Regulation of HEF1 Expression and Phosphorylation by TGF-β1 and Cell Adhesion
2002; Elsevier BV; Volume: 277; Issue: 42 Linguagem: Inglês
10.1074/jbc.m202263200
ISSN1083-351X
AutoresMingzhe Zheng, Paula J. McKeown‐Longo,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoTransforming growth factor-β1 (TGF-β1) is a multipotential cytokine, which regulates remodeling of tissue extracellular matrix during early tumorigenesis and wound healing. Human enhancer of filamentation-1 (HEF1), a multifunctional docking protein, is involved in integrin-based signaling, which affects cell motility, growth, and apoptosis. Our studies reveal that TGF-β1 is a potent inducer of HEF1 gene transcription in human dermal fibroblasts. TGF-β1 promoted HEF1 expression in a dose-dependent manner and resulted in a 16-fold increase in HEF1 protein level. TGF-β1 had no effect on the stability of either HEF1 protein or mRNA. The TGF-β1-induced HEF1 expression was independent of cell adhesion and resistant to cytoskeleton disruption. TGF-β1 increased levels of both p105 and p115 HEF1 in adherent fibroblasts. Digestion with specific phosphatases indicated that the p115HEF1 resulted from serine/threonine phosphorylation of p105HEF1. The appearance of the p115HEF1 as well as tyrosine phosphorylation of p105HEF1 required cell adhesion and/or an organized cytoskeleton. Anin vitro kinase assay indicated that p105HEF1 was a substrate for Src. PP1, a specific Src kinase inhibitor, was able to block adhesion-dependent tyrosine phosphorylation of p105HEF1. These findings suggest that TGF-β1 regulatesHEF1 gene expression and that HEF1 phosphorylation is dependent on cell adhesion and Src kinase activity. Transforming growth factor-β1 (TGF-β1) is a multipotential cytokine, which regulates remodeling of tissue extracellular matrix during early tumorigenesis and wound healing. Human enhancer of filamentation-1 (HEF1), a multifunctional docking protein, is involved in integrin-based signaling, which affects cell motility, growth, and apoptosis. Our studies reveal that TGF-β1 is a potent inducer of HEF1 gene transcription in human dermal fibroblasts. TGF-β1 promoted HEF1 expression in a dose-dependent manner and resulted in a 16-fold increase in HEF1 protein level. TGF-β1 had no effect on the stability of either HEF1 protein or mRNA. The TGF-β1-induced HEF1 expression was independent of cell adhesion and resistant to cytoskeleton disruption. TGF-β1 increased levels of both p105 and p115 HEF1 in adherent fibroblasts. Digestion with specific phosphatases indicated that the p115HEF1 resulted from serine/threonine phosphorylation of p105HEF1. The appearance of the p115HEF1 as well as tyrosine phosphorylation of p105HEF1 required cell adhesion and/or an organized cytoskeleton. Anin vitro kinase assay indicated that p105HEF1 was a substrate for Src. PP1, a specific Src kinase inhibitor, was able to block adhesion-dependent tyrosine phosphorylation of p105HEF1. These findings suggest that TGF-β1 regulatesHEF1 gene expression and that HEF1 phosphorylation is dependent on cell adhesion and Src kinase activity. transforming growth factor β1 Src homology bovine serum albumin phenylmethylsulfonyl fluoride focal adhesion kinase Dulbecco's modified Eagle's medium protein-tyrosine phosphatase monoclonal antibody fibroblast growth factor platelet-derived growth factor epidermal growth factor calf intestinal alkaline phosphatase polyclonal antibody 6-carboxyfluorescein 6-carboxy-tetramethylrhodamine human enhancer of filamentation 1 Remodeling of the tissue extracellular matrix occurs as a component of a number of physiological