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• W2023503727 abstract "Stimulation of growth factor signaling has been implicated in the development of invasive phenotype and p21-activated kinase (PAK1) activation in human breast epithelial cancer cells. To further explore the roles of PAK1 in the invasive behavior of breast cancer cells, in the present study we investigated the influence of inhibition of PAK1 activity on the reorganization of cytoskeleton components that control motility and invasiveness of cells, using a highly invasive breast cancer MDA-MB435 as a model system. Our results demonstrate that overexpression of a kinase dead K299R PAK1 mutant leads to suppression of motile phenotypes as well as invasiveness of cells both in the absence or presence of exogenous heregulin-β1. In addition, these phenotypic changes were accompanied by a blockade of disassembly of focal adhesion points, stabilization of stress fibers, and enhanced cell spreading and were dependent on the presence of the kinase dead domain but independent of the presence of the Rac/cdc42 intact (Cdc42/Rac interactive binding) domain of PAK1. We also demonstrated that in K299R PAK1-expressing cells, F-actin filaments were stabilized by persistent co-localization with the actin-binding proteins tropomyosin and caldesmon. Extension of these studies to invasive breast cancer MDA-MB231 cells illustrated that conditional expression of kinase-defective K299R PAK1 was also accompanied by persistent cell spreading, multiple focal adhesion points, and reduced invasiveness. Furthermore, inhibition of PAK1 activity in breast cancer cells was associated with a reduction in c-Jun N-terminal kinase activity, inhibition of DNA binding activity of transcription factor AP-1, and suppression of in vivo transcription driven by AP-1 promoter (known to be involved in breast cancer invasion). These findings suggest that PAK1 downstream pathways have a role in the development and maintenance of invasive phenotypes in breast cancer cells. Stimulation of growth factor signaling has been implicated in the development of invasive phenotype and p21-activated kinase (PAK1) activation in human breast epithelial cancer cells. To further explore the roles of PAK1 in the invasive behavior of breast cancer cells, in the present study we investigated the influence of inhibition of PAK1 activity on the reorganization of cytoskeleton components that control motility and invasiveness of cells, using a highly invasive breast cancer MDA-MB435 as a model system. Our results demonstrate that overexpression of a kinase dead K299R PAK1 mutant leads to suppression of motile phenotypes as well as invasiveness of cells both in the absence or presence of exogenous heregulin-β1. In addition, these phenotypic changes were accompanied by a blockade of disassembly of focal adhesion points, stabilization of stress fibers, and enhanced cell spreading and were dependent on the presence of the kinase dead domain but independent of the presence of the Rac/cdc42 intact (Cdc42/Rac interactive binding) domain of PAK1. We also demonstrated that in K299R PAK1-expressing cells, F-actin filaments were stabilized by persistent co-localization with the actin-binding proteins tropomyosin and caldesmon. Extension of these studies to invasive breast cancer MDA-MB231 cells illustrated that conditional expression of kinase-defective K299R PAK1 was also accompanied by persistent cell spreading, multiple focal adhesion points, and reduced invasiveness. Furthermore, inhibition of PAK1 activity in breast cancer cells was associated with a reduction in c-Jun N-terminal kinase activity, inhibition of DNA binding activity of transcription factor AP-1, and suppression of in vivo transcription driven by AP-1 promoter (known to be involved in breast cancer invasion). These findings suggest that PAK1 downstream pathways have a role in the development and maintenance of invasive phenotypes in breast cancer cells. p21-activated kinase c-Jun N-terminal kinase tropomyosin caldesmon Dulbecco's modified Eagle's medium fetal calf serum hemagglutinin green fluorescence protein Cdc42/Rac interactive binding heregulin β1 cytomegalovirus activation protein Breast cancer is one of the most common malignancies in the United States, affecting