization with N-cadherin and controls vascular permeability and endothelial cell migration.     

Honokiol Inhibits Non-Small Cell Lung Cancer Cell Migration by Targeting PGE2-Mediated Activation of β-Catenin Signaling

Lung cancer remains a leading cause of death due to its metastasis to distant organs. We have examined the effect of honokiol, a bioactive constituent from the Magnolia plant, on human non-small cell lung cancer (NSCLC) cell migration and the molecular mechanisms underlying this effect. Using an in vitro cell migration assay, we found that treatment of A549, H1299, H460 and H226 NSCLC cells with honokiol resulted in inhibition of migration of these cells in a dose-dependent manner, which was associated with a reduction in the levels of cyclooxygenase-2 (COX-2) and prostaglandin E₂ (PGE₂). Celecoxib, a COX-2 inhibitor, also inhibited cell migration. Honokiol inhibited PGE₂-enhanced migration of NSCLC cells, inhibited the activation of NF-κB/p65, an upstream regulator of COX-2, in A549 and H1299 cells, and treatment of cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, also inhibited migration of NSCLC cells. PGE₂ has been shown to activate β-catenin signaling, which contributes to cancer cell migration. Therefore, we checked the effect of honokiol on β-catenin signaling. It was observed that treatment of NSCLC cells with honokiol degraded cytosolic β-catenin, reduced nuclear accumulation of β-catenin and down-regulated matrix metalloproteinase (MMP)-2 and MMP-9, which are the down-stream targets of β-catenin and play a crucial role in cancer cell metastasis. Honokiol enhanced: (i) the levels of casein kinase-1α, glycogen synthase kinase-3β, and (ii) phosphorylation of β-catenin on critical residues Ser⁴⁵, Ser^33/37 and Thr⁴¹. These events play important roles in degradation or inactivation of β-catenin. Treatment of celecoxib also reduced nuclear accumulation of β-catenin in NSCLC cells. FH535, an inhibitor of Wnt/β-catenin pathway, inhibited PGE₂-enhanced cell migration of A549 and H1299 cells. These results indicate that honokiol inhibits non-small cell lung cancer cells migration by targeting PGE₂-mediated activation of β-catenin signaling.     

CD148 Tyrosine Phosphatase Promotes Cadherin Cell Adhesion

CD148 is a transmembrane tyrosine phosphatase that is expressed at cell junctions. Recent studies have shown that CD148 associates with the cadherin/catenin complex and p120 catenin (p120) may serve as a substrate. However, the role of CD148 in cadherin cell-cell adhesion remains unknown. Therefore, here we addressed this issue using a series of stable cells and cell-based assays. Wild-type (WT) and catalytically inactive (CS) CD148 were introduced to A431D (lacking classical cadherins), A431D/E-cadherin WT (expressing wild-type E-cadherin), and A431D/E-cadherin 764AAA (expressing p120-uncoupled E-cadherin mutant) cells. The effects of CD148 in cadherin adhesion were assessed by Ca²⁺ switch and cell aggregation assays. Phosphorylation of E-cadherin/catenin complex and Rho family GTPase activities were also examined. Although CD148 introduction did not alter the expression levels and complex formation of E-cadherin, p120, and β-catenin, CD148 WT, but not CS, promoted cadherin contacts and strengthened cell-cell adhesion in A431D/E-cadherin WT cells. This effect was accompanied by an increase in Rac1, but not RhoA and Cdc42, activity and largely diminished by Rac1 inhibition. Further, we demonstrate that CD148 reduces the tyrosine phosphorylation of p120 and β-catenin; causes the dephosphorylation of Y529 suppressive tyrosine residue in Src, a well-known CD148 site, increasing Src activity and enhancing the phosphorylation of Y228 (a Src kinase site) in p120, in E-cadherin contacts. Consistent with these findings, CD148 dephosphorylated both p120 and β-catenin in vitro. The shRNA-mediated CD148 knockdown in A431 cells showed opposite effects. CD148 showed no effects in A431D and A431D/E-cadherin 764AAA cells. In aggregate, these findings provide the first evidence that CD148 promotes E-cadherin adhesion by regulating Rac1 activity concomitant with modulation of p120, β-catenin, and Src tyrosine phosphorylation. This effect requires E-cadherin and p120 association.     