and pathological processes including wound healing as well as tumorigenesis. Changes in tumor stroma, including deposition of extracellular matrix, activation of fibroblasts and inflammatory cells, and recruitment of endothelial cells have been proposed to mimic steps of wound healing (1Dvorak H.F. N. Engl. J. Med. 1986; 315: 1650-1659Crossref PubMed Scopus (3480) Google Scholar, 2Skobe M. Fusenig N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1050-1055Crossref PubMed Scopus (196) Google Scholar, 3Olumi A.F. Grossfeld G.D. Hayward S.W. Carroll P.R. Tlsty T.D. Cunha G.R. Cancer Res. 1999; 59: 5002-5011PubMed Google Scholar, 4Hudson J.D. Shoaibi M.A. Maestro R. Carnero A. Hannon G.J. Beach D.H. J. Exp. Med. 1999; 190: 1375-1382Crossref PubMed Scopus (566) Google Scholar). During wound healing, transforming growth factor-β1 (TGF-β1),1 a growth regulatory peptide is released from platelets in response to tissue injury. TGF-β1 plays a complex role in the healing of wounds through its ability to regulate growth, motility, and differentiation of various cell types (5Sporn M.B. Roberts A.B. J. Clin. Invest. 1993; 92: 2565-2566Crossref PubMed Scopus (89) Google Scholar). The effects of TGF-β1 are in part mediated through changes in the expression of a number of genes, which regulate extracellular matrix deposition as well as cell adhesion and motility. Genetic targets of TGF-β1 include matrix molecules such as collagen and fibronectin, metalloproteases, protease inhibitors, and the integrin family of adhesion receptors (6Ignotz R.A. Massague J. J. Biol. Chem. 1986; 261: 4337-4345Abstract Full Text PDF PubMed Google Scholar, 7Ignotz R.A. Massague J. Cell. 1987; 51: 189-197Abstract Full Text PDF PubMed Scopus (369) Google Scholar). TGF-β1-dependent matrix remodeling also occurs during tumor progression, where TGF-β1 secreted by the tumor is thought to stimulate fibroblast-dependent changes in the stromal matrix that are necessary for angiogenesis and tumor growth (8Hanahan D. Weinberg R.A. Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (21908) Google Scholar, 9Rennov-Jessen L. Petersen O.W. Bissell M.J. Physiol. Rev. 1996; 76: 69-125Crossref PubMed Scopus (638) Google Scholar).Human enhancer of filamentation 1, HEF1, is a multidomain docking protein of the Cas family, which is believed to participate in integrin-based signaling pathways (10O'Neill G.M. Fashena S.J. Golemis E.A Trends Cell Biol. 2000; 10: 111-119Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). HEF1 was initially identified as a human protein that confers changes in actin organization in yeast (11Law S.F. Estojak J. Wang B. Mysliwiec T. Kruh G. Golemis E.A. Mol. Cell. Biol. 1996; 16: 3327-3337Crossref PubMed Scopus (221) Google Scholar) as well as changes in the shape of mammalian cells (10O'Neill G.M. Fashena S.J. Golemis E.A Trends Cell Biol. 2000; 10: 111-119Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 12Fashena S.J. Einarson M.B. O'Neill G.M. Patriotis C. Golemis E.A. J. Cell Sci. 2002; 115: 99-111PubMed Google Scholar). The protein sequence and domain structure of HEF1 are similar to that of p130Cas, with both proteins possessing an amino-terminal SH3 domain, multiple potential SH2-binding sites in the central substrate domain, and a carboxyl-terminal dimerization module (11Law S.F. Estojak J. Wang B. Mysliwiec T. Kruh G. Golemis E.A. Mol. Cell. Biol. 1996; 16: 3327-3337Crossref PubMed Scopus (221) Google Scholar). Ligation of β1 integrins in hematopoietic or lymphocytic cells causes tyrosine phosphorylation of HEF1 (13Sattler M. Salgia R. Shrikhande G. Verma S. Uemura N. Law