one in nine women. Localized breast cancer prior to metastasis can be cured by surgery. The high mortality rate associated with breast cancer, however, is related to its ability to metastasize beyond the mammary gland and invade distant sites while the primary tumor is small and undetected. Thus, tumor cell migration/invasion is an important factor in formation of solid tumors and is necessary for their spread to distinct organs. The process of malignancy requires, among other steps, changes in growth factor pathways and increased migration. The exposure of cells to growth factors has been shown to cause cytoskeleton reorganization, formation of lamellipodia, membrane ruffling, and altered cell morphology, and accordingly, such exposure is implicated in stimulating cell migration and invasion (1.Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5185) Google Scholar). Most eukaryotic cells possess the capacity to move over or through a substrate, and cell migration plays a key role in both normal physiology and in a disease setting, including invasion and metastasis (2.Mitchison T.J. Cramer L.P. Cell. 1996; 84: 371-379Abstract Full Text Full Text PDF PubMed Scopus (1299) Google Scholar). In many tissues, the motility function of cells is normally repressed but can be activated by appropriate stimuli, oncogenic transformation, or both. In fact, one of the earliest responses of cells to many extracellular growth factors is rapid reorganization of their cytoskeleton and cell shape. The leading edge of a motile cell is composed of thin protrusions of membrane that continuously extend and retract mediating the initial stage of cell movement and determining the direction of advance. The underlying cytoskeleton of a leading edge is composed of actin-filament bundles (in filopodia) or meshworks (in lamellipodia) oriented toward the membrane (3.Lauffenburger D.A. Horwitz A.F. Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3254) Google Scholar). Cell migration also involves changes in cytoskeleton actin stress fibers that end in focal adhesions, which are places where the plasma membrane is attached to the substratum. Focal adhesions are specialized sites of adhesion for cells in culture associated with well defined actin stress fibers, and they represent the link between the outside extracellular matrix components and the cytoplasmic integrins coupled with cytoskeletal proteins. Numerous proteins involved in signal transduction are known to be concentrated at these sites. According to a recent model, intracellular components of the focal adhesion complex and actin filaments associate with integrins upon integrin engagement with the extracellular matrix. In the presence of active Rho, these complexes and actin cluster together by actin-myosin contraction, which leads to focal adhesion and stress fiber formation. The focal adhesion points play an important role in the regulation of cell motility, which involves cyclic formation of cell adhesion and disassembly of actin filaments and focal adhesion complexes (1.Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5185) Google Scholar).The small GTPases, including Cdc42, Rac1, and RhoA, have been implicated in the regulation of morphology, the formation of filopodia/lamellopodia, membrane ruffles, and stress fibers, and motility of mammalian cells (1.Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5185) Google Scholar). More specifically, Rac1 induces cortical actin polymerization seen as the process of membrane ruffling and lamellipodia (4.Ridley A.J. Paterson H.F. Johston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3050) Google Scholar). RhoA induces stress fiber formation possibly by catalyzing the formation of focal adhesion (5.Ridley A. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3797) Google Scholar), and cdc42 induces the formation of peripheral actin microspikes and filopodia (6.Kozma R,. Ahmed S. Best A. Lim L. Mol. Cell. Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (880) Google Scholar). With regard to the mechanism by which the small GTPases initiate and regulate the formation of cytoskeletal structures, recently a family of serine/theonine kinases known as p21-activated kinases (PAKs)1 have been identified as GTPase targets (7.Sells M.A. Knaus U.G. Bagrodia S. Ambrose D.M. Bokoch G.M. Chernoff J. Curr. Biol. 1997; 7: 202-210Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar). Activation of PAK1 has been shown to result in phenotypic changes reminiscent of those produced by GTPases. If activated either by overexpression of dominant active PAK1 mutants or by stimulation of cells with growth factors, PAK1 causes the accumulation of F-actin and the formation of lamellipodia and filopodia. Furthermore, activated PAK1 has been also shown to be co-localized with F-actin at the leading edge of the cells (7.Sells M.A. Knaus U.G. Bagrodia S. Ambrose D.M. Bokoch G.M. Chernoff J. Curr. Biol. 1997; 7: 202-210Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar, 8.Sells M.A. Boyd J.T. Chernoff J. J. Cell Biol. 1999; 145: 837-849Crossref PubMed Scopus (327) Google Scholar). Activation of PAK1 has been shown to be accompanied by the disassembly of stress fibers and focal adhesion complexes (9.Manser E. Huang H.Y. Loo T.H. Chen G. Dong J.M. Leung T. Lim L. Mol. Cell. Biol. 1997; 17: 1129-1143Crossref PubMed Google Scholar), both of which are widely believed to be required for increased motility. Because PAK1 is a downstream target of cdc42 and Rac1, which are known to activate extracellular signal-regulated kinase, JNK, p38 kinases, and the transcription factor nuclear factor-κB (10.Coso O.A. Chiariello M., Yu, J-C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1559) Google Scholar, 11.Minden A. Lin A.,. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1444) Google Scholar, 12.Sulciner D.J. Irani K., Yu, Z.X. Ferrans V.J. Goldschmidt-Clermont P. Finkel T. Mol. Cell. Biol. 1996; 16: 7115-7121Crossref PubMed Google Scholar), it has been proposed that PAK1 or a related kinase may mediate the actions of cdc42 and Rac1. Indeed, PAK1 has been shown to activate JNK and extracellular signal-regulated kinase kinases (13.Brown J.L. Stowers L. Baer M. Trejo J. Coughlin S. Chant J. Curr. Biol. 1996; 6: 598-605Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 14.Yablonski D. Kane L.P. Quian D. Weiss A. EMBO J. 1998; 17: 5647-5657Crossref PubMed Scopus (109) Google Scholar). Invasion of T-lymphocytes and fibroblast has been shown to involve activated Rac1 and Tiam-1 (15.Michiels F. Habets G.G. Stam J.C. van der Kammen R.A. Collard J.G. Nature. 1995; 25: 338-340Crossref Scopus (506) Google Scholar), a Rac1 exchange factor. In addition to the PAK1-JNK pathway, other effectors for Rac1 such as POR1 have also been implicated in actin rearrangements (16.Joneson T. McDonough M. Bar-Sagi D. Van Aelst L. Science. 1996; 274: 1374-1376Crossref PubMed Scopus (232) Google Scholar, 17.Keely P.J. Westwick J.K. Whitehead I.P. Der C.J. Parise L.V. Nature. 1997; 390: 632-636Crossref PubMed Scopus (649) Google Scholar). Using well differentiated mammary breast cancer cells Keely et al. (17.Keely P.J. Westwick J.K. Whitehead I.P. Der C.J. Parise L.V. Nature. 1997; 390: 632-636Crossref PubMed Scopus (649) Google Scholar) showed that PAK1 as well as JNK1 activities are not required to induce motility and invasion in cells that are overexpressing active mutants of Rac1. These studies dissociate Cdc42 and Rac1-induced actin polymerization (ruffles formation) from PAK and JNK activation, raising the possibility of existence of a divergent pathway, which involves another Rac1 effector, POR1. It was shown that Rac1 mutants that are capable of binding PAK1 but not POR1 (and thus activate JNK) are also able to induce a motile phenotytpe in these cells (16.Joneson T. McDonough M. Bar-Sagi D. Van Aelst L. Science. 1996; 274: 1374-1376Crossref PubMed Scopus (232) Google Scholar). However, the same Rac mutants behave in an opposite manner when used in different cell systems (17.Keely P.J. Westwick J.K. Whitehead I.P. Der C.J. Parise L.V. Nature. 