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• W2000237287 abstract "Two characteristics of highly malignant cells are their increased motility and secretion of proteinases allowing these cells to penetrate surrounding basement membranes and metastasize. Activation of 21-kDa activated kinases (PAKs) is an important mechanism for increasing cell motility. Recently, we reported that binding of receptor-recognized forms of the proteinase inhibitor α2-macroglobulin (α2M*) to GRP78 on the cell surface of 1-LN human prostate cancer cells induces mitogenic signaling and cellular proliferation. In the current study, we have examined the ability of α2M* to activate PAK-1 and PAK-2. Exposure of 1-LN cells to α2M* caused a 2- to 3-fold increase in phosphorylated PAK-2 and a similar increase in its kinase activity toward myelin basic protein. By contrast, the phosphorylation of PAK-1 was only negligibly affected. Silencing the expression of the GRP78 gene, using either of two different mRNA sequences, greatly attenuated the appearance of phosphorylated PAK-2 in α2M*-stimulated cells. Treatment of 1-LN cells with α2M* caused translocation of PAK-2 in association with NCK to the cell surface as evidenced by the coimmunoprecipitation of PAK-2 and NCK in the GRP78 immunoprecipitate from plasma membranes. α2M*-induced activation of PAK-2 was inhibited by prior incubation of the cells with specific inhibitors of tyrosine kinases and phosphatidylinositol 3-kinase. PAK-2 activation was accompanied by significant increases in the levels of phosphorylated LIMK and phosphorylated cofilin. Silencing the expression of the PAK-2 gene greatly attenuated the phosphorylation of LIMK. In conclusion, we show for the first time the activation of PAK-2 in 1-LN prostate cancer cells by a proteinase inhibitor, α2-macroglobulin. These studies suggest a mechanism by which α2M* enhances the metastatic potential of these cells. Two characteristics of highly malignant cells are their increased motility and secretion of proteinases allowing these cells to penetrate surrounding basement membranes and metastasize. Activation of 21-kDa activated kinases (PAKs) is an important mechanism for increasing cell motility. Recently, we reported that binding of receptor-recognized forms of the proteinase inhibitor α2-macroglobulin (α2M*) to GRP78 on the cell surface of 1-LN human prostate cancer cells induces mitogenic signaling and cellular proliferation. In the current study, we have examined the ability of α2M* to activate PAK-1 and PAK-2. Exposure of 1-LN cells to α2M* caused a 2- to 3-fold increase in phosphorylated PAK-2 and a similar increase in its kinase activity toward myelin basic protein. By contrast, the phosphorylation of PAK-1 was only negligibly affected. Silencing the expression of the GRP78 gene, using either of two different mRNA sequences, greatly attenuated the appearance of phosphorylated PAK-2 in α2M*-stimulated cells. Treatment of 1-LN cells with α2M* caused translocation of PAK-2 in association with NCK to the cell surface as evidenced by the coimmunoprecipitation of PAK-2 and NCK in the GRP78 immunoprecipitate from plasma membranes. α2M*-induced activation of PAK-2 was inhibited by prior incubation of the cells with specific inhibitors of tyrosine kinases and phosphatidylinositol 3-kinase. PAK-2 activation was accompanied by significant increases in the levels of phosphorylated LIMK and phosphorylated cofilin. Silencing the expression of the PAK-2 gene greatly attenuated the phosphorylation of LIMK. In conclusion, we show for the first time the activation of PAK-2 in 1-LN prostate cancer cells by a proteinase inhibitor, α2-macroglobulin. These studies suggest a mechanism