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Hyaluronan-mediated CD44 Interaction with RhoGEF and Rho Kinase Promotes Grb2-associated Binder-1 Phosphorylation and Phosphatidylinositol 3-Kinase Signaling Leading to Cytokine (Macrophage-Colony Stimulating Factor) Production and Breast Tumor Progression

2003; Elsevier BV; Volume: 278; Issue: 32 Linguagem: Inglês

10.1074/jbc.m301885200

ISSN

1083-351X

Autores

Lilly Bourguignon, Patrick A. Singleton, Hongbo Zhu, Falko Diedrich,

Tópico(s)

Glycosylation and Glycoproteins Research

Resumo

In this study we have examined CD44 (a hyaluronan (HA) receptor) interaction with a RhoA-specific guanine nucleotide exchange factor (p115RhoGEF) in human metastatic breast tumor cells (MDA-MB-231 cell line). Immunoprecipitation and immunoblot analyses indicate that both CD44 and p115RhoGEF are expressed in MDA-MB-231 cells and that these two proteins are physically associated as a complex in vivo. The binding of HA to MDA-MB-231 cells stimulates p115RhoGEF-mediated RhoA signaling and Rho kinase (ROK) activity, which, in turn, increases serine/threonine phosphorylation of the adaptor protein, Gab-1 (Grb2-associated binder-1). Phosphorylated Gab-1 promotes PI 3-kinase recruitment to CD44v3. Subsequently, PI 3-kinase is activated (in particular, α, β, γ forms but not the δ form of the p110 catalytic subunit), AKT signaling occurs, the cytokine (macrophage-colony stimulating factor (M-CSF)) is produced, and tumor cell-specific phenotypes (e.g. tumor cell growth, survival and invasion) are up-regulated. Our results also demonstrate that HA/CD44-mediated oncogenic events (e.g. AKT activation, M-CSF production and breast tumor cell-specific phenotypes) can be effectively blocked by a PI 3-kinase inhibitor (LY294002). Finally, we have found that overexpression of a dominant-negative form of ROK (by transfection of MBA-MD-231 cells with the Rho-binding domain cDNA of ROK) not only inhibits HA/CD44-mediated RhoA-ROK activation and Gab-1 phosphorylation but also down-regulates oncogenic signaling events (e.g. Gab-1·PI 3-kinase-CD44v3 association, PI 3-kinase-mediated AKT activation, and M-CSF production) and tumor cell behaviors (e.g. cell growth, survival, and invasion). Taken together, these findings strongly suggest that CD44 interaction with p115RhoGEF and ROK plays a pivotal role in promoting Gab-1 phosphorylation leading to Gab-1·PI 3-kinase membrane localization, AKT signaling, and cytokine (M-CSF) production during HA-mediated breast cancer progression. In this study we have examined CD44 (a hyaluronan (HA) receptor) interaction with a RhoA-specific guanine nucleotide exchange factor (p115RhoGEF) in human metastatic breast tumor cells (MDA-MB-231 cell line). Immunoprecipitation and immunoblot analyses indicate that both CD44 and p115RhoGEF are expressed in MDA-MB-231 cells and that these two proteins are physically associated as a complex in vivo. The binding of HA to MDA-MB-231 cells stimulates p115RhoGEF-mediated RhoA signaling and Rho kinase (ROK) activity, which, in turn, increases serine/threonine phosphorylation of the adaptor protein, Gab-1 (Grb2-associated binder-1). Phosphorylated Gab-1 promotes PI 3-kinase recruitment to CD44v3. Subsequently, PI 3-kinase is activated (in particular, α, β, γ forms but not the δ form of the p110 catalytic subunit), AKT signaling occurs, the cytokine (macrophage-colony stimulating factor (M-CSF)) is produced, and tumor cell-specific phenotypes (e.g. tumor cell growth, survival and invasion) are up-regulated. Our results also demonstrate that HA/CD44-mediated oncogenic events (e.g. AKT activation, M-CSF production and breast tumor cell-specific phenotypes) can be effectively blocked by a