1997; 390: 632-636Crossref PubMed Scopus (649) Google Scholar).It is increasingly accepted that alterations in the reorganization and stability of actin filaments may contribute to increased cell migration and to cell transformation, anchorage-independent growth, and invasiveness. Tropomyosin (TM) and caldesmon (CaD) are two essential proteins required for the reorganization of the actin cytoskeleton mediated by stabilization of microfilaments (18.Sobue K. Sellers J.R. J. Biol. Chem. 1991; 266: 12115-12118Abstract Full Text PDF PubMed Google Scholar, 19.Tanaka J. Waranabe T. Nakamura N. Sobue K. J. Cell Sci. 1993; 104: 595-606Crossref PubMed Google Scholar). Although TM was initially discovered as a helical protein that binds to actin grooves in muscle, it is now clear that Ca2+-dependent contractility in nonmuscle cells is also controlled by TM in conjunction with CaD (20.Huber P.A. Fraser I.D. Marston S.B. Biochem. J. 1995; 312: 617-625Crossref PubMed Scopus (26) Google Scholar). CaD is an actin- and calmodulin-binding protein that is distributed at intervals along stress fibers (18.Sobue K. Sellers J.R. J. Biol. Chem. 1991; 266: 12115-12118Abstract Full Text PDF PubMed Google Scholar, 21.Yamashiro-Matsumura S. Matsumura F. J. Cell Biol. 1988; 106: 1973-1983Crossref PubMed Scopus (69) Google Scholar). Recent studies have shown relationships between 1) overexpression of CaD and the stabilization of actin microfilament bundles by enhancing the half-life of TM (22.Warren K.S. Shutt D.C. McDermott J.P. Lin J.L. Soll D.R. Lin J.J. Cell Motil. Cytoskeleton. 1996; 34: 215-229Crossref PubMed Scopus (34) Google Scholar); 2) suppression of TM1 and the anchorage-independent growth of hamster embryo cells (23.Boyd J. Risinger J.I. Wiseman R.W. Merrick B.A.,. Selkirk J.K. Barrett J.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11534-11538Crossref PubMed Scopus (82) Google Scholar); 3) modulation of the TM isoforms and tumorigenic transformation (24.Franzen B. Linder S. Alaiya A.A. Eriksson E. Fujioka K.,. Bergman A.C. Jornvall H. Auer G. Electrophoresis. 1997; 18: 582-587Crossref PubMed Scopus (80) Google Scholar, 25.Bhattacharya B. Prasad G.L. Valverius E.M. Salomon D.S. Cooper H.L. Cancer Res. 1990; 50: 2105-2112PubMed Google Scholar); and 4) CaD/v-erbB interaction and actin stress fiber disassembly (26.McManus M.J. Lingle W.L. Salisbury J.L. Maihle N.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11351-11356Crossref PubMed Scopus (27) Google Scholar). Despite the widely acknowledged roles of TM and CaD on one hand and PAK1 on the other hand in actin stabilization, the potential relationship between these cytoskeleton regulators and invasiveness remains elusive.Recently, we showed that stimulation of a noninvasive human breast cancer cell by heregulin leads to the activation of PAK1 kinases, redistribution of PAK1 at the leading edges of cells, development of lamellipodia/filopodia and stress fibers, and increased invasiveness of breast cancer cells (27.Adam L. Vadlamudi R. Kondapaka S.B. Chernoff J. Mendelsohn J. Kumar R. J. Biol. Chem. 1998; 273: 28238-28246Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). To further explore the role of PAK1 in the invasive behavior of breast cancer cells, in this study we investigated the influence of suppression of the PAK1 activity on the reorganization of cytoskeleton components that control motility and in vitro invasiveness of cells using the highly invasive MDA-MB435 and MDA-MB231 breast cancer cell model systems. Our results demonstrated that overexpression of catalytically defective K299R PAK1 leads to biogenesis of stable focal adhesions and stress fibers with matrix-independent enhanced cell attachment and spreading properties. All these biological effects were dependent on the presence of the kinase defective site and independent on the presence of an intact Rac/cdc42 binding domain. In addition, these phenotypic changes were accompanied by persistent suppression of JNK activity, DNA binding activity of transcription factor AP-1, and in vivotranscription driven by collagenase promoter. These findings suggest that the PAK1-dependent pathway have a role in the maintenance of invasive behavior of breast cancer cells.DISCUSSIONBreast cancer is one of the most common malignancies in western countries. The high mortality rate associated with breast cancer, however, is related to its ability to metastasize beyond the mammary gland and invade distant sites, whereas the primary tumor is small and undetected. Thus, tumor cell migration/invasion is an important factor in the formation of solid tumors, and it is necessary for their spread to distinct organs. The progression of breast cancer cells to a more invasive phenotype is believed