FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2061261084> ?p ?o ?g. }

• W2061261084 endingPage "34554" @default.
• W2061261084 startingPage "34548" @default.
• W2061261084 abstract "Normal cells are dependent upon integrin-mediated adhesion to the extracellular matrix for cell proliferation and survival. Integrins regulate these processes partially through control of extracellular signal-regulated kinases 1 and 2 (ERK1/2). A trait of malignant cells is their ability to undergo anchorage-independent growth. Melanomas are tumors arising from normal melanocytes that, if undetected at an early stage, are highly invasive and poorly treatable. Proliferation of melanoma cells and melanocytes is dependent upon ERK1/2 signaling, and mutation of B-Raf, a component of the ERK1/2 pathway, is commonly found in melanomas. We addressed the role of integrin-mediated adhesion in ERK1/2 signaling in human melanoma cells and primary melanocytes. Basal ERK1/2 activity was low, and growth factor activation was adhesion-dependent in normal human melanocytes. By contrast in mutant B-Raf-expressing melanoma cells (SK-MEL-24 and SK-MEL-28), the ERK1/2 pathway was constitutively active, and adhesion-dependent regulation of ERK1/2 activity was by-passed. Furthermore, in melanoma cells, ERK1/2 translocated to the nucleus and regulated transcription events in an adhesion-independent manner. Expression of mutant V599E B-Raf in normal melanocytes was sufficient to promote adhesion-independent ERK1/2 signaling. These results indicate that alterations in the adhesion requirement for ERK1/2 signaling in melanocytes are associated with the acquisition of malignant cell behavior. Normal cells are dependent upon integrin-mediated adhesion to the extracellular matrix for cell proliferation and survival. Integrins regulate these processes partially through control of extracellular signal-regulated kinases 1 and 2 (ERK1/2). A trait of malignant cells is their ability to undergo anchorage-independent growth. Melanomas are tumors arising from normal melanocytes that, if undetected at an early stage, are highly invasive and poorly treatable. Proliferation of melanoma cells and melanocytes is dependent upon ERK1/2 signaling, and mutation of B-Raf, a component of the ERK1/2 pathway, is commonly found in melanomas. We addressed the role of integrin-mediated adhesion in ERK1/2 signaling in human melanoma cells and primary melanocytes. Basal ERK1/2 activity was low, and growth factor activation was adhesion-dependent in normal human melanocytes. By contrast in mutant B-Raf-expressing melanoma cells (SK-MEL-24 and SK-MEL-28), the ERK1/2 pathway was constitutively active, and adhesion-dependent regulation of ERK1/2 activity was by-passed. Furthermore, in melanoma cells, ERK1/2 translocated to the nucleus and regulated transcription events in an adhesion-independent manner. Expression of mutant V599E B-Raf in normal melanocytes was sufficient to promote adhesion-independent ERK1/2 signaling. These results indicate that alterations in the adhesion requirement for ERK1/2 signaling in melanocytes are associated with the acquisition of malignant cell behavior. Adhesion to the extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; ERK1/2, extracellular signal-regulated kinases 1 and 2; MAP, mitogen-activated protein; MKP2, MAP kinase phosphatase-2; MEK, MAP protein kinase/extracellular signal-regulated kinase kinase; FGF, fibroblast growth factor; BSA, bovine serum albumin; PBS, phosphate-buffered saline; CMV, cytomegalovirus. regulates a variety of cellular processes, including proliferation and survival (1Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3829) Google Scholar). Integrins are the main receptors that recognize fibronectin, laminin, and collagen within the ECM (2Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6955) Google Scholar). Engagement and clustering of integrins