PI 3-kinase inhibitor (LY294002). Finally, we have found that overexpression of a dominant-negative form of ROK (by transfection of MBA-MD-231 cells with the Rho-binding domain cDNA of ROK) not only inhibits HA/CD44-mediated RhoA-ROK activation and Gab-1 phosphorylation but also down-regulates oncogenic signaling events (e.g. Gab-1·PI 3-kinase-CD44v3 association, PI 3-kinase-mediated AKT activation, and M-CSF production) and tumor cell behaviors (e.g. cell growth, survival, and invasion). Taken together, these findings strongly suggest that CD44 interaction with p115RhoGEF and ROK plays a pivotal role in promoting Gab-1 phosphorylation leading to Gab-1·PI 3-kinase membrane localization, AKT signaling, and cytokine (M-CSF) production during HA-mediated breast cancer progression. CD44 (an hyaluronan (HA) 1The abbreviations used are: HA, hyaluronan; ROK, Rho kinase, also called Rho-associated kinase; PI, phosphatidylinositol; aa, amino acid(s); Gab-1, Grb2-associated binder-1; IRS, insulin receptor substrate; GFP, green fluorescent protein; GTPγS, guanosine 5′-3-O-(thio)triphosphate; GST, glutathione S-transferase; M-CSF, macrophage-colony stimulating factor; DMEM, Dulbecco's modified Eagle's medium; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PIP2, phosphatidylinositol 1,4,5-diphosphate; PIP3, phosphatidylinositol 1,4,5-trisphosphate; PtdIns, phosphatidylinositol; EGF, epidermal growth factor; GEF, guanine nucleotide exchange factor; RGS, regulator of G-protein signaling domain; DH, dbl homology domain; PH, pleckstrin homology domain; RB, Rho-binding domain; RANKL, receptor activator of nuclear factor κB ligand; RANK, RANKL receptor; ERM, ezrin/radixin/moesin. receptor) belongs to a family of multifunctional transmembrane glycoproteins expressed in a number of tissues and cells, including breast tissues and cells (1Bourguignon L.Y.W. J. Mammary Gland Biol. Neoplasia. 2000; 6: 287-297Crossref Scopus (144) Google Scholar, 2Dall P. Heider K.-H. Sinn H.-P. Skroch-Angel P. Adolf G. Kaufmann M. Herrlich P. Ponta H. Int. J. Cancer. 1995; 60: 471-477Crossref PubMed Scopus (157) Google Scholar, 3Iida N. Bourguignon L.Y.W. J. Cell. Physiol. 1995; 162: 127-133Crossref PubMed Scopus (128) Google Scholar, 4Kaufmann M. Heider K.H. Sinn H.P. von Minckwitz G. Ponta H. Herrlich P. Lancet. 1995; 345: 615-619Abstract PubMed Google Scholar, 5Sy M.S. Mori H. Liu D. Curr. Opin. Oncol. 1997; 9: 108-112Crossref PubMed Scopus (52) Google Scholar, 6Kalish E. Iida N. Moffat F.L. Bourguignon L.Y.W. Front. Biosci. 1999; 4: 1-8Crossref PubMed Google Scholar). It is encoded by a single gene that contains 19 exons (7Screaton G.R. Bell M.V. Jackson D.G. Cornelis F.B. Gerth U. Bell J.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12160-12164Crossref PubMed Scopus (982) Google Scholar). Of the 19 exons, 12 exons can be alternatively spliced (7Screaton G.R. Bell M.V. Jackson D.G. Cornelis F.B. Gerth U. Bell J.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12160-12164Crossref PubMed Scopus (982) Google Scholar). Most often, breast tumor cells and tissues express several different CD44 spliced variant (CD44v) isoforms in addition to CD44s (the standard form) and CD44E (the epithelial form) (7Screaton G.R. Bell M.V. Jackson D.G. Cornelis F.B. Gerth U. Bell J.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12160-12164Crossref PubMed Scopus (982) Google Scholar). Because various CD44 isoforms mediate different functions, much attention has been paid to the changes in CD44v isoform production during malignant transformation (1Bourguignon L.Y.W. J. Mammary Gland Biol. Neoplasia. 2000; 6: 287-297Crossref Scopus (144) Google Scholar, 2Dall P. Heider K.-H. Sinn H.