to be influenced by the migration of cells from the primary site of tumor, incorporating the ability of cancer cells to invade through basement membrane and reestablish themselves at distant sites. Because a successful invasion and metastasis of breast tumor is conditional for cell migration from the primary tumor, cell motility must play a pivotal role in the development and maintenance of the invasive phenotype of breast cancer cells. Therefore, understanding cell motility is likely to delineate pathways that may be an integral part of metastasis. Recently, we showed that PAK1 had a role in cell motility and invasiveness of human breast cancer cells stimulated by heregulin-β1 (27.Adam L. Vadlamudi R. Kondapaka S.B. Chernoff J. Mendelsohn J. Kumar R. J. Biol. Chem. 1998; 273: 28238-28246Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). To better understand the role of PAK1 in the reorganization of cytoskeleton components that control motility and in vitro invasiveness of cells, here we investigated the influence of inhibition of PAK1 activity on the modulation of invasive phenotypes in breast cancer cells. We utilized two distinct highly invasive breast cancer cell model systems, one involving stable overexpression of kinase dead K299R PAK1 in MDA-MB435 cells and another where there is a conditional regulation of the expression of K299R PAK1 in MDA-MB231 cells.The results presented here indicate that overexpression of kinase dead PAK1 in invasive breast cancer cells leads to significant morphological changes and reduction of in vitro invasiveness. Our conclusion that suppression of PAK1 activity modulated the invasion of breast cancer cells was supported by the following lines of evidence: (i) expression of K299R PAK1 mutant blocked the transmigration of cells across a porous membrane, (ii) K299R PAK1 mutant blocked the formation of ruffles and filopodia induced by other stimuli, (iii) inhibition of PAK1 by K299R was accompanied by persistent cell spreading and an increase in the number and maturity of the focal complexes, (iv) K299R expression resulted in the appearance of thicker actin cables with prolonged co-localization of tropomyosin or caldesmon, and (v) the K299R PAK1-mediated blockade of ruffles and filopodia formation and of migration could not be reversed by HRG. Taken together, these data provide evidence that PAK1-regulated reorganization of cytoskeleton structures may be required to maintain the invasive phenotype of breast cancer cells.Among the various pathways leading to actin reorganization, the Rho subfamily of the small GTPases, cdc42 and Rac1, has been implicated in the regulation of morphology and motility of mammalian cells. Recent studies have identified the PAKs as a target of activated GTPases, as activation of PAK1 has been shown to result in the phenotypic changes reminiscent of those produced by Rac and/or cdc42 (36.Manser E. Leung T. Salihuddin H. Zhao Z.-S. Li L. Nature. 1994; 367: 40-46Crossref PubMed Scopus (1292) Google Scholar). Furthermore, a role of PAK in promoting cell migration has been also suggested by the physical interaction between PAK1 and actin in response to upstream stimuli (7.Sells M.A. Knaus U.G. Bagrodia S. Ambrose D.M. Bokoch G.M. Chernoff J. Curr. Biol. 1997; 7: 202-210Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar, 27.Adam L. Vadlamudi R. Kondapaka S.B. Chernoff J. Mendelsohn J. Kumar R. J. Biol. Chem. 1998; 273: 28238-28246Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). The propensity of malignant breast cancer cells to invade must depend on their ability to migrate from the primary tumor site. Our observation that kinase dead PAK1 predominantly localizes to the fairly stable focal points supports a role for PAK1 in promoting cell motility, probably by its known function to dissolve the focal adhesions (31.Frost J.A. Khokhlatchev A. Stippec S. White M.A. Cobb M.H. J. Biol. Chem. 1998; 273: 28191-28198Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). However, because the K299R kinase dead PAK1 mutant still contains binding sites for Rac/cdc42, which are known to promote phenotypic changes similar to those caused by PAK1, it is possible that the observed effects could have been reflections of ineffective transduction of Rac/cdc42 