by which α2M* enhances the metastatic potential of these cells. Cancer of the prostate is the most commonly diagnosed malignancy of men (1Jemal A. Thomas A. Murray T. Thun M. CA Cancer J. Clin. 2002; 52: 23-47Crossref PubMed Scopus (2917) Google Scholar). In the development of prostate cancer, deregulation of cell growth control often is accompanied by acquisition of androgen independence, a poor prognostic indicator (2Heinlein C.A. Chang C. Endocr. Rev. 2004; 25: 276-308Crossref PubMed Scopus (1277) Google Scholar, 3Montano X. Djamgoz M. FEBS Lett. 2004; 571: 1-8Crossref PubMed Scopus (59) Google Scholar). Growth factors, including epidermal growth factor, insulin-like growth factor, and fibroblast growth factor play a role in the progression of androgen-independent prostate cancer (2Heinlein C.A. Chang C. Endocr. Rev. 2004; 25: 276-308Crossref PubMed Scopus (1277) Google Scholar, 3Montano X. Djamgoz M. FEBS Lett. 2004; 571: 1-8Crossref PubMed Scopus (59) Google Scholar). These growth factors induce mitogenic cellular responses by activating their specific receptors. Ligand binding to these receptors induces the autophosphorylation of the receptor on specific tyrosine residues resulting in the assembly of multiprotein complexes, which activate the Ras/MAPK 1The abbreviations used are: MAPK, mitogen-activated protein kinase; α2M, α2-macroglobulin; α2M*, “activated” α2-macroglobuin, which binds to cell surface receptors; PAK-1, p21-activated protein kinase-1 (α) and p21-activated protein kinase-2 (γ); GRP78, glucose-regulated proteins 78; PI, phosphatidylinositol-dependent protein kinase; HBSS, Hanks' balanced salt solution; ECF; enhanced chemifluorescence; MBP, myelin basic protein; DMEM, Dulbecco's modified Eagle's medium; dsRNA, double-stranded RNA; FBS, fetal bovine serum; PBD, p21 binding domain. 1The abbreviations used are: MAPK, mitogen-activated protein kinase; α2M, α2-macroglobulin; α2M*, “activated” α2-macroglobuin, which binds to cell surface receptors; PAK-1, p21-activated protein kinase-1 (α) and p21-activated protein kinase-2 (γ); GRP78, glucose-regulated proteins 78; PI, phosphatidylinositol-dependent protein kinase; HBSS, Hanks' balanced salt solution; ECF; enhanced chemifluorescence; MBP, myelin basic protein; DMEM, Dulbecco's modified Eagle's medium; dsRNA, double-stranded RNA; FBS, fetal bovine serum; PBD, p21 binding domain. and PI 3-kinase signaling pathways (4Schlessinger J. Cell. 2000; 103: 193-200Abstract Full Text Full Text PDF PubMed Scopus (3487) Google Scholar). In addition to increased activation of signaling pathways that promote cellular proliferation and/or suppression of apoptosis, increased motility is often seen in malignantly transformed cells. This increase in motility, along with increased secretion of proteinases, especially matrix metalloproteinases, enables highly metastatic cancer cells to penetrate surrounding basement membranes and invade blood vessels and lymphatics. One mechanism that promotes increased motility of malignant cells is activation of members of the 21-kDa activated kinase (PAK) family.These proteins are Ser/Thr kinases that mediate Rac and Cdc42 GTPase-dependent signaling (see reviews in Refs. 5Sells M.A. Chernoff J. Trends Cell Biol. 1997; 7: 162-167Abstract Full Text PDF PubMed Scopus (264) Google Scholar, 6Daniels R.H. Bokoch G.M. Trends Biochem. Sci. 1999; 24: 350-355Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 7Vadlamudi R.K. Kumar R. Cancer Metastasis Rev. 2003; 22: 385-393Crossref PubMed Scopus (65) Google Scholar, 8Bokoch G.M. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (872) Google Scholar and references therein). The mammalian PAK family consists of six members, including PAK-1 and PAK-2. PAK-1 is tissue-specific in its expression, whereas PAK-2 is ubiquitously expressed. The catalytic activity of PAKs is regulated by the binding of active GTPases to the conserved p21 binding motif in the NH2-terminal domain leading to the relief of autoinhibitory interactions with the COOH-terminal catalytic domain (5Sells M.A. Chernoff J. Trends Cell Biol. 1997; 7: 162-167Abstract Full Text PDF PubMed Scopus (264) Google Scholar, 6Daniels R.H. Bokoch G.M. Trends Biochem. Sci. 1999; 24: 350-355Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 7Vadlamudi R.K. Kumar R. Cancer Metastasis Rev. 2003; 22: 385-393Crossref PubMed Scopus (65) Google Scholar, 8Bokoch G.M. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (872) Google Scholar). PAK-2 is also activated by caspase or caspase-like proteinases, which generate constitutively active p34 PAK-2, the COOH-terminal catalytic domain (9Walter B.N. Huang Z. Jakobi R. Tuazon P.T. Alnemri P.T. Traugh J.A. J. Biol. Chem. 1998; 273: 28733-28739Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Activated full-length PAK-2 stimulates cell survival and growth in response to various stress stimulants, whereas its proteolytic fragment, p34 protein, stimulates cell death (9Walter B.N. Huang Z. Jakobi R. Tuazon P.T. Alnemri P.T. Traugh J.A. J. Biol. Chem. 1998; 273: 28733-28739Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 10Jakobi R. McCarthy C.C. Koeppel M.A. Stringer D.K. J. Biol. Chem. 2003; 278: 38675-38685Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Stimulation of cell survival by activated full-length PAK-2 is partly mediated by phosphorylation and inhibition of pro-apoptotic Bad (8Bokoch G.M. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (872) Google Scholar, 9Walter B.N. Huang Z. Jakobi R. Tuazon P.T. Alnemri P.T. Traugh J.A. J. Biol. Chem. 1998; 273: 28733-28739Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 10Jakobi R. McCarthy C.C. Koeppel M.A. Stringer D.K. J. Biol. Chem. 2003; 278: 38675-38685Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The activation of PAK-2 in response to irradiation or cytosine β-d-arabinoside is dependent on protein-tyrosine kinase and PI 3-kinase activity (11Roig J. Thazon P.T. Zipfel P.A. Pendergast A.M. Traugh J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14346-14351Crossref PubMed Scopus (42) Google Scholar). PAK-1 mediates signals from the Ras/MAPK and PI 3-kinase signaling pathways to promote cell transformation. PAKs also play important roles in modulating the ability of cancer cells to move and metastasize (5Sells M.A. Chernoff J. Trends Cell Biol. 1997; 7: 162-167Abstract Full Text PDF PubMed Scopus (264) Google Scholar, 6Daniels R.H. Bokoch G.M. Trends Biochem. Sci. 1999; 24: 350-355Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 7Vadlamudi R.K. Kumar R. Cancer Metastasis Rev. 2003; 22: 385-393Crossref PubMed Scopus (65) Google Scholar, 8Bokoch G.M. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (872) Google Scholar). A number of highly metastatic human breast cancer lines exhibit constitutively elevated PAK-1 or PAK-2 activity (12Salh B. Marrota A. Wagey R. Sayed M. Pelech S. Int. J. Cancer. 2002; 98: 148-154Crossref PubMed Scopus (65) Google Scholar).α2-Macroglobulin (α2M) is a broad specificity proteinase inhibitor that binds to cell surface receptors when activated by proteinases (13Krieger M. Herz J. Annu. Rev. Biochem. 1994; 63: 601-637Crossref PubMed Scopus (1057) Google Scholar). The activated form of α2M (α2M*) is also produced by direct reaction of internal thiol esters present in each of its four identical subunits with small amines or ammonia (13Krieger M. Herz J. Annu. Rev. Biochem. 