-P. Skroch-Angel P. Adolf G. Kaufmann M. Herrlich P. Ponta H. Int. J. Cancer. 1995; 60: 471-477Crossref PubMed Scopus (157) Google Scholar, 3Iida N. Bourguignon L.Y.W. J. Cell. Physiol. 1995; 162: 127-133Crossref PubMed Scopus (128) Google Scholar, 4Kaufmann M. Heider K.H. Sinn H.P. von Minckwitz G. Ponta H. Herrlich P. Lancet. 1995; 345: 615-619Abstract PubMed Google Scholar, 5Sy M.S. Mori H. Liu D. Curr. Opin. Oncol. 1997; 9: 108-112Crossref PubMed Scopus (52) Google Scholar, 6Kalish E. Iida N. Moffat F.L. Bourguignon L.Y.W. Front. Biosci. 1999; 4: 1-8Crossref PubMed Google Scholar). Specifically, there is accumulating evidence that the induction and overexpression of CD44v isoforms are associated with the invasion and progression of breast carcinomas and tumor cell lines (1Bourguignon L.Y.W. J. Mammary Gland Biol. Neoplasia. 2000; 6: 287-297Crossref Scopus (144) Google Scholar, 2Dall P. Heider K.-H. Sinn H.-P. Skroch-Angel P. Adolf G. Kaufmann M. Herrlich P. Ponta H. Int. J. Cancer. 1995; 60: 471-477Crossref PubMed Scopus (157) Google Scholar, 3Iida N. Bourguignon L.Y.W. J. Cell. Physiol. 1995; 162: 127-133Crossref PubMed Scopus (128) Google Scholar, 4Kaufmann M. Heider K.H. Sinn H.P. von Minckwitz G. Ponta H. Herrlich P. Lancet. 1995; 345: 615-619Abstract PubMed Google Scholar, 5Sy M.S. Mori H. Liu D. Curr. Opin. Oncol. 1997; 9: 108-112Crossref PubMed Scopus (52) Google Scholar, 6Kalish E. Iida N. Moffat F.L. Bourguignon L.Y.W. Front. Biosci. 1999; 4: 1-8Crossref PubMed Google Scholar). These CD44v isoforms appear to confer the malignant properties of abnormal adhesion, growth, migration, and invasion (1Bourguignon L.Y.W. J. Mammary Gland Biol. Neoplasia. 2000; 6: 287-297Crossref Scopus (144) Google Scholar, 2Dall P. Heider K.-H. Sinn H.-P. Skroch-Angel P. Adolf G. Kaufmann M. Herrlich P. Ponta H. Int. J. Cancer. 1995; 60: 471-477Crossref PubMed Scopus (157) Google Scholar, 3Iida N. Bourguignon L.Y.W. J. Cell. Physiol. 1995; 162: 127-133Crossref PubMed Scopus (128) Google Scholar, 4Kaufmann M. Heider K.H. Sinn H.P. von Minckwitz G. Ponta H. Herrlich P. Lancet. 1995; 345: 615-619Abstract PubMed Google Scholar, 5Sy M.S. Mori H. Liu D. Curr. Opin. Oncol. 1997; 9: 108-112Crossref PubMed Scopus (52) Google Scholar, 6Kalish E. Iida N. Moffat F.L. Bourguignon L.Y.W. Front. Biosci. 1999; 4: 1-8Crossref PubMed Google Scholar). Furthermore, CD44 has been shown to interact with extracellular matrix components (e.g. hyaluronan (HA) at the N terminus of the extracellular domain) (8Turley E.A. Nobel P.W. Bourguignon L.Y.W. J. Biol. Chem. 2002; 277: 4589-4592Abstract Full Text Full Text PDF PubMed Scopus (877) Google Scholar, 9Liao H.X. Lee D.M. Levesque M.C. Haynes B.F. J. Immunol. 1995; 155: 3938-3945PubMed Google Scholar, 10Yang B. Yang B.L. Savani R.C. Turley E.A. EMBO J. 1994; 13: 286-296Crossref PubMed Scopus (338) Google Scholar) and to contain specific binding sites for the cytoskeletal proteins (e.g. ankyrin and ERM) within the 70-amino acid C terminus of its cytoplasmic domain (11Lokeshwar V.B. Fregien N. Bourguignon L.Y.W. J. Cell Biol. 1994; 126: 1099-1109Crossref PubMed Scopus (200) Google Scholar, 12Bourguignon L.Y.W. Curr. Top. Membr. 1996; 43: 293-312Crossref Scopus (24) Google Scholar, 13Bourguignon L.Y.W. Zhu D. Zhu H. Front. Biosci. 1998; 3: 637-649Crossref PubMed Scopus (107) Google Scholar, 14Zhu D. Bourguignon L.Y.W. Cell Motil. Cytoskel. 1998; 39: 209-222Crossref PubMed Scopus (0) Google Scholar, 15Zhu D. Bourguignon L.Y.W. J. Cell. Physiol. 2000; 183: 182-195Crossref PubMed Scopus (59) Google Scholar, 16Bretscher A. Curr. Opin. Cell Biol. 1999; 11: 109-116Crossref PubMed Scopus (331) Google Scholar). Several mechanisms for the regulation of HA/CD44-mediated function have been suggested. These include modifications by an additional exon-coded structure (via an alternative splicing process) (1Bourguignon L.Y.W. J. Mammary Gland Biol. Neoplasia. 