signals. However, transfection of double PAK1 mutant H83, 86L, K299R (which has mutation in CRIB site and PAK1 kinase domain) also demonstrated increased actin bundling, cell spreading,and stabilization of focal points. Furthermore, expression of the PAK1 autoinhibitory domain (PAK 83–149) also exhibited changes in cell shape (to a lesser degree) similar to K299R PAK1. Taken together, these results indicated that PAK1 kinase activity is involved in the cell shape changes, increased focal points and F-actin bundling in MDA-MB-435 cells. These findings suggest a role of PAK1 kinase activity in the motility of human epithelial cancer cells.Recently, kinase active PAK1 has been shown to cause excessive dissolution of focal adhesions and stress fibers (9.Manser E. Huang H.Y. Loo T.H. Chen G. Dong J.M. Leung T. Lim L. Mol. Cell. Biol. 1997; 17: 1129-1143Crossref PubMed Google Scholar, 31.Frost J.A. Khokhlatchev A. Stippec S. White M.A. Cobb M.H. J. Biol. Chem. 1998; 273: 28191-28198Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar) and directional haptotaxis in fibroblasts (8.Sells M.A. Boyd J.T. Chernoff J. J. Cell Biol. 1999; 145: 837-849Crossref PubMed Scopus (327) Google Scholar). Another notable finding in this study was the essential role of PAK1 in actin dynamics, as catalytically inactive PAK1 may impair the formation of dynamic actin-containing structures necessary for cell migration and the formation of the focal points. Some of the possible mechanisms for the observed decreased migration potential may include a change in F-actin conformation induced by increasing the actin bundling or stability or blocking the F-actin capping. Caldesmon and tropomyosin are two major actin-binding proteins that can modulate the stability of actin filament (32.Zhao Z.-S. Manser E. Chen Q. Chong C. Leung T. Lim T. Mol. Cell. Biol. 1998; 18: 2153-2163Crossref PubMed Google Scholar). It has been shown that caldesmon coupled with tropomyosin not only blocks the binding of human fascin to actin (37.Ishikawa R. Yamashiro S. Kohama K. Matsumura F. J. Biol. Chem. 1998; 273: 26991-26997Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) but also dissociates fascin from actin (38.Matsumura F. Yamashiro-Matsumura S. J. Biol. Chem. 1986; 261: 4655-4659Abstract Full Text PDF PubMed Google Scholar), implying that caldesmon together with tropomyosin regulates the assembly of actin bundles. There is also an increasing body of evidence showing that caldesmon stabilizes microfilaments in nonmuscle cells (33.Matsumura F. Yamashiro S Curr. Opin. Cell Biol. 1993; 5: 70-76Crossref PubMed Scopus (108) Google Scholar), probably by increasing actin binding of tropomyosin (39.Smith C.W. Pritchard K. Marston S.B. J. Biol. Chem. 1987; 262: 116-122Abstract Full Text PDF PubMed Google Scholar, 40.Novy R.E. Lin J.L. J. Biol. Chem. 1991; 266: 16917-16924Abstract Full Text PDF PubMed Google Scholar). In nonmuscle cells, caldesmon, an actin-binding protein, may have important regulatory functions in cell motility, analogous to those suggested for the regulation of smooth muscle contraction. Dissociation of caldesmon from microfilaments may destabilize the structure of microfilaments. Something similar may happen during cell motility where there is a dramatic cytoskeleton rearrangement of the stress fibers connecting the cells in the focal points (attachment and spreading) with subsequent dissolution and reformation of more dynamic actin-containing structures such as ruffles and lamellopodias. It remains to be established how PAK1 contributes to the interaction of the actin with tropomyosin and caldesmon. Several substrates have been recently found to be phosphorylated by different members of the PAK family with functional impact on the contraction properties of actin filaments. More contraction of actin filaments (stress fibers) causes less motility in nonmuscle cells (41.Aspenstrom P. Curr. Opin. Cell Biol. 1999; 11: 95-102Crossref PubMed Scopus (284) Google Scholar). Recent studies have shown that PAK3 is able to phosphorylate caldesmon independently of the induction of muscle contraction (42.Van Eyk J.E. Arrell D.K. Foster D.B. Strauss J.D. Heinonen T.Y.K. Furmaniak-Kazmierczak E. Cote G.P. Mak A.S. J. Biol. Chem. 1998; 273: 23433-23439Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Evidence also suggests that myosin light chain kinase binds to cellular actin filaments and is localized to actinomyosin-containing stress fibers in nonmuscle cells. In a very elegant study on smooth muscle cells that used GFP-labeled myosin light chain kinase the filament-bound kinase was shown to be sufficient for Ca2+-dependent phosphorylation of myosin regulatory light chain and contraction of stress fiber (43.Lin P.