1994; 63: 601-637Crossref PubMed Scopus (1057) Google Scholar). Binding of α2M* to macrophages (14Misra U.K. Chu C.T. Rubenstein D.S. Gawdi G. Pizzo S.V. Biochem. J. 1993; 290: 885-891Crossref PubMed Scopus (68) Google Scholar, 15Misra U.K. Chu C.T. Gawdi G. Pizzo S.V. J. Biol. Chem. 1994; 269: 18303-18306Abstract Full Text PDF PubMed Google Scholar), rheumatoid synovial fibroblasts (16Misra U.K. Gonzalez-Gronow M. Gawdi G. Pizzo S.V. J. Biol. Chem. 1997; 272: 497-502Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), and 1-LN prostate cancer cells triggers increases in the levels of intracellular inositol 1,3,4-trisphosphate and cytosolic-free calcium [Ca2+]i and is followed by activation of components of the Ras/MAPK and PI 3-kinase signaling cascades (17Misra U.K. Pizzo S.V. Cell. Signal. 2004; 16: 487-496Crossref PubMed Scopus (32) Google Scholar, 18Asplin I.R. Misra U.K. Gawdi G. Gonzalez-Gronow M. Pizzo S.V. Arch. Biochem. Biophys. 2000; 383: 135-141Crossref PubMed Scopus (27) Google Scholar, 19Misra U.K. Gonzalez-Gronow M. Gawdi G. Wang F. Pizzo S.V. Cell. Signal. 2004; 16: 929-938Crossref PubMed Scopus (84) Google Scholar, 20Misra U.K. Gonzalez-Gronow M. Gawdi G. Hart J.P. Johnson C.E. Pizzo S.V. J. Biol. Chem. 2002; 277: 42082-42087Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). As a consequence of these events, α2M* up-regulates DNA synthesis and cellular proliferation (17Misra U.K. Pizzo S.V. Cell. Signal. 2004; 16: 487-496Crossref PubMed Scopus (32) Google Scholar, 18Asplin I.R. Misra U.K. Gawdi G. Gonzalez-Gronow M. Pizzo S.V. Arch. Biochem. Biophys. 2000; 383: 135-141Crossref PubMed Scopus (27) Google Scholar, 19Misra U.K. Gonzalez-Gronow M. Gawdi G. Wang F. Pizzo S.V. Cell. Signal. 2004; 16: 929-938Crossref PubMed Scopus (84) Google Scholar, 20Misra U.K. Gonzalez-Gronow M. Gawdi G. Hart J.P. Johnson C.E. Pizzo S.V. J. Biol. Chem. 2002; 277: 42082-42087Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Based on these and other observations, we hypothesized that α2M* functions like a growth factor, and its receptor functions as a growth factor receptor (13Krieger M. Herz J. Annu. Rev. Biochem. 1994; 63: 601-637Crossref PubMed Scopus (1057) Google Scholar). Low density lipoprotein receptor-related protein-1 was identified in the 1990s as an α2M* receptor. Subsequent studies in our laboratory suggested that a receptor distinct from low density lipoprotein receptor-related protein-1 must account for α2M*-dependent signal transduction (14Misra U.K. Chu C.T. Rubenstein D.S. Gawdi G. Pizzo S.V. Biochem. J. 1993; 290: 885-891Crossref PubMed Scopus (68) Google Scholar, 15Misra U.K. Chu C.T. Gawdi G. Pizzo S.V. J. Biol. Chem. 1994; 269: 18303-18306Abstract Full Text PDF PubMed Google Scholar, 16Misra U.K. Gonzalez-Gronow M. Gawdi G. Pizzo S.V. J. Biol. Chem. 1997; 272: 497-502Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 17Misra U.K. Pizzo S.V. Cell. Signal. 2004; 16: 487-496Crossref PubMed Scopus (32) Google Scholar, 18Asplin I.R. Misra U.K. Gawdi G. Gonzalez-Gronow M. Pizzo S.V. Arch. Biochem. Biophys. 2000; 383: 135-141Crossref PubMed Scopus (27) Google Scholar, 19Misra U.K. Gonzalez-Gronow M. Gawdi G. Wang F. Pizzo S.V. Cell. Signal. 