Direct Interaction between AR and PAK6 in Androgen-Stimulated PAK6 Activation

A p21-activated kinase 6 (PAK6) was previously identified to be an androgen receptor (AR) interacting protein through a yeast two-hybrid screening. We used hormone responsive prostate cancer LAPC4 and LNCap cell lines as models to study the signaling events associated with androgen stimulation and PAK6. An androgen-stimulated PAK6 kinase activation was observed in LAPC4 cells expressing endogenous PAK6 and in LNCap cells ectopically expressing a wild type PAK6. This activation was likely mediated through a direct interaction between AR and PAK6 since siRNA knock-down of AR in LAPC4 cells downregulated androgen-stimulated PAK6 activation. In addition, LNCap cells expressing a non-AR-interacting PAK6 mutant exhibited dampened androgen-stimulated kinase activation. As a consequence of androgen-stimulated activation, PAK6 was phosphorylated at multiple serine/threonine residues including the AR-interacting domain of PAK6. Furthermore, androgen-stimulation promoted prostate cancer cell motility and invasion were demonstrated in LNCap cells ectopically expressing PAK6-WT. In contrast, LNCap expressing non-AR-interacting mutant PAK6 did not respond to androgen stimulation with increased cell motility and invasion. Our results demonstrate that androgen-stimulated PAK6 activation is mediated through a direct interaction between AR and PAK6 and PAK6 activation promotes prostate cancer cells motility and invasion.     

Two species of human CRK cDNA encode proteins with distinct biological activities.

Two distinct human CRK cDNAs, designated CRK-I and CRK-II, were isolated from human embryonic lung cells by polymerase chain reaction and by screening of a human placenta cDNA library, respectively. CRK-I differed from CRK-II in that it lacked a 170-nucleotide sequence, suggesting that CRK-I and CRK-II were the products of alternative splicing. The amino acid sequences deduced from these two cDNAs differed in the carboxyl termini and contained one SH2 and either one or two SH3 domains. RNAse protection analysis demonstrated both CRK-I and CRK-II mRNAs in various human cells. Three CRK proteins, of 42, 40, and 28 kDa, were identified in human embryonic lung cells by means of antibodies against the SH2 region and the SH3 region of the bacterially expressed CRK-I protein. Transient expression of CRK-I and CRK-II cDNAs in COS7 cells showed that the former encoded the 28-kDa protein and the latter encoded the 40- and 42-kDa proteins. All human cell lines so far examined expressed the 40-kDa protein; however, expression of the 28- and the 42-kDa proteins was variable. In a comparison of the biological activity of the two human CRK proteins, both proteins were stably expressed in rat 3Y1 cells. All cell lines expressing CRK-I protein showed altered morphology, proliferated in soft agar, and grew as massive tumors in nude mice. Although CRK-II-expressing cells showed a slight morphologic change, they did not make colonies in soft agar or grow in nude mice. These results demonstrate that the two species of human CRK cDNA encode proteins which differ in their biological activities.     

p85 beta-PIX is required for cell motility through phosphorylations of focal adhesion kinase and p38 MAP kinase