2000; 6: 287-297Crossref Scopus (144) Google Scholar, 2Dall P. Heider K.-H. Sinn H.-P. Skroch-Angel P. Adolf G. Kaufmann M. Herrlich P. Ponta H. Int. J. Cancer. 1995; 60: 471-477Crossref PubMed Scopus (157) Google Scholar, 3Iida N. Bourguignon L.Y.W. J. Cell. Physiol. 1995; 162: 127-133Crossref PubMed Scopus (128) Google Scholar, 4Kaufmann M. Heider K.H. Sinn H.P. von Minckwitz G. Ponta H. Herrlich P. Lancet. 1995; 345: 615-619Abstract PubMed Google Scholar, 5Sy M.S. Mori H. Liu D. Curr. Opin. Oncol. 1997; 9: 108-112Crossref PubMed Scopus (52) Google Scholar, 6Kalish E. Iida N. Moffat F.L. Bourguignon L.Y.W. Front. Biosci. 1999; 4: 1-8Crossref PubMed Google Scholar), variable N/O-linked glycosylation on the extracellular domain of the CD44 (17Lokeshwar V.B. Bourguignon L.Y.W. J. Biol. Chem. 1991; 266: 17983-17989Abstract Full Text PDF PubMed Google Scholar, 18Lokeshwar V.B. Iida N. Bourguignon L.Y.W. J. Biol. Chem. 1996; 271: 23853-23864Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), and selective interactions of the cytoplasmic domain of the CD44 with ankyrin (11Lokeshwar V.B. Fregien N. Bourguignon L.Y.W. J. Cell Biol. 1994; 126: 1099-1109Crossref PubMed Scopus (200) Google Scholar, 15Zhu D. Bourguignon L.Y.W. J. Cell. Physiol. 2000; 183: 182-195Crossref PubMed Scopus (59) Google Scholar) and various signaling molecules (e.g. the Src family tyrosine kinases (14Zhu D. Bourguignon L.Y.W. Cell Motil. Cytoskel. 1998; 39: 209-222Crossref PubMed Scopus (0) Google Scholar, 19Bourguignon L.Y.W. Zhu H. Shao L. Chen Y.W. J. Biol. Chem. 2001; 276: 7327-7336Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar), p185HER2 (20Bourguignon L.Y.W. Zhu H. Chu A. Iida N. Zhang L. Hung M.C. J. Biol. Chem. 1997; 272: 27913-27918Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 21Bourguignon L.Y.W. Zhu H. Zhou B. Diedrich F. Singleton P.A. Hung M.C. J. Biol. Chem. 2001; 276: 48679-48692Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), Rho kinase (ROK) (22Bourguignon L.Y.W. Zhu H. Shao L. Zhu D. Chen Y.W. Cell Motil. Cytoskel. 1999; 43: 269-287Crossref PubMed Scopus (145) Google Scholar), transforming growth factor-β receptor kinases (23Bourguignon L.Y.W. Singleton P. Zhu H. Zhou B. J. Biol. Chem. 2002; 277: 39703-39712Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar), and the guanine nucleotide exchange factors Tiam1 (24Bourguignon L.Y.W. Zhu H. Shao L. Chen Y.W. J. Biol. Chem. 2000; 275: 1829-1838Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar) and Vav2 (21Bourguignon L.Y.W. Zhu H. Zhou B. Diedrich F. Singleton P.A. Hung M.C. J. Biol. Chem. 2001; 276: 48679-48692Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar)). In addition, CD44 has been shown to be involved in the production of cytokines (e.g. interleukin 8 (1Bourguignon L.Y.W. J. Mammary Gland Biol. Neoplasia. 2000; 6: 287-297Crossref Scopus (144) Google Scholar) and fibroblast growth factor-2 (13Bourguignon L.Y.W. Zhu D. Zhu H. Front. Biosci. 1998; 3: 637-649Crossref PubMed Scopus (107) Google Scholar)) and hormones (e.g. parathyroid hormone-related protein (23Bourguignon L.Y.W. Singleton P. Zhu H. Zhou B. J. Biol. Chem. 2002; 277: 39703-39712Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar)) in breast tumor cells. These findings clearly indicate that CD44 plays a pivotal role in activating oncogenic signaling and HA-mediated breast tumor cell function. Members of the Rho subclass of the Ras superfamily (small molecular weight GTPases, e.g. RhoA, Rac1, and Cdc42) are known to transduce signals regulating many cellular processes (25Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5220) Google Scholar). The rationale for our focusing on RhoGTPase is based on previous reports suggesting that CD44-associated cytoskeletal proteins (e.g. ankyrin and ERM) and that tumor cell-specific phenotypes are dependent on RhoGTPase signaling events (16Bretscher A. Curr. Opin. Cell Biol. 1999; 11: 109-116Crossref PubMed Scopus (331) Google Scholar, 22Bourguignon L.Y.W. Zhu H. Shao L. Zhu D. Chen Y.W. Cell Motil. Cytoskel. 