S.F. Golemis E.A. Griffin J.D. J. Biol. Chem. 1997; 272: 14320-14326Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 14Manie S.N. Beck A.R. Astier A Law S.F. Canty T. Hirai H. Druker B.J. Avraham H. Haghayeghi N. Sattler M. Salgia R. Griffin J.D. Golemis E.A. Freedman A.S. J. Biol. Chem. 1997; 272: 4230-4236Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 15Minegishi M. Tachibana K. Sato T. Iwata S. Nojima Y. Morimoto C. J. Exp. Med. 1996; 184: 1365-1375Crossref PubMed Scopus (147) Google Scholar). HEF1 is a substrate for several tyrosine kinases, including FAK, RAFTK, and Src family members (16Astier A Manie S.N. Avraham H. Hirai H. Law S.F. Zhang Y-Z., Fu, Y. Druker B.J. Haghayeghi N. Freedman A.S. Avraham S. J. Biol. Chem. 1997; 272: 19719-19724Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 17deJong R. van Wijk A. Haataja L. Heisterkamp N. Groffen J. J. Biol. Chem. 1997; 272: 32649-32655Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 18Zhang Z. Baron R. Horne W.C. J. Biol. Chem. 2000; 275: 37219-37223Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In adherent cells, HEF1 localizes to focal adhesions, where it may modulate adhesion-based signaling (11Law S.F. Estojak J. Wang B. Mysliwiec T. Kruh G. Golemis E.A. Mol. Cell. Biol. 1996; 16: 3327-3337Crossref PubMed Scopus (221) Google Scholar, 19Kanda H. Mimura T. Hamasaki K. Yamamoto K. Yazaki Y. Hirai H. Nojima Y. Immunology. 1999; 97: 56-61Crossref PubMed Scopus (17) Google Scholar). Recent studies suggest that phosphorylated HEF1 can function as a downstream effector of FAK to promote integrin-dependent cell motility (20van Seventer G.A. Salmen H.J. Law S.F. O'Neill G.M. Mullen M.M. Franz M. Kanner S.B. Golemis E.A. van Seventer J.M. Eur. J. Immunol. 2001; 31: 1417-1427Crossref PubMed Scopus (76) Google Scholar, 21Law S.F. O'Neill G.M. Fashena S.J. Einarson M.B. Golemis E.A. Mol. Cell. Biol. 2000; 20: 5184-5195Crossref PubMed Scopus (79) Google Scholar, 22Ohashi Y. Iwata S. Kamiguchi K. Morimoto C. J. Immunol. 1999; 163: 3727-3734PubMed Google Scholar). In the breast cancer cell line, MCF-7, HEF1 is post-translationally processed during the cell cycle and during apoptosis, giving rise to specific forms (p105, p115, p55, and p28) which differentially localize to both focal adhesions as well as the nucleus (21Law S.F. O'Neill G.M. Fashena S.J. Einarson M.B. Golemis E.A. Mol. Cell. Biol. 2000; 20: 5184-5195Crossref PubMed Scopus (79) Google Scholar, 23Law S.F. Zhang Y.-Z. Klein-Szanto A.J.P. Golemis E.A. Mol. Cell. Biol. 1998; 18: 3540-3551Crossref PubMed Scopus (94) Google Scholar). Molecular genetic experiments have indicated that HEF1 overexpression leads to an increase in cell motility and apoptosis, consistent with a role for HEF1 in regulating integrin signaling (12Fashena S.J. Einarson M.B. O'Neill G.M. Patriotis C. Golemis E.A. J. Cell Sci. 2002; 115: 99-111PubMed Google Scholar, 20van Seventer G.A. Salmen H.J. Law S.F. O'Neill G.M. Mullen M.M. Franz M. Kanner S.B. Golemis E.A. van Seventer J.M. Eur. J. Immunol. 2001; 31: 1417-1427Crossref PubMed Scopus (76) Google Scholar, 21Law S.F. O'Neill G.M. Fashena S.J. Einarson M.B. Golemis E.A. Mol. Cell. Biol. 2000; 20: 5184-5195Crossref PubMed Scopus (79) Google Scholar, 22Ohashi Y. Iwata S. Kamiguchi K. Morimoto C. J. Immunol. 1999; 163: 3727-3734PubMed Google Scholar).Earlier studies have proposed that HEF1 is expressed primarily in cells of lymphoid and epithelial origin (11Law S.F. Estojak J. Wang B. Mysliwiec T. Kruh G. Golemis E.A. Mol. Cell. Biol. 1996; 16: 3327-3337Crossref PubMed Scopus (221) Google Scholar, 23Law S.F. Zhang Y.