leads to the formation of focal adhesions that link to the actin cytoskeleton, initiation of cell spreading, and the establishment of a platform that regulates signaling events (3Assoian R.K. Schwartz M.A. Curr. Opin. Genet. Dev. 2001; 11: 48-53Crossref PubMed Scopus (270) Google Scholar). The ERK1/2 cascade impacts on cell proliferation and survival, and this pathway is regulated by adhesion (4Cobb M.H. Prog. Biophys. Mol. Biol. 1999; 71: 479-500Crossref PubMed Scopus (762) Google Scholar, 5Howe A.K. Aplin A.E. Juliano R.L. Curr. Opin. Genet. Dev. 2002; 12: 30-35Crossref PubMed Scopus (249) Google Scholar). Growth factors activate the ERK1/2 pathway by binding and activating cell surface receptors. Activated receptors recruit adaptor proteins leading to enhanced GTP loading of Ras, which recruits Raf family kinases to the membrane where the latter are activated. Three isoforms of Raf have been identified, A-Raf, B-Raf, and Raf-1 (also known as C-Raf) (6Chong H. Vikis H.G. Guan K.-L. Cell. Signaling. 2003; 15: 463-469Crossref PubMed Scopus (346) Google Scholar). Knockout studies have demonstrated important differences in their roles (7Wojnowski L. Zimmer A.M. Beck T.W. Hahn H. Bernal R. Rapp U.R. Zimmer A. Nat. Genet. 1997; 16: 293-297Crossref PubMed Scopus (254) Google Scholar, 8Pritchard C.A. Bolin L. Slattery R. Murray R. McMahon M. Curr. Biol. 1996; 6: 614-617Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Raf isoforms initiate a kinase cascade that sequentially activates mitogen-activated protein kinase/ERK kinases 1 and 2 (MEK1/2) and ERK1/2. We and others (reviewed in Refs. 3Assoian R.K. Schwartz M.A. Curr. Opin. Genet. Dev. 2001; 11: 48-53Crossref PubMed Scopus (270) Google Scholar and 5Howe A.K. Aplin A.E. Juliano R.L. Curr. Opin. Genet. Dev. 2002; 12: 30-35Crossref PubMed Scopus (249) Google Scholar) have shown that loss of adhesion impairs growth factor-mediated ERK1/2 activation and ERK translocation to the nucleus in fibroblast and endothelial cells. Adhesion effects on the ERK1/2 pathway provide a possible mechanistic basis for adhesion control of cell proliferation and survival. In the G1 phase of the cell cycle, cyclin D1 couples to cyclin-dependent kinases 4 and 6 to regulate retinoblastoma hyperphosphorylation. Induction of cyclin D1 is dependent upon both adhesion and growth factors (9Zhu X. Ohtsubo M. Bohmer R.M. Roberts J.M. Assoian R.K. J. Cell Biol. 1996; 133: 391-403Crossref PubMed Scopus (407) Google Scholar), and ERK1/2 signaling regulates cyclin D1 at the transcriptional level (10Lavoie J.N. L'Allemain G. Brunet A. Muller R. Pouyssegur J. J. Biol. Chem. 1996; 271: 20608-20616Abstract Full Text Full Text PDF PubMed Scopus (1089) Google Scholar, 11Kerkhoff E. Rapp U. Mol. Cell. Biol. 1997; 17: 2576-2586Crossref PubMed Scopus (152) Google Scholar). Whether ERK1/2 activation is sufficient for cyclin D1 induction in the absence of integrin engagement remains unclear. In one case, activation of ERK1/2 in suspended cells by inducible expression of Raf-1 was insufficient to induce cyclin D1 and subsequent G1 cell cycle events (12Le Gall M. Grall D. Chambard J.C. Pouyssegur J. Van Obberghen-Schilling E. Oncogene. 1998; 17: 1271-1277Crossref PubMed Scopus (44) Google Scholar). However, in other work (13Roovers K. Davey G. Zhu X. Bottazzi M.E. Assoian R.K. Mol. Biol. Cell. 1999; 10: 3197-3204Crossref PubMed Scopus (188) Google Scholar), inducible expression of active MEK and resulting activation of ERK1/2 induced cyclin D1 in non-adherent cells. The difference in effects on G1 cell cycle events between these two studies is likely due to the extent of adhesion-dependent regulation of activated ERK1/2 nucleocytoplasmic trafficking (14Aplin A.E. Stewart S.A. Assoian R.K. Juliano R.L. J. Cell Biol. 2001; 153: 273-282Crossref PubMed Scopus (223) Google Scholar). ERK1/2-mediated protein phosphorylation and regulation of gene