2004; 16: 929-938Crossref PubMed Scopus (84) Google Scholar, 20Misra U.K. Gonzalez-Gronow M. Gawdi G. Hart J.P. Johnson C.E. Pizzo S.V. J. Biol. Chem. 2002; 277: 42082-42087Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). These events require the presence of a small number of sites (∼1500/cells) demonstrating very high ligand affinity (Kd 50–100 pm) for cellular binding of α2M* or its receptor binding domain. This second receptor, initially termed the α2M* signaling receptor, was later isolated from murine peritoneal macrophages and 1-LN human prostate cancer cells and identified as cell surface-associated GPR78, a heat shock protein of the HSP70 family (20Misra U.K. Gonzalez-Gronow M. Gawdi G. Hart J.P. Johnson C.E. Pizzo S.V. J. Biol. Chem. 2002; 277: 42082-42087Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). This molecular chaperone has been highly characterized for its ability to promote cell survival during endoplasmic reticulum (ER) stress (see reviews in Refs. 21Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1919) Google Scholar, 22Lee A.S. Trends Biochem. Sci. 2001; 26: 504-510Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar, 23Harding H.P. Calfon M. Urano F. Novoa I. Ron D. Annu. Rev. Cell. Dev. Biol. 2002; 18: 575-579Crossref PubMed Scopus (800) Google Scholar, 24Zhang K. Kaufman R.J. J. Biol. Chem. 2004; 279: 25935-25938Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar, 25Rutkowski D.T. Kaufman R.J. Trends Cell Biol. 2004; 14: 20-28Abstract Full Text Full Text PDF PubMed Scopus (1173) Google Scholar, 26Hendershot L.M. Mount Sinai J. Med. 2004; 71: 289-297PubMed Google Scholar and references therein). GRP78 is involved in many cellular processes, including antigen presentation, translocation of newly synthesized polypeptides across the ER membrane, and their subsequent folding, maturation, transport, or retrotranslocation (21Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1919) Google Scholar, 22Lee A.S. Trends Biochem. Sci. 2001; 26: 504-510Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar, 23Harding H.P. Calfon M. Urano F. Novoa I. Ron D. Annu. Rev. Cell. Dev. Biol. 2002; 18: 575-579Crossref PubMed Scopus (800) Google Scholar, 24Zhang K. Kaufman R.J. J. Biol. Chem. 2004; 279: 25935-25938Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar, 25Rutkowski D.T. Kaufman R.J. Trends Cell Biol. 2004; 14: 20-28Abstract Full Text Full Text PDF PubMed Scopus (1173) Google Scholar, 26Hendershot L.M. Mount Sinai J. Med. 2004; 71: 289-297PubMed Google Scholar). An increased expression of GPR78 is a part of the unfolded protein response required to alleviate ER stress, maintain ER function, and protect cells against cell death (21Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1919) Google Scholar, 22Lee A.S. Trends Biochem. Sci. 2001; 26: 504-510Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar, 23Harding H.P. Calfon M. Urano F. Novoa I. Ron D. Annu. Rev. Cell. Dev. Biol. 2002; 18: 575-579Crossref PubMed Scopus (800) Google Scholar, 24Zhang K. Kaufman R.J. J. Biol. Chem. 2004; 279: 25935-25938Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar, 25Rutkowski D.T. Kaufman R.J. Trends Cell Biol. 2004; 14: 20-28Abstract Full Text Full Text PDF PubMed Scopus (1173) Google Scholar, 26Hendershot L.M. Mount Sinai J. Med. 2004; 71: 289-297PubMed Google Scholar). GRP78 is constitutively expressed, but its synthesis can be up-regulated by a variety of stressful conditions (21Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1919) Google Scholar, 22Lee A.S. Trends Biochem. Sci. 2001; 26: 504-510Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar, 23Harding H.P. Calfon M. Urano F. Novoa I. Ron D. Annu. Rev. Cell. Dev. Biol. 2002; 18: 575-579Crossref PubMed Scopus (800) Google Scholar, 24Zhang K. Kaufman R.J. J. Biol. Chem. 2004; 279: 25935-25938Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar, 25Rutkowski D.T. Kaufman R.J. Trends Cell Biol. 2004; 14: 20-28Abstract Full Text Full Text PDF PubMed Scopus (1173) Google Scholar, 26Hendershot L.M. Mount Sinai J. Med. 2004; 71: 289-297PubMed Google Scholar, 28Adam L. Vadlamudi R. Kondapaka S. Chernoff B.J. Mendelsohn J. Kumar R. J. Biol. Chem. 1998; 273: 28238-28246Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 29Hart J.P. Gunn M.D. Pizzo S.V. J. Immunol. 