-Z. Klein-Szanto A.J.P. Golemis E.A. Mol. Cell. Biol. 1998; 18: 3540-3551Crossref PubMed Scopus (94) Google Scholar). In the present study we have found that p115 and p105 HEF1 are expressed in dermal fibroblasts and that their expression is regulated by TGF-β1. TGF-β1 up-regulated HEF1 mRNA transcription and induced a rapid increase in total HEF1 synthesis, which resulted in a 16-fold increase in HEF1 protein levels within 4 h. Tyrosine phosphorylation of p105HEF1 was dependent on Src kinase and cell adhesion. Serine/threonine phosphorylation of HEF1 was also dependent on cell adhesion. Disruption of either cell adhesion or actin organization resulted in a rapid dephosphorylation of both tyrosine and serine/threonine residues. These findings demonstrate that in dermal fibroblasts, the levels of phosphorylated HEF1 are coordinately regulated by both growth factors and cell adhesion.DISCUSSIONIn this study, we have found that TGF-β1 is a potent inducer of expression of the Cas family member, HEF1, in human dermal fibroblasts. An increase of more than 16-fold in HEF1 protein levels was detected when quiescent fibroblasts were treated with 1 ng/ml of TGF-β1 for 4 h. TGF-β1 induced HEF1 expression by up-regulating HEF1 mRNA transcription and had no effect on either HEF1 protein or mRNA turnover. Regulation of HEF1 gene expression by TGF-β1 was unaffected by either actin-altering drugs or by cell suspension suggesting that adhesion- or cell shape-based signaling pathways are not involved in the regulation of HEF1 expression by TGF-β1. In contrast, the induction of the plasminogen-activator inhibitor gene by TGF-β1 has recently been shown to be regulated by adhesion (37Kutz S.M. Hordines J. McKeown-Longo P.J. Higgins P.J. J. Cell Sci. 2001; 114: 3905-3914Crossref PubMed Google Scholar). Varied effects of TGF-β1 on HEF1 expression have been reported previously. In H-9 lymphoblastoid cells as well as A549 lung carcinoma cells, TGF-β1 induced a rapid decrease in HEF1 levels which was dependent on proteosome-mediated degradation (38Liu X. Elia A.E. Law S.F. Golemis E.A. Farley J.R. Wang T. EMBO J. 2000; 19: 6759-6769Crossref PubMed Scopus (64) Google Scholar). In the A549 cells, the initial loss of endogenous HEF1 was followed by a modest (4-fold) increase in HEF1 protein which was due to an increase in gene expression (38Liu X. Elia A.E. Law S.F. Golemis E.A. Farley J.R. Wang T. EMBO J. 2000; 19: 6759-6769Crossref PubMed Scopus (64) Google Scholar). In contrast, we found no decrease in HEF1 protein levels following TGF-β1 treatment in human skin fibroblasts (Fig. 3). It has been reported that expression of HEF1 in human breast carcinoma cells is cell cycle-regulated and rapidly enhanced upon induction of cell growth (23Law S.F. Zhang Y.-Z. Klein-Szanto A.J.P. Golemis E.A. Mol. Cell. Biol. 1998; 18: 3540-3551Crossref PubMed Scopus (94) Google Scholar). The results presented here demonstrate that there are only slight effects on HEF1 expression by treatment of serum-starved fibroblasts with EGF, acidic FGF, basic FGF, or PDGF, which are known mitogens for fibroblasts (Fig.1). Apparently, mitogenic signals are insufficient to induce high levels of HEF1. Furthermore, TGF-β1-induced total HEF1 production is not changed in suspended fibroblasts in which cell proliferation is interrupted (Fig. 10, panels A and B). These findings suggest that the function of TGF-β1-induced HEF1 in fibroblasts may be unrelated to cell growth.Previous studies using hematopoetic cells have shown that ligation of the β1 integrin with fibronectin or with antibodies to the integrin receptor results in the tyrosine