transcription also elicits pro-survival signals (15Lewis T.S. Hunt J.B. Aveline L.D. Jonscher K.R. Louie D.F. Yeh J.M. Nahreini T.S. Resing K.A. Ahn N.G. Mol. Cell. 2000; 6: 1343-1354Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 16Schulze A. Lehmann K. Jefferies H.B.J. McMahon M. Downward J. Genes Dev. 2001; 15: 981-994Crossref PubMed Scopus (222) Google Scholar). As with cell cycle studies, different effects have been observed upon recovering ERK1/2 activation in suspended cells on protection from anoikis, a form of apoptosis induced by loss of adhesion or adhesion to an inappropriate ECM. Enhancing ERK1/2 activity in non-adherent conditions through expression of Raf-1 in breast cancer cells or by activation of the EGF receptor in keratinocytes leads to protection from anoikis (16Schulze A. Lehmann K. Jefferies H.B.J. McMahon M. Downward J. Genes Dev. 2001; 15: 981-994Crossref PubMed Scopus (222) Google Scholar, 17Jost M. Huggett T.M. Kari C. Rodeck U. Mol. Biol. Cell. 2001; 12: 1519-1527Crossref PubMed Scopus (93) Google Scholar, 18Le Gall M. Chambard J.C. Breittmayer J.P. Grall D. Pouyssegur J. Van Obberghen-Schilling E. Mol. Biol. Cell. 2000; 11: 1103-1112Crossref PubMed Scopus (156) Google Scholar). Other studies in endothelial and epithelial cell lines indicate that activation of ERK1/2 is insufficient or only partially protects cells from anoikis (19Aoudjit F. Vuori K. J. Cell Biol. 2001; 152: 633-643Crossref PubMed Scopus (254) Google Scholar, 20Khwaja A. Rodriguez-Viciana P. Wennstrom S. Warne P.H. Downward J. EMBO J. 1997; 16: 2783-2793Crossref PubMed Scopus (940) Google Scholar). Susceptibility to anoikis despite high ERK1/2 activation has been shown to correlate with impaired trafficking of ERK1/2 to the nucleus (21Danilkovitch A. Donley S. Skeel A. Leonard E.J. Mol. Cell. Biol. 2000; 20: 2218-2227Crossref PubMed Scopus (99) Google Scholar, 22Lai J.-M. Wu S. Huang D.-Y. Chang Z.-F. Mol. Cell. Biol. 2002; 22: 7581-7592Crossref PubMed Scopus (41) Google Scholar). Together, these studies highlight the need for increased understanding of adhesion-dependent regulation of ERK1/2 signaling events and how this control impacts on cell proliferation and survival. During malignant conversion, cells become self-sufficient in proliferation and survival signals (23Hanahan D. Weinberg R.A. Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (22628) Google Scholar). ERK1/2 are constitutively active in a variety of tumor cell types (24Hoshino R. Chatani Y. Yamori T. Tsuruo T. Oka H. Yoshida O. Shimada Y. Ari-i S. Wada H. Fujimoto J. Kohno M. Oncogene. 1999; 18: 813-822Crossref PubMed Scopus (614) Google Scholar). However, the extent to which ERK1/2 remain highly active and promote transcriptional events in the absence of adhesion has not been well established in malignant cells. We focused on melanoma cell lines and their normal precursors, melanocytes, because mutations in B-Raf, a component in the ERK1/2 pathway, are associated with two-thirds of melanomas (25Davies H. Bignell G.R. Cox C. Stephens P. Edkins S. Clegg S. Teague J. Woffendin H. Garnett M.J. Bottomley W. Davis N. Dicks E. Ewing R. Floyd Y. Gray K. Hall S. Hawes R. Hughes J. Kosmidou V. Menzies A. Mould C. Parker A. Stevens C. Watt S. Hooper S. Wilson R. Jayatilake H. Gusterson B.A. Cooper C. Shipley J. Hargrave D. Pritchard-Jones K. Maitland N. Chenevix-Trench G. Riggins G.J. Bigner D.D. Palmieri G. Cossu A. Flanagan A. Nicholson A. Ho J.W. Leung S.Y. Yuen S.T. Weber B.L. Seigler H.F. Darrow T.L. Paterson H. Marais R. Marshall C.J. Wooster R. Stratton M.R. Futreal P.A. Nature. 2002; 417: 949-954Crossref PubMed Scopus (8397) Google Scholar, 26Rajagopalan H. Bardelli A. Lengauer C. Kinzler K.W. Vogelstein B. Velculescu V.E. Nature. 