Lysophosphatidic acid (LPA) mediates diverse biological responses, including cell migration, through the activation of G-protein-coupled receptors. Recently, we have shown that LPA stimulates p21-activated kinase (PAK) that is critical for focal adhesion kinase (FAK) phosphorylation and cell motility. Here, we provide the direct evidence that p85 beta-PIX is required for cell motility of NIH-3T3 cells by LPA through FAK and p38 MAP kinase phosphorylations. LPA induced p85 beta-PIX binding to FAK in NIH-3T3 cells that was inhibited by pretreatment of the cells with phosphoinositide 3-kinase inhibitor, LY294002. Furthermore, the similar inhibition of the complex formation was also observed, when the cells were transfected with either p85 beta-PIX mutant that cannot bind GIT or dominant negative mutants of Rac1 (N17Rac1) and PAK (PAK-PID). Transfection of the cells with specific p85 beta-PIX siRNA led to drastic inhibition of LPA-induced FAK phosphorylation, peripheral redistribution of p85 beta-PIX with FAK and GIT1, and cell motility. p85 beta-PIX was also required for p38 MAP kinase phosphorylation induced by LPA. Finally, dominant negative mutant of Rho (N19Rho)-transfected cells did not affect PAK activation, while the cells stably transfected with p85 beta-PIX siRNA or N17Rac1 showed the reduction of LPA-induced PAK activation. Taken together, the present data suggest that p85 beta-PIX, located downstream of Rac1, is a key regulator for the activations of FAK or p38 MAP kinase and plays a pivotal role in focal complex formation and cell motility induced by LPA.     

The Cdc42 Effector Kinase PAK4 Localizes to Cell-Cell Junctions and Contributes to Establishing Cell Polarity

The serine/threonine kinase PAK4 is a Cdc42 effector whose role is not well understood; overexpression of PAK4 has been associated with some cancers, and there are reports that correlate kinase level with increased cell migration in vitro. Here we report that PAK4 is primarily associated with cell-cell junctions in all the cell lines we tested, and fails to accumulate at focal adhesions or at the leading edge of migrating cells. In U2OS osteosarcoma and MCF-7 breast cancer cell lines, PAK4 depletion did not affect collective cell migration, but affected cell polarization. By contrast, Cdc42 depletion (as reported by many studies) caused a strong defect in junctional assembly in multiple cells lines. We also report that the depletion of PAK4 protein or treatment of cells with the PAK4 inhibitor PF-3758309 can lead to defects in centrosome reorientation (polarization) after cell monolayer wounding. These experiments are consistent with PAK4 forming part of a conserved cell-cell junctional polarity Cdc42 complex. We also confirm β-catenin as a target for PAK4 in these cells. Treatment of cells with PF-3758309 caused inhibition of β-catenin Ser-675 phosphorylation, which is located predominantly at cell-cell junctions.     

A role for p21-activated kinase in endothelial cell migration

The serine/threonine p21-activated kinase (PAK) is an effector for Rac and Cdc42, but its role in regulating cytoskeletal organization has been controversial. To address this issue, we investigated the role of PAK in migration of microvascular endothelial cells. We found that a dominant negative (DN) mutant of PAK significantly inhibited cell migration and increased stress fibers and focal adhesions. The DN effect mapped to the most NH(2)-terminal proline-rich SH3-binding sequence. Observation of a green fluorescent protein-tagged alpha-actinin construct in living cells revealed that the DN construct had no effect on membrane ruffling, but dramatically inhibited stress fiber and focal contact motility and turnover. Constitutively active PAK inhibited migration equally well and also increased stress fibers and focal adhesions, but had a somewhat weaker effect on their dynamics. In contrast to their similar effects on motility, DN PAK decreased cell contractility, whereas active PAK increased contractility. Active PAK also increased myosin light chain (MLC) phosphorylation, as indicated by staining with an antibody to phosphorylated MLC, whereas DN PAK had little effect, despite the increase in actin stress fibers. These results demonstrate that although PAK is not required for extension of lamellipodia, it has substantial effects on cell adhesion and contraction. These data suggest a model in which PAK plays a role coordinating the formation of new adhesions at the leading edge with contraction and detachment at the trailing edge.     