phosphorylation of HEF1 within 30 min (13Sattler M. Salgia R. Shrikhande G. Verma S. Uemura N. Law S.F. Golemis E.A. Griffin J.D. J. Biol. Chem. 1997; 272: 14320-14326Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 14Manie S.N. Beck A.R. Astier A Law S.F. Canty T. Hirai H. Druker B.J. Avraham H. Haghayeghi N. Sattler M. Salgia R. Griffin J.D. Golemis E.A. Freedman A.S. J. Biol. Chem. 1997; 272: 4230-4236Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 15Minegishi M. Tachibana K. Sato T. Iwata S. Nojima Y. Morimoto C. J. Exp. Med. 1996; 184: 1365-1375Crossref PubMed Scopus (147) Google Scholar), implicating a role for HEF1 in "outside-in" signaling through the integrin. In adherent cells, HEF1 can form complexes with Abl, as well as Src kinases, and overexpressing of either kinase has been shown to up-regulate tyrosine phorphorylation of HEF1 (11Law S.F. Estojak J. Wang B. Mysliwiec T. Kruh G. Golemis E.A. Mol. Cell. Biol. 1996; 16: 3327-3337Crossref PubMed Scopus (221) Google Scholar, 18Zhang Z. Baron R. Horne W.C. J. Biol. Chem. 2000; 275: 37219-37223Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Evidence presented here indicates that HEF1 is a substrate for Src but not Abl kinase and that Src kinase is required for the adhesion-dependent tyrosine phosphorylation of HEF1. These results also indicate that Src, rather than Abl, plays an important role in adhesion-regulated tyrosine phosphorylation of HEF1 in human dermal fibroblasts and that HEF1 is part of the integrin signaling pathway.In this study, we have shown that the appearance of the p115 species of HEF1 is also regulated by cell adhesion. Earlier studies in MCF-7 cells have suggested that the p115 species of HEF1 may represent a serine/threonine phosphorylated form of HEF1 (23Law S.F. Zhang Y.-Z. Klein-Szanto A.J.P. Golemis E.A. Mol. Cell. Biol. 1998; 18: 3540-3551Crossref PubMed Scopus (94) Google Scholar). Our results support this earlier finding and extend it to show that the conversion of p105HEF1 to p115HEF1 is adhesion-dependent. Conversion of p105HEF1 to p115HEF1 occurred as a consequence of adhesion of cells to numerous matrix molecules and was sensitive to drugs that disrupt actin stress fibers. This suggests that serine/threonine phosphorylation of HEF1 arises as a consequence of cytoskeleton organization rather than from ligation of a specific integrin receptor. Serine/threonine phosphorylation of HEF1 can be distinguished from integrin-dependent tyrosine phosphorylation of other focal adhesion proteins. Fibronectin-dependent adhesion quickly induced tyrosine phosphorylation of p130Cas to a maximal level within 15 min (Fig. 6), in a manner similar to integrin-dependent tyrosine phosphorylation of tensin, paxillin, and FAK (31Nojima Y. Morino N. Mimura T. Hasasaki K. Furuya H. Sakai R. Sato T. Tachibana K. Miromoto C. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 15398-15402Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 34Bockholt S.M. Burridge K. J. Biol. Chem. 1993; 268: 14565-14567Abstract Full Text PDF PubMed Google Scholar, 40Burridge K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1175) Google Scholar, 41Bhattacharya S., Fu, C. Bhattacharya J. Greenberg S. J. Biol. Chem. 1995; 270: 16781-16787Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In contrast, the serine/threonine phosphorylation of HEF1 occurred between 1 and 2 h after adhesion (Fig. 6), suggesting that activation of serine/threonine kinases occurs subsequent to the phosphorylation of HEF1 on tyrosine.Treatment of cells with latrunculin A resulted in a rapid loss of phosphorylated species of