1999; 43: 269-287Crossref PubMed Scopus (145) Google Scholar, 26Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita S. Tsukita S. J. Cell Biol. 1996; 135: 36-51Crossref Scopus (511) Google Scholar). Overexpression of certain RhoGTPases in human tumors often correlates with poor prognosis (28Fritz G. Just I. Kaina B. Int. J. Cancer. 1999; 81: 682-687Crossref PubMed Scopus (581) Google Scholar, 29Suwa H. Ohshio G. Imamura T. Watanabe G. Arii S. Imamura M. Narumiya S. Hiai H. Fukumoto M. Br. J. Cancer. 1998; 77: 147-152Crossref PubMed Scopus (251) Google Scholar). In particular, coordinated RhoGTPase signaling is considered to be a possible mechanism underlying cell proliferation and motility, an obvious prerequisite for metastasis (16Bretscher A. Curr. Opin. Cell Biol. 1999; 11: 109-116Crossref PubMed Scopus (331) Google Scholar, 25Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5220) Google Scholar, 26Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita S. Tsukita S. J. Cell Biol. 1996; 135: 36-51Crossref Scopus (511) Google Scholar, 28Fritz G. Just I. Kaina B. Int. J. Cancer. 1999; 81: 682-687Crossref PubMed Scopus (581) Google Scholar, 29Suwa H. Ohshio G. Imamura T. Watanabe G. Arii S. Imamura M. Narumiya S. Hiai H. Fukumoto M. Br. J. Cancer. 1998; 77: 147-152Crossref PubMed Scopus (251) Google Scholar, 30Sahai E. Olson M.F. Marshall C.J. EMBO J. 2001; 20: 755-766Crossref PubMed Scopus (333) Google Scholar, 31Yoshioka K. Nakamori S. Itoh K. Cancer Res. 1999; 59: 2004-2010PubMed Google Scholar). Presently, very little information is available regarding how they are activated by cell surface receptors. To date, at least 30 different guanine nucleotide exchange factors (GEFs) have been identified (32Cerione R.A. Zheng Y. Curr. Opin. Cell Biol. 1996; 8: 216-222Crossref PubMed Scopus (466) Google Scholar). Our recent results indicate that the interaction between CD44v3 isoform and two GEFs, such as Tiam1 (24Bourguignon L.Y.W. Zhu H. Shao L. Chen Y.W. J. Biol. Chem. 2000; 275: 1829-1838Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar) and Vav2 (21Bourguignon L.Y.W. Zhu H. Zhou B. Diedrich F. Singleton P.A. Hung M.C. J. Biol. Chem. 2001; 276: 48679-48692Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), up-regulates Rac1 signaling and cytoskeleton-mediated metastatic tumor progression. As part of our continued effort to identify CD44 isoform-linked GEFs, which correlate with certain metastatic behaviors, a new candidate molecule, named p115RhoGEF, has been identified. p115RhoGEF, which is the human homolog of the mouse protein Lsc, is involved in RhoA activation (33Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (675) Google Scholar); structurally, it contains numerous functional domains and structural motifs found in signal transduction proteins and oncoproteins. These motifs include N-terminal RGS (regulator of G protein signaling) domain, a dbl homology domain (DH), and a pleckstrin homology domain (PH) (33Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (675) Google Scholar, 34Fukuhara S. Murga C. Zohar M. Igishi T. Gutkind J.S. J. Biol. Chem. 1999; 274: 5868-5879Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). In particular, the sequence between