Cdc42 and Rac1 are necessary for autotaxin-induced tumor cell motility in A2058 melanoma cells

Autotaxin (ATX) is a strong motogen that can increase invasiveness and angiogenesis. In the present study, we investigated the signal transduction mechanism of ATX-induced tumor cell motility. Unlike N19RhoA expressing cells, the cells expressing N17Cdc42 or N17Rac1 showed reduced motility against ATX. ATX activated Cdc42 and Rac1 and increased complex formation between these small G proteins and p21-activated kinase (PAK). Furthermore, ATX phosphorylated focal adhesion kinase (FAK) that was not shown in cells expressing dominant negative mutants of Cdc42 or Rac1. Collectively, these data strongly indicate that Cdc42 and Rac1 are essential for ATX-induced tumor cell motility in A2058 melanoma cells, and that PAK and FAK might be also involved in the process.     

Activation of p21-activated kinase 1 is required for lysophosphatidic acid-induced focal adhesion kinase phosphorylation and cell motility in human melanoma A2058 cells.

Lysophosphatidic acid (LPA), one of the naturally occurring phospholipids, stimulates cell motility through the activation of Rho family members, but the signaling mechanisms remain to be elucidated. In the present study, we investigated the roles of p21-activated kinase 1 (PAK1) on LPA-induced focal adhesion kinase (FAK) phosphorylation and cell motility. Treatment of human melanoma cells A2058 with LPA increased phosphorylation and activation of PAK1, which was blocked by treatment with pertussis toxin and by inhibition of phosphoinositide 3-kinase (PI3K) with an inhibitor LY294002 or by overexpression of catalytically inactive mutant of PI3Kgamma, indicating that LPA-induced PAK1 activation was mediated via a Gi protein and the PI3Kgamma signaling pathway. In addition, we demonstrated that Rac1/Cdc42 signals acted as upstream effector molecules of LPA-induced PAK activation. However, Rho-associated kinase, MAP kinase kinase 1/2 or phospholipase C might not be involved in LPA-induced PAK1 activation or cell motility stimulation. Furthermore, PAK1 was necessary for FAK phosphorylation by LPA, which might cause cell migration, as transfection of the kinase deficient mutant of PAK1 or PAK auto-inhibitory domain significantly abrogated LPA-induced FAK phosphorylation. Taken together, these findings strongly indicated that PAK1 activation was necessary for LPA-induced cell motility and FAK phosphorylation that might be mediated by sequential activation of Gi protein, PI3Kgamma and Rac1/Cdc42.     

Actopaxin (α-Parvin) Phosphorylation Is Required for Matrix Degradation and Cancer Cell Invasion

Dysregulation of cell adhesion and motility is known to be an important factor in the development of tumor malignancy. Actopaxin (α-parvin) is a paxillin, integrin-linked kinase, and F-actin binding focal adhesion protein with several serine phosphorylation sites in the amino terminus that contribute to the regulation of cell spreading and migration. Here, phosphorylation of actopaxin is shown to contribute to the regulation of matrix degradation and cell invasion. Osteosarcoma cells stably expressing wild type (WT), nonphosphorylatable (Quint), and phosphomimetic (S4D/S8D) actopaxin demonstrate that actopaxin phosphorylation is necessary for efficient Src and matrix metalloproteinase-driven degradation of extracellular matrix. Rac1 was found to be required for actopaxin-induced matrix degradation whereas inhibition of myosin contractility promoted degradation in the phosphomutant-expressing Quint cells, indicating that a balance of Rho GTPase signaling and regulation of cellular tension are important for the process. Furthermore, actopaxin forms a complex with the Rac1/Cdc42 GEF β-PIX and Rac1/Cdc42 effector PAK1, to regulate actopaxin-dependent matrix degradation. Actopaxin phosphorylation is elevated in the invasive breast cancer cell line MDA-MB-231 compared with normal breast epithelial MCF10A cells. Expression of the nonphosphorylatable Quint actopaxin in MDA-MB-231 cells inhibits cell invasion whereas overexpression of WT actopaxin promotes invasion in MCF10A cells. Taken together, this study demonstrates a new role for actopaxin phosphorylation in matrix degradation and cell invasion via regulation of Rho GTPase signaling.     