2002; 418: 934Crossref PubMed Scopus (1070) Google Scholar). The most common mutation in B-Raf is a valine to glutamic acid substitution at residue 599 (V599E) located within the activation loop. This phosphomimetic substitution results in constitutive B-Raf activity and activation of ERK1/2 when overexpressed in COS-7 fibroblasts (25Davies H. Bignell G.R. Cox C. Stephens P. Edkins S. Clegg S. Teague J. Woffendin H. Garnett M.J. Bottomley W. Davis N. Dicks E. Ewing R. Floyd Y. Gray K. Hall S. Hawes R. Hughes J. Kosmidou V. Menzies A. Mould C. Parker A. Stevens C. Watt S. Hooper S. Wilson R. Jayatilake H. Gusterson B.A. Cooper C. Shipley J. Hargrave D. Pritchard-Jones K. Maitland N. Chenevix-Trench G. Riggins G.J. Bigner D.D. Palmieri G. Cossu A. Flanagan A. Nicholson A. Ho J.W. Leung S.Y. Yuen S.T. Weber B.L. Seigler H.F. Darrow T.L. Paterson H. Marais R. Marshall C.J. Wooster R. Stratton M.R. Futreal P.A. Nature. 2002; 417: 949-954Crossref PubMed Scopus (8397) Google Scholar). We show that basal ERK1/2 activity is low in normal human melanocytes, and growth factor activation of ERK1/2 is impaired in non-adherent cells as compared with cells adherent to fibronectin. In contrast, SK-MEL-24 and SK-MEL-28 melanoma cells that harbor mutant V599E B-Raf display constitutively high ERK1/2 activation in serum-free conditions. Furthermore, ERK1/2 activity, translocation to the nucleus, and regulation of transcriptional events are maintained in cells in the absence of integrin-mediated adhesion. Expression of V599E B-Raf in primary melanocytes is sufficient to promote ERK1/2 signaling independent of adhesion. The data indicate that adhesion regulates ERK1/2 activation in melanocytes and that this control is by-passed by mutation of B-Raf in malignant melanoma cells. Isolation of Primary Melanocytes—Primary human melanocytes were isolated from neonatal foreskins, as previously described (27Scott G. Cassidy L. Busacco A. J. Invest. Dermatol. 1997; 108: 147-153Abstract Full Text PDF PubMed Scopus (71) Google Scholar). Foreskin tissue was incubated with dispase to separate the epidermis from the dermis. Separated epidermal sheets were incubated with trypsin for 20 min at 37 °C and vortexed, and the cells were pelleted by centrifugation. Cells were cultured in MCDB 153 medium (Sigma, St. Louis, MO) containing 0.2% (w/v) sodium bicarbonate, 0.5% (v/v) fetal bovine serum, 0.05% (v/v) bovine pituitary extract, 0.5 μg/ml hydrocortisone, 10 μg/ml insulin, 1 ng/ml basic FGF, and 8 nm 12-O-tetradecanoylphorbol-13-acetate. All procedures were carried out according to the Research Subject Review Board at the University of Rochester. Cell Culture—The human melanoma SK-MEL-24 and SK-MEL-28 cell lines were purchased from ATCC (Manassas, VA). Cells were grown in Eagle's minimal essential medium containing Earle's salts, 2 mm glutamine, 1 mm sodium pyruvate, 0.1 mm nonessential amino acids, 1.5% (w/v) sodium bicarbonate, and 10% fetal bovine serum. COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Transfection—Primary melanocytes were transfected with Trans-It reagent (Mirus, Madison WI). Melanoma cell lines were transfected with Maxfect (Molecula Research Laboratories, Herndon, VA). Lipo-fectAMINE reagent (Invitrogen, Carlsbad, CA) was used for transfecting COS-7 cells. All transfections were performed according to the manufacturers' protocols. The BRAF V599E containing plasmid was donated kindly by Dr. Richard Marais, Institute of Cancer Research, London (25Davies H. Bignell G.R. Cox C. Stephens P. Edkins S. Clegg S. Teague J. Woffendin H. Garnett M.J. Bottomley W. Davis N. Dicks E. Ewing R. Floyd Y. Gray K. Hall S. Hawes R. Hughes J. Kosmidou V. Menzies A. Mould C. Parker A. Stevens C. Watt S. Hooper S. Wilson R. Jayatilake H. Gusterson B.A. Cooper C. Shipley J. Hargrave D. Pritchard-Jones K. Maitland N. Chenevix-Trench G. Riggins G.J. Bigner D.D. Palmieri G. Cossu A. Flanagan A. Nicholson A. Ho J.W. Leung S.Y. Yuen S.T. Weber B.L. Seigler H.F. Darrow T.L. Paterson H. Marais R. Marshall C.J. Wooster R. Stratton M.R. Futreal