amino acids (aa) 421 and aa 635 contains significant sequence homology to the dbl homology domain (DH) of many proteins that exhibit GDP/GTP exchange activity for specific members of the Ras superfamily of GTP-binding proteins (32Cerione R.A. Zheng Y. Curr. Opin. Cell Biol. 1996; 8: 216-222Crossref PubMed Scopus (466) Google Scholar). The p115RhoGEF has been clearly shown to act as a GDP/GTP exchange protein for the Rho subfamily of GTPases, including RhoA (33Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (675) Google Scholar). In addition, p115RhoGEF (similar to PDZ-RhoGEF) contains an RGS domain, which interacts with Gα13 and regulates the ability of DH to carry out GDP/GTP exchange activity for RhoA (33Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (675) Google Scholar, 34Fukuhara S. Murga C. Zohar M. Igishi T. Gutkind J.S. J. Biol. Chem. 1999; 274: 5868-5879Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). In the C-terminal region, p115RhoGEF contains one PH domain that is commonly detected in signaling molecules and cytoskeletal proteins (35Shaw G. Bioassays. 1996; 18: 35-46Crossref PubMed Scopus (253) Google Scholar, 36Hemmings B.A. Science. 1997; 275: 1899Crossref PubMed Scopus (67) Google Scholar). Activation of RhoGTPases such as RhoA has been shown to produce specific structural changes in the plasma membrane associated with actin filament bundling, stress fiber formation, and acto-myosin-based cytoskeletal function (25Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5220) Google Scholar). Several different enzymes have been identified as possible downstream targets for RhoA signaling. One such enzyme is Rho kinase (ROK, also called Rho-associated kinase), which is a serine-threonine kinase known to interact with Rho in a GTP-dependent manner (37Matsui T. Amano M. Yamamoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (938) Google Scholar, 38Leung T. Chen X.Q. Manser E. Lim L. Mol. Cell. Biol. 1996; 16: 5313-5327Crossref PubMed Google Scholar, 39Amano M. Ito M. Kimura K. Fukata Y. Chihara K. Nakano T. Matsuura Y. Kaibuchi K. J. Biol. Chem. 1996; 271: 20246-20249Abstract Full Text Full Text PDF PubMed Scopus (1676) Google Scholar). ROK is composed of four functional domains, including a kinase domain (catalytic site), a coiled-coil domain, a Rho-binding (RB) domain, and a PH domain (40Amano M. Chihara K. Kimura K Fukata Y. Nakamura N. Matsuura Y. Kaibuchi K. Science. 1997; 275: 1308-1311Crossref PubMed Scopus (948) Google Scholar). This enzyme has been shown to regulate cytoskeleton function by phosphorylating several important cytoskeletal regulators, including myosin-binding subunit of myosin phosphatase (41Kimura N. Ito M. Amano M. Chihara K. Fukata Y. Nakafuku M. Yamamori B. Feng J. Nakano T. Okawa K. Iwamatsu K. Kaibuchi K. Science. 1996; 273: 245-248Crossref PubMed Scopus (2435) Google Scholar), calponin (42Kaneko T. Amano M. Maeda A. Goto H. Takahashi K. Ito M. Kaibuchi K. Biochem. Biophys. Res. Commun. 2000; 273: 110-116Crossref PubMed Scopus (106) Google Scholar), adducin (43Fukata Y. Oshiro N. Kinoshita N. Kawano Y. Matsuoka Y. Bennett V. Matsuura Y. Kaibuchi K. J. Cell Biol. 1999; 145: 347-361Crossref PubMed Scopus (255) Google Scholar), and Lin 11/Isl-1/Mec-3 (LIM) kinase (44Amano T. Tanabe K. Eto T. Narumiya S. Mizuno K. Biochem. J. 2001; 354: 149-159Crossref PubMed Scopus (137) Google Scholar). ROK is also involved in the “cross-talk” between Ras and Rho signaling leading to cellular transformation (30Sahai E. Olson M.F. Marshall C.J. EMBO J. 2001; 20: 755-766Crossref PubMed Scopus (333) Google Scholar). We have demonstrated that ROK phosphorylates the cytoplasmic domain of the CD44v3, 8–10 isoform and up-regulates the interaction between the CD44v3, 8–10 isoform and the cytoskeletal protein, ankyrin, during HA/CD44-regulated tumor cell migration (22Bourguignon L.Y.W. Zhu H. Shao L. Zhu D. Chen Y.W. Cell Motil. Cytoskel. 