2004; 172: 70-78Crossref PubMed Scopus (41) Google Scholar) that perturb protein folding and assembly within the ER, including glucose deprivation, acidosis, and hypoxia (21Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1919) Google Scholar, 22Lee A.S. Trends Biochem. Sci. 2001; 26: 504-510Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar, 23Harding H.P. Calfon M. Urano F. Novoa I. Ron D. Annu. Rev. Cell. Dev. Biol. 2002; 18: 575-579Crossref PubMed Scopus (800) Google Scholar, 24Zhang K. Kaufman R.J. J. Biol. Chem. 2004; 279: 25935-25938Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar, 25Rutkowski D.T. Kaufman R.J. Trends Cell Biol. 2004; 14: 20-28Abstract Full Text Full Text PDF PubMed Scopus (1173) Google Scholar, 26Hendershot L.M. Mount Sinai J. Med. 2004; 71: 289-297PubMed Google Scholar). Poorly vascularized solid tumors demonstrate both hypoxia and acidosis, and these cells may be viewed as highly stressed.The presence of GRP78 on the cell surface has only recently been appreciated. Constitutive cell surface expression on normal cells is low, but various treatments up-regulate its cell surface expression (18Asplin I.R. Misra U.K. Gawdi G. Gonzalez-Gronow M. Pizzo S.V. Arch. Biochem. Biophys. 2000; 383: 135-141Crossref PubMed Scopus (27) Google Scholar). For example, we have demonstrated in vivo up-regulation of GRP78 on the surface of antigen-presenting cells when mice are exposed to various stimuli (27Bhattarcharjee G. Misra U.K. Gawdi G. Cianciolo G. Pizzo S.V. J. Cell Biochem. 2001; 82: 260-270Crossref PubMed Scopus (14) Google Scholar). These events appear to have consequences with respect to α2M*-mediated antigen presentation (28Adam L. Vadlamudi R. Kondapaka S. Chernoff B.J. Mendelsohn J. Kumar R. J. Biol. Chem. 1998; 273: 28238-28246Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Normal fibroblasts do not show cell surface expression of GRP78, and α2M* treatment does not trigger signaling responses by these cells. Rheumatoid synovial fibroblasts, however, express GRP78 on their cell surface and signal briskly when exposed to α2M* (16Misra U.K. Gonzalez-Gronow M. Gawdi G. Pizzo S.V. J. Biol. Chem. 1997; 272: 497-502Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). GRP78 also is expressed to a high degree on the surface of a number of cancer cells, including the highly metastatic 1-LN human prostate cancer cell line (29Hart J.P. Gunn M.D. Pizzo S.V. J. Immunol. 2004; 172: 70-78Crossref PubMed Scopus (41) Google Scholar). By contrast, GRP78 cell surface localization and α2M*-dependent signal transduction do not occur with PC-3 cells, a cell line of low malignant potential, and the parent line for the 1-LN cell line (see, for example, Refs. 17Misra U.K. Pizzo S.V. Cell. Signal. 2004; 16: 487-496Crossref PubMed Scopus (32) Google Scholar, 18Asplin I.R. Misra U.K. Gawdi G. Gonzalez-Gronow M. Pizzo S.V. Arch. Biochem. Biophys. 2000; 383: 135-141Crossref PubMed Scopus (27) Google Scholar, 19Misra U.K. Gonzalez-Gronow M. Gawdi G. Wang F. Pizzo S.V. Cell. Signal. 2004; 16: 929-938Crossref PubMed Scopus (84) Google Scholar). Recent studies have demonstrated antibodies against GRP78 in the sera of prostate cancer patients, and the presence of these antibodies is highly correlated with increased metastatic potential and a poor prognosis (30Mintz P.J. Kim J. Do K.-A. Wang X. Zinner R.G. Cristofanilli M. Arap A.M. Hong W.K. Troncoso P. Logothetis C.J. Pasqualini R. Arap W. Nat. Biotechnol. 