JAK2 tyrosine kinase phosphorylates PAK1 and regulates PAK1 activity and functions

The serine-threonine kinase PAK1 is activated by small GTPase-dependent and -independent mechanisms and promotes cell survival. However, the role of tyrosyl phosphorylation in the regulation of PAK1 function is poorly understood. In this study, we have shown that the prolactin-activated tyrosine kinase JAK2 phosphorylates PAK1 in vivo. Wild type, but not kinase-dead, JAK2 directly phosphorylates PAK1 in cells and in an in vitro kinase assay. PAK1 tyrosines 153, 201, and 285 were identified as sites of JAK2 tyrosyl phosphorylation by mass spectrometry and two-dimensional peptide mapping. Mutation of PAK1 tyrosines 153, 201, and 285 to phenylalanines individually or in combination implicated these PAK1 tyrosines in the regulation of PAK1 kinase activity. Tyrosyl phosphorylation by JAK2 significantly increases PAK1 kinase activity, whereas similar phosphorylation of the PAK1 Y153F,Y201F,Y285F mutant has no effect on PAK1 activity. Tyrosyl phosphorylation of wild type PAK1 decreases apoptosis induced by serum deprivation and staurosporine treatment and increases cell motility. In contrast, these parameters are unaltered in the PAK1 Y153F,Y201F,Y285F mutant. Our findings indicate that JAK2 phosphorylates PAK1 at these specific tyrosines and that this phosphorylation plays an important role in cell survival and motility.     

Migration-stimulating factor (MSF) is over-expressed in non-small cell lung cancer and promotes cell migration and invasion in A549 cells over-expressing MSF

Migration-stimulating factor (MSF), an oncofetal truncated isoform of fibronectin, is a potent stimulator of cell invasion. However, its distribution and motogenic role in non-small cell lung cancer (NSCLC) have never been identified. In this study, real-time PCR and immunohistochemical staining (IHC) were performed to detect MSF mRNA and protein levels in tumor tissues and matched adjacent tumor-free tissues. Furthermore, to examine the effect of MSF on invasiveness, MSF was upregulated in A549 cells. The invasiveness and viability of A549 cells were then determined using a transwell migration assay and the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assays, respectively. The expression level of MSF in NSCLC tissue was markedly higher than in matched adjacent tumor-free tissue. Additionally, the level of MSF protein expression in stage III and IV NSCLC samples was higher than in stage I and II NSCLC samples. More importantly, we also demonstrated that migration and invasion of A549 cells increased substantially after upregulating MSF, although proliferation remained unchanged. Meanwhile, we found no correlation between increasing motility and invasiveness of MSF-overexpressing cells and expression levels and activities of matrix metalloprotease MMP-2 and MMP-9. Our current study shows that MSF plays a role in migration and invasion of A549 cells and suggests that MSF may be a potential biomarker of NSCLC progression.     

Phosphorylation of actopaxin regulates cell spreading and migration

Actopaxin is an actin and paxillin binding protein that localizes to focal adhesions. It regulates cell spreading and is phosphorylated during mitosis. Herein, we identify a role for actopaxin phosphorylation in cell spreading and migration. Stable clones of U2OS cells expressing actopaxin wild-type (WT), nonphosphorylatable, and phosphomimetic mutants were developed to evaluate actopaxin function. All proteins targeted to focal adhesions, however the nonphosphorylatable mutant inhibited spreading whereas the phosphomimetic mutant cells spread more efficiently than WT cells. Endogenous and WT actopaxin, but not the nonphosphorylatable mutant, were phosphorylated in vivo during cell adhesion/spreading. Expression of the nonphosphorylatable actopaxin mutant significantly reduced cell migration, whereas expression of the phosphomimetic increased cell migration in scrape wound and Boyden chamber migration assays. In vitro kinase assays demonstrate that extracellular signal-regulated protein kinase phosphorylates actopaxin, and treatment of U2OS cells with the MEK1 inhibitor UO126 inhibited adhesion-induced phosphorylation of actopaxin and also inhibited cell migration.