1999; 43: 269-287Crossref PubMed Scopus (145) Google Scholar). Most recently, the PH domain of ROK is found to be involved in the direct binding to CD44- and HA-mediated Ca2+ signaling in endothelial cell function (45Singleton P.A. Bourguignon L.Y.W. Cell Motil. Cytoskel. 2002; 53: 293-316Crossref PubMed Scopus (71) Google Scholar). Thus, ROK is clearly one of the important signaling molecules required for membrane-cytoskeleton interaction, Ca2+ regulation, and HA/CD44-mediated cell function (22Bourguignon L.Y.W. Zhu H. Shao L. Zhu D. Chen Y.W. Cell Motil. Cytoskel. 1999; 43: 269-287Crossref PubMed Scopus (145) Google Scholar, 45Singleton P.A. Bourguignon L.Y.W. Cell Motil. Cytoskel. 2002; 53: 293-316Crossref PubMed Scopus (71) Google Scholar). In the pathogenesis of cancer, Grb-2-associated binder-1 (Gab-1) and phosphatidylinositol 3-kinase (PI 3-kinase) are key mediators in regulating oncogenesis (for review see Ref. 46Katso R. Okkenhaug K. Ahmadi K. White S. Timms J. Waterfield M.D. Annu. Rev. Cell Dev. Biol. 2001; 17: 615-675Crossref PubMed Scopus (1005) Google Scholar). Specifically, Gab-1 (a member of the insulin receptor substrate (IRS) family) functions as one of the major adapter molecules downstream of growth factor signaling (47Hogado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 8: 560-564Crossref Scopus (601) Google Scholar, 48Sun X.J. Rothenberg P. Kahn C.R. Backer J.M. Araki E. Wilden P.A. Cahill D.A. Goldstein B.J. White M.F. Nature. 1991; 352: 73-77Crossref PubMed Scopus (1285) Google Scholar). Gab-1 also possesses multiple phosphorylation sites that could act as docking sites for PI 3-kinase known to consist of a catalytic subunit p110 (α, β, and δ) and regulatory subunit p85 (α, β, and p55γ) or the catalytic subunit p110γ and the regulatory subunit p101 (for reviews see Refs. 46Katso R. Okkenhaug K. Ahmadi K. White S. Timms J. Waterfield M.D. Annu. Rev. Cell Dev. Biol. 2001; 17: 615-675Crossref PubMed Scopus (1005) Google Scholar, 49Vanhaesbroeck B. leevers S.J. Panayatou G. Waterfield M.D. Trends. Biochem. Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (833) Google Scholar, 50Fruman D.A. Meyers R. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1319) Google Scholar, 51Rameh L. Cantley L. J. Biol. Chem. 1999; 274: 8347-8350Abstract Full Text Full Text PDF PubMed Scopus (852) Google Scholar). One recent study found a positive link between HA-CD44 interaction and Gab-1-associated PI 3-kinase activation during the stimulation of cellular transformation by a Met-hepatocyte growth factor oncoprotein (Tpr-Met) (52Kamikura D.M. Khoury H. Maroun C. Naujokas M.A. Park M. Mol. Cell. Biol. 2000; 20: 3482-3496Crossref PubMed Scopus (49) Google Scholar). HA and CD44 also promote PI 3-kinase signaling in a tumor cell-specific manner. For example, PI 3-kinase participates in CD44-mediated survival pathway in colon carcinoma cells (53Bates R.C. Edwards N.S. Burn G.F. Fisher D.E. Cancer Res. 2001; 61: 5275-5283PubMed Google Scholar). HA activates PI 3 kinase-AKT pathways leading to cell motility and cell survival-signaling pathways (54Sohara Y. Ishiguro N. Machida K. Kurata H. Thant A.A. Senga T. Matsuda S. Kimata K. Iwata H. Hamaguchi M. Mol. Biol. Cell. 2001; 12: 1859-1868Crossref PubMed Scopus (89) Google Scholar). The active mutant of p110 subunit of PI 3-kinase exerts its action on the cleavage of CD44 during cancer cell migration (55Kawano Y. Okamoto I. Murakami D. Itoh H. Yoshida M. Ueda S. Saya H.J. J. Biol. Chem. 2000; 275: 