-J. Luby-Phelps K. Stull J.T. J. Biol. Chem. 1997; 272: 7412-7420Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). One of the recently identified PAK1 substrates is indeed myosin light chain kinase, and its phosphorylation by PAK1 in vitro andin vivo was shown to decrease myosin light chain activity, leading to a decrease in cell spreading and thus less actin-myosin contraction (44.Sanders L.C. Matsumura F. Bokoch G.M. de Lanerolle P. Science. 1999; 283: 2083-2085Crossref PubMed Scopus (500) Google Scholar). Our finding that the kinase dead mutant K299R PAK1 increased the cell spreading and the appearance of stress fibers suggests a possible role of PAK1 in microfilaments remodeling via the myosin light chain kinase pathway. However our results do not distinguish if these effects are because of the increase in actin-myosin contraction, stabilization of actin by actin-binding proteins, or both. Future studies will be needed to determine the precise nature of interplay between Rac, cdc42, and RhoA effectors in these processes.The evidence that overexpression of the kinase dead K299R PAK1 mutant in breast cancer cells has the capacity to inhibit the DNA binding activity and of AP-1-dependent transcription in vivo is of special interest, as it strongly suggests that PAK1-induced signaling may modulate the expression of genes, such as matrix metalloproteinases (45.Westermarck J. Kahari V.M. FASEB J. 1999; 13: 781-793Crossref PubMed Scopus (1390) Google Scholar), which are relevant for invasion via the AP-1-dependent pathway. This hypothesis is further supported by our preliminary studies showing a reduction of gelatinolytic activity in the conditioned medium that had K299R PAK1-overexpressing MDA-MB435 cells (data not shown). Because PAK1 is considered to be an activator of JNK and p38 MAPK (46.Bagrodia S. Derijard B. Davis R.J. Cerione R.A. J. Biol. Chem. 1995; 270: 27995-27998Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar), it is possible that the JNK/p38 downstream events influence the expression of gene products with functions in motility/invasion of breast cancer cells. Taken together, our findings of regulation of actin reorganization, cell migration and invasion, and gene regulation functions of breast cancer cells by PAK1 open a new avenue of investigation closely linking PAK1, cytoskeleton-signaling, transcription of genes involved in metastasis, and breast cancer invasiveness. In addition, our present finding may lead to strategies targeting PAK1 as a potential inhibitor of invasion and metastasis of breast cancer cells. Breast cancer is one of the most common malignancies in the United States, affecting one in nine women. Localized breast cancer prior to metastasis can be cured by surgery. The high mortality rate associated with breast cancer, however, is related to its ability to metastasize beyond the mammary gland and invade distant sites while the primary tumor is small and undetected. Thus, tumor cell migration/invasion is an important factor in formation of solid tumors and is necessary for their spread to distinct organs. The process of malignancy requires, among other steps, changes in growth factor pathways and increased migration. The exposure of cells to growth factors has been shown to cause cytoskeleton reorganization, formation of lamellipodia, membrane ruffling, and altered cell morphology, and accordingly, such exposure is implicated in stimulating cell migration" @default.
• W2023503727 created "2016-06-24" @default.
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• W2023503727 date "2000-04-01" @default.
• W2023503727 modified "2023-10-13" @default.
• W2023503727 title "Regulation of Microfilament Reorganization and Invasiveness of Breast Cancer Cells by Kinase Dead p21-activated Kinase-1" @default.
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