HEF1. Tyrosine phosphorylation was lost within 30 min, while p115HEF was decreased 50% by 30 min. Similar results were seen when cells were placed in suspension. This rapid loss of phosphorylated species of HEF1 suggests that disruption of actin fibers within the cell results in an increase in both tyrosine and serine/threonine phosphatase activity. Replating of suspended cells onto a fibronectin substrate resulted in a complete restoration of p115HEF1 levels within 2 h. These findings indicate that the interconversion between p105HEF1 and p115 HEF1 may be caused by the cytoskeletal regulation of both serine/threonine kinases and phosphatases. TGF-β1 did not alter the kinetics of appearance of p115HEF1 and tyrosine-phosphorylated p105HEF1 nor the ratio of p115versus p105HEF1 and tyrosine-phosphorylated p105HEF1versus p105HEF1 protein, suggesting that TGF-β1 affects only HEF1 levels and not post-translational modifications by the kinases or phosphatases.The functional significance of the serine/threonine phosphorylation of HEF1 is not well understood. HEF1 is differentially proteolyzed by caspases to yield a 55-kDa and a 28-kDa fragment during mitosis and apoptosis, respectively (21Law S.F. O'Neill G.M. Fashena S.J. Einarson M.B. Golemis E.A. Mol. Cell. Biol. 2000; 20: 5184-5195Crossref PubMed Scopus (79) Google Scholar, 23Law S.F. Zhang Y.-Z. Klein-Szanto A.J.P. Golemis E.A. Mol. Cell. Biol. 1998; 18: 3540-3551Crossref PubMed Scopus (94) Google Scholar). It has been proposed that specific HEF1 fragments have distinct activities as the 55 kDa associates with the mitotic spindle while the 28-kDa fragment causes apoptosis. The caspase cleavage site, which generates the 55-kDa fragment is immediately adjacent to a serine-rich region, raising the possibility that differential phosphorylation of serines might help regulate the sensitivity of HEF1 to caspase-mediated degradation. Another role for phosphorylated HEF1 has been proposed in a recent study, which has shown that p115HEF1 can complex with smad3 and inhibit smad3-mediated gene responses to TGF-β1 (38Liu X. Elia A.E. Law S.F. Golemis E.A. Farley J.R. Wang T. EMBO J. 2000; 19: 6759-6769Crossref PubMed Scopus (64) Google Scholar). Therefore, p115HEF1 may also function to negatively regulate TGF-β1 signaling pathways, which require smad3.The relationship of HEF1 to TGF-β1 function in dermal fibroblasts is not known. A role for HEF1 in growth control or apoptosis does not seem likely as TGF-β1 does not normally regulate these processes in differentiated fibroblasts. As HEF1 has been shown to be part of the signaling pathway regulated by the β1 integrin, one might speculate that HEF1 levels are coordinately increased to facilitate changes in intracellular signaling pathways, which might accompany the TGF-β1-induced changes in matrix composition. TGF-β1-mediated changes in fibronectin matrix are known to promote the differentiation of fibroblasts to myofibroblasts (42Serini G. Bochaton-Piallat M.L. Ropraz P. Geinoz A. Borsi L. Zardi L. Gabbiani G. J. Cell Biol. 1998; 142: 873-881Crossref PubMed Scopus (668) Google Scholar, 43Vaughan M.B. Howard E.W. Tomasek J.J. Exp. Cell Res. 2000; 257: 180-189Crossref PubMed Scopus (397) Google Scholar), a process, which is associated with an increase in actin stress fiber formation as well as cellular contractility. The known effects of HEF on cell shape as well as integrin function suggest that HEF1 may participate in the coordination of signals from the ECM with the cytoskeleton to effect contractile-based changes in fibroblast function during wound healing and/or tumor progression. TGF-β1-promoted increases in cellular contractility participate in wound contraction as well as enhance fibronectin matrix deposition (44Allen-Hoffman B.L. Crankshaw C.L. Mosher D.F. Mol. Cell. Biol. 1988; 8: 4234-4242Crossref PubMed Scopus (55) Google Scholar, 45Reed M.J. Vernon R.B. Abrass I.B. Sage E.H. J. Cell. Physiol. 