2003; 21: 57-63Crossref PubMed Scopus (290) Google Scholar, 31Arap M.A. Lahdenranta J. Mintz P.J. Hajitou A. Sarkis A.S. Arap W. Pasqualini R. Cancer Cell. 2004; 6: 275-284Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar). The appearance of the normally cryptic GRP78 protein on the cell surface in high concentration may be a critical factor in the development of autoantibodies to GRP78.The circulating concentration of α2M is 1–5 μm, and α2M* comprises about 200–500 nm of this pool (32Pizzo S.V. Wu S.M. Colman R.W. Hirsh J. Marder V.J. Salzman E.W. Hemostasis and Thromboses. Basic Principles and Clinical Practice. 4th. Ed. Lippincott, Williams & Wilkins, Baltimore, MD2000: 367-386Google Scholar). It has been estimated that about 1 g of α2M turns over daily (32Pizzo S.V. Wu S.M. Colman R.W. Hirsh J. Marder V.J. Salzman E.W. Hemostasis and Thromboses. Basic Principles and Clinical Practice. 4th. Ed. Lippincott, Williams & Wilkins, Baltimore, MD2000: 367-386Google Scholar). Prostate cancer cells also produce prostate cancer-specific antigen, a proteinase that binds readily to α2M (33Otto A. Bar J. Bienmeier G. J. Urol. 1998; 159: 297-303Crossref PubMed Scopus (62) Google Scholar, 34Zhang W.-M. Finne P. Leinonen J. Vesalainen S. Nordling S. Rannikko S. Stenman U.-H. Clin. Chem. 1998; 44: 2471-2479Crossref PubMed Scopus (69) Google Scholar). Thus highly aggressive prostate cancer may secrete prostate cancer-specific antigen, which by binding to α2M generates α2M* further increasing the concentration of α2M* in the tumor microenvironment. Furthermore, tumors may be viewed as existing under ER stress and tumors protect themselves from ER stress by expressing unfolded protein response, of which enhanced GRP78 synthesis is a biomarker (13Krieger M. Herz J. Annu. Rev. Biochem. 1994; 63: 601-637Crossref PubMed Scopus (1057) Google Scholar, 14Misra U.K. Chu C.T. Rubenstein D.S. Gawdi G. Pizzo S.V. Biochem. J. 1993; 290: 885-891Crossref PubMed Scopus (68) Google Scholar, 15Misra U.K. Chu C.T. Gawdi G. Pizzo S.V. J. Biol. Chem. 1994; 269: 18303-18306Abstract Full Text PDF PubMed Google Scholar, 16Misra U.K. Gonzalez-Gronow M. Gawdi G. Pizzo S.V. J. Biol. Chem. 1997; 272: 497-502Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 17Misra U.K. Pizzo S.V. Cell. Signal. 2004; 16: 487-496Crossref PubMed Scopus (32) Google Scholar, 18Asplin I.R. Misra U.K. Gawdi G. Gonzalez-Gronow M. Pizzo S.V. Arch. Biochem. Biophys. 2000; 383: 135-141Crossref PubMed Scopus (27) Google Scholar). A small pool of newly synthesized GRP78 translocates to cell surface from the ER in association with MTJ-1 (35Misra U.K. Gonzalez-Gronow M. Gawdi G. Pizzo S.V. J. Immunol. 2005; 174: 2092-2097Crossref PubMed Scopus (94) Google Scholar). Therefore, it could be envisaged that, under these conditions, a substantial amount of α2M* would be available to bind to cell surface GRP78 thus triggering the activation of mitogenic signaling-dependent cell proliferation. Because it is known that PAKs can be activated via PI 3-kinase signaling (5Sells M.A. Chernoff J. Trends Cell Biol. 1997; 7: 162-167Abstract Full Text PDF PubMed Scopus (264) Google Scholar, 6Daniels R.H. Bokoch G.M. Trends Biochem. Sci. 1999; 24: 350-355Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 7Vadlamudi R.K. Kumar R. Cancer Metastasis Rev. 2003; 22: 385-393Crossref PubMed Scopus (65) Google Scholar, 8Bokoch G.M. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (872) Google Scholar, 11Roig J. Thazon P.T. Zipfel P.A. Pendergast A.M. Traugh J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14346-14351Crossref PubMed Scopus (42) Google Scholar) and membrane localization (5Sells M.A. Chernoff J. Trends Cell Biol. 1997; 7: 162-167Abstract Full Text PDF PubMed Scopus (264) Google Scholar, 6Daniels R.H. Bokoch G.M. Trends Biochem. Sci. 1999; 24: 350-355Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 7Vadlamudi R.K. Kumar R. Cancer Metastasis Rev. 2003; 22: 385-393Crossref PubMed Scopus (65) Google Scholar, 8Bokoch G.M. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (872) Google Scholar, 36Lu W. Mayer B.J. Oncogene. 1999; 18: 797-806Crossref PubMed Scopus (71) Google Scholar), we suggest that activated PAKs may mediate α2M*-induced affects on 1-LN prostate cancer cells. Here we demonstrated that α2M* mediates PAK-2 activation in 1-LN cells, but PAK-1 is only negligibly affected. We then examined the effect of treating 1-LN cells with α2M* on the mechanism of activation of PAK-2, Rac-1, LIMK, and cofilin. We report that exposure of 1-LN cells to α2M* induces autophosphorylation of PAK-2, activation of the kinase activity of PAK-2 toward myelin basic protein in a tyrosine kinase and PI 3-kinase-dependent manner, and recruitment of PAK-2 to plasma membrane via the adaptor protein NCK. Rac-1 is also activated. We further demonstrate activation of LIMK and cofilin, which are essential for regulating cytoskeletal organization.EXPERIMENTAL PROCEDURESMaterials—Culture media were purchased from Invitrogen. Antibodies against PAK-1, PAK-2, and Bad, as well as the phosphorylated forms of PAK-1, PAK-2, LIMK, cofilin, and Bad (Ser112 or Ser136), were procured from Cell Signaling Technology, Inc. (Beverly, MA). Myelin basic protein and actin antibodies were from Sigma. Anti-GRP78 antibodies were purchased from Stressgen (Victoria, BC, Canada). [γ-33P]ATP (specific activity, 3000 Ci/mmol) was purchased from PerkinElmer Life Sciences. The sources for the inhibitors used have been described previously. α2M* was prepared as described previously. Other reagents used in the study were of analytical reagent quality and were procured locally.Effect of α2M* Stimulation on Activation of PAK-1 and PAK-2 in 1-LN Cells—The highly metastatic human prostate carcinoma cell line 1-LN, derived from less metastatic PC-3 cells, was a kind gift from Dr. Philip Walther (Duke University Medical Center, Durham, NC). Confluent 1-LN cells obtained after overnight incubation in 6-well plates (4 × 106 cells/well) were washed twice with HBSS, with a volume of the HBSS added to the monolayers. One set of cells was stimulated with different concentrations of α2M*, and cells were incubated as above for different time periods. The other set of cells was stimulated with different concentrations of α2M* for 10 min. At the end of incubation, medium was aspirated, and the cells were lysed in lysis buffer containing 20 mm Tris·HCl (pH 8.6), 0.1 m NaCl, 1 mm EDTA, 50 mm NaF, 30 mm sodium pyrophosphate, 1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml leupeptin, and 0.5% Nonidet P-40 for 10 min on ice. The DNA strands were broken by passing the lysates through a 27-gauge needle and syringe several times. The lysates were centrifuged at 800 × g for 5 min at 4 °C to remove cell debris. The supernatants were transferred to clean tubes, and their protein contents were determined (37Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213462) Google Scholar). Equal amounts of lysate proteins were electrophoresed according to Laemmli (38Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206048) Google Scholar). Proteins from gel (10%) were transferred to a Hybond P® membrane and immunoblotted with antibodies against phosphorylated and unph" @default.
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• W2000237287 title "Binding of Activated α2-Macroglobulin to Its Cell Surface Receptor GRP78 in 1-LN Prostate Cancer Cells Regulates PAK-2-dependent Activation of LIMK" @default.
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