29628-29635Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). These findings suggest PI 3-kinase activation is closely coupled with HA-mediated CD44 signaling. Because both Rho signaling and PI 3-kinase activation play an important role in regulating breast tumor progression, we have focused in this study on the relationship between these two signaling pathways during HA/CD44-mediated breast tumor progression. A unique mechanism is described concerning CD44 interaction with p115RhoGEF and ROK that stimulates Gab-1 phosphorylation/membrane localization and PI 3-kinase-AKT activation leading to HA/CD44-regulated cytokine production and tumor cell behaviors required for breast cancer progression. Cell Culture—The breast tumor cell line MDA-MB-231 was obtained from the American Type Culture Collection (ATCC) and grown in Eagle's minimum essential medium supplemented with Earle's salt solution, essential and non-essential amino acids, vitamins, and 10% fetal bovine serum. Antibodies and Reagents—Monoclonal rat anti-human CD44 antibody (clone: 020; isotype: IgG2b; obtained from CMB-Tech, Inc., San Francisco, CA) used in this study recognizes a common determinant of the CD44 class of glycoproteins. Both rabbit anti-CD44v3 antibody and rabbit anti-Rho kinase (ROK) were prepared according to the procedures described previously (22Bourguignon L.Y.W. Zhu H. Shao L. Zhu D. Chen Y.W. Cell Motil. Cytoskel. 1999; 43: 269-287Crossref PubMed Scopus (145) Google Scholar). For the preparation of polyclonal rabbit anti-p115RhoGEF antibody, specific synthetic peptides (∼15–17 amino acids unique for the ROK or p115RhoGEF sequence) were prepared by the Peptide Laboratories using an Advanced Chemtech automatic synthesizer (model ACT350). All polyclonal antibodies were prepared using conventional DEAE-cellulose chromatography and tested to be monospecific (by immunoblot assays). Mouse monoclonal anti-green fluorescent protein (GFP) and mouse monoclonal anti-FLAG (M2) were purchased from BD Pharmingen and Sigma, respectively. Rabbit anti-phospho-threonine antibody and rabbit anti-phospho-serine antibody were obtained from Zymed Laboratories Inc.. Monoclonal mouse anti-p110α, mouse anti-p110β, mouse anti-p110γ and mouse anti-p110δ were purchased from Santa Cruz Biotechnology. Several other immunoreagents, including mouse anti-AKT-1 (protein kinase B), rabbit anti-phospho-AKT-1 (threonine 308), and rabbit anti-Gab-1, were purchased from Upstate Biotechnology, Inc. The specific inhibitor of PI 3-kinase (LY294002) was obtained from Calbiochem. Rooster comb hyaluronan (HA) was purchased from Sigma. High molecular mass HA polymers (∼106 Da) were purified by gel filtration column chromatography using a Sephacryl S1000 column as described previously (18Lokeshwar V.B. Iida N. Bourguignon L.Y.W. J. Biol. Chem. 1996; 271: 23853-23864Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The purity of high molecular weight HA polymers used in our experiments was further verified by anion exchange high performance liquid chromatography. No small HA fragments was detected in these preparations. Cloning, Expression, and Purification of CD44 Cytoplasmic Domain (CD44cyt) from E. coli—The cytoplasmic domain of human CD44 (CD44cyt) was cloned into pFLAG-AST using the PCR-based cloning strategy (15Zhu D. Bourguignon L.Y.W. J. Cell. Physiol. 2000; 183: 182-195Crossref PubMed Scopus (59) Google Scholar). Using human CD44 cDNA as template, one PCR primer pair (left, FLAG-EcoRI; right, FLAG-XbaI) was designed to amplify complete CD44 cytoplasmic domain. The amplified DNA fragments were one-step-cloned into a pCR2.1 vector and sequenced. Then, the DNA fragments were cut out by doub

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