1994; 158: 169-179Crossref PubMed Scopus (161) Google Scholar). During tumor progression, TGF-β1 secreted by tumor cells may have a paracrine function to stimulate tumor stroma. Increased-matrix fibronectin and fibronectin synthesis in stroma is associated with breast tumors and melanoma (30Bittner M. Meltzer P. Chen Y. Jiang Y. Seftor E. Hendrix M. Radmacher M. Simon R. Yakhini Z. Ben-Dor A. Sampas N. Dougherty E. Wang E. Marincola F. Gooden C. Lueders J. Glatfelter A. Pollock P. Carpten J. Gillanders E. Leja D. Dietrich K. Beaudry C. Berens M. Alberts D. Sondak V. Nature. 2000; 406: 536-540Crossref PubMed Scopus (1697) Google Scholar,39D'Ovidio M.C. Mastracchio A. Marzullo A. Ciabatta M. Pini B. Uccini S. Zardi L. Ruco L.P. Eur. J. Cancer. 1998; 34: 1081-1085Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Recent experiments suggest that the TGF-β1-mediated increase in matrix production by stromal cells may facilitate melanoma survival and metastasis (36Berking C. Takemoto R. Schaider H. Showe L. Satyamoorthy K.P. Robbins P. Herlyn M. Cancer Res. 2001; 61: 8306-8316PubMed Google Scholar). Further studies will be needed to determine the role of HEF1 in mediating the effects of TGF-β1 on dermal fibroblasts. Remodeling of the tissue extracellular matrix occurs as a component of a number of physiological and pathological processes including wound healing as well as tumorigenesis. Changes in tumor stroma, including deposition of extracellular matrix, activation of fibroblasts and inflammatory cells, and recruitment of endothelial cells have been proposed to mimic steps of wound healing (1Dvorak H.F. N. Engl. J. Med. 1986; 315: 1650-1659Crossref PubMed Scopus (3480) Google Scholar, 2Skobe M. Fusenig N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1050-1055Crossref PubMed Scopus (196) Google Scholar, 3Olumi A.F. Grossfeld G.D. Hayward S.W. Carroll P.R. Tlsty T.D. Cunha G.R. Cancer Res. 1999; 59: 5002-5011PubMed Google Scholar, 4Hudson J.D. Shoaibi M.A. Maestro R. Carnero A. Hannon G.J. Beach D.H. J. Exp. Med. 1999; 190: 1375-1382Crossref PubMed Scopus (566) Google Scholar). During wound healing, transforming growth factor-β1 (TGF-β1),1 a growth regulatory peptide is released from platelets in response to tissue injury. TGF-β1 plays a complex role in the healing of wounds through its ability to regulate growth, motility, and differentiation of various cell types (5Sporn M.B. Roberts A.B. J. Clin. Invest. 1993; 92: 2565-2566Crossref PubMed Scopus (89) Google Scholar). The effects of TGF-β1 are in part mediated through changes in the expression of a number of genes, which regulate extracellular matrix deposition as well as cell adhesion and motility. Genetic targets of TGF-β1 include matrix molecules such as collagen and fibronectin, metalloproteases, protease inhibitors, and the integrin family of adhesion receptors (6Ignotz R.A. Massague J. J. Biol. Chem. 1986; 261: 4337-4345Abstract Full Text PDF PubMed Google Scholar, 7Ignotz R.A. Massague J. Cell. 1987; 51: 189-197Abstract Full Text PDF PubMed Scopus (369) Google Scholar). TGF-β1-dependent matrix remodeling also occurs during tumor progression, where TGF-β1 secreted by the tumor is thought to stimulate fibroblast-dependent changes in the stromal matrix that are necessary for angiogenesis and tumor growth (8Hanahan D. Weinberg R.A. Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (21908) Google Scholar, 9Rennov-Jessen L. Petersen O.W. Bissell M.J. Physiol. Rev. 1996; 76: 69-125Crossref PubMed Scopus (638) Google Scholar). Human enhancer of filamentation 1, HEF1, is a multidomain docking protein of the Cas family, which is believed to participate in integrin-based signaling pathways (10O'Neill G.M. Fashena S.J. Golemis E.A Trends Cell Biol. 2000; 10: 111-119Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). HEF1 was initially identified as a human protein that confers changes in actin organization in yeast (11Law S.F. Estojak J. Wang B. Mysliwiec T. Kruh G. Golemis E.A. Mol. Cell. Biol. 1996; 16: 3327-3337Crossref PubMed Scopus (221) Google Scholar) as well as changes in the shape of mammalian cells (10O'Neill G.M. Fashena S.J. Golemis E.A Trends Cell Biol. 2000; 10: 111-119Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 12Fashena S.J. Einarson M.B. O'Neill G.M. Patriotis C. Golemis E.A. J. Cell Sci. 2002; 115: 99-111PubMed Google Scholar). The protein sequence and domain structure of HEF1 are similar to that of p130Cas, with both proteins possessing an amino-terminal SH3 domain, multiple potential SH2-binding sites in the central substrate domain, and a carboxyl-terminal dimerization module (11Law S.F. Estojak J. Wang B. Mysliwiec T. Kruh G. Golemis E.A. Mol. Cell. Biol. 1996; 16: 3327-3337Crossref PubMed Scopus (221) Google Scholar). Ligation of β1 integrins in hematopoietic or lymphocytic cells causes tyrosine phosphorylation of HEF1 (13Sattler M. Salgia R. Shrikhande G. Verma S. Uemura N. Law S.F. Golemis E.A. Griffin J.D. J. Biol. Chem. 1997; 272: 14320-14326Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 14Manie S.N. Beck A.R. Astier A Law S.F. Canty T. Hirai H. Druker B.J. Avraham H. Haghayeghi N. Sattler M. Salgia R. Griffin J.D. Golemis E.A. Freedman A.S. J. Biol. Chem. 1997; 272: 4230-4236Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 15Minegishi M. Tachibana K. Sato T. Iwata S. Nojima Y. Morimoto C. J. Exp. Med. 1996; 184: 1365-1375Crossref PubMed Scopus (147) Google Scholar). HEF1 is a substrate for several tyrosine kinases, including FAK, RAFTK, and Src family members (16Astier A Manie S.N. Avraham H. Hirai H. Law S.F. Zhang Y-Z., Fu, Y. Druker B.J. Haghayeghi N. Freedman A.S. Avraham S. J. Biol. Chem. 1997; 272: 19719-19724Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 17deJong R. van Wijk A. Haataja L. Heisterkamp N. Groffen J. J. Biol. 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Einarson M.B. Golemis E.A. Mol. Cell. Biol. 2000; 20: 5184-5195Crossref PubMed Scopus (79) Google Scholar, 22Ohashi Y. Iwata S. Kamiguchi K. Morimoto C. J. Immunol. 1999; 163: 3727-3734PubMed Google Scholar). In the breast cancer cell line, MCF-7, HEF1 is post-translationally processed during the cell cycle and during apoptosis, giving rise to specific forms (p105, p115, p55, and p28) which differentially localize to both focal adhesions as well as the nucleus (21Law S.F. O'Neill G.M. Fashena S.J. Einarson M.B. Golemis E.A. Mol. Cell. Biol. 2000; 20: 5184-5195Crossref PubMed Scopus (79) Google Scholar, 23Law S.F. Zhang Y.-Z. Klein-Szanto A.J.P. Golemis E.A. Mol. Cell. Biol. 1998; 18: 3540-3551Crossref PubMed Scopus (94) Google Scholar). Molecular genetic experiments have indicated that HEF1 overexpression leads to an increase in cell motility and apoptosis, consistent with a role for HEF1 in regulating integrin signaling (12Fashena S.J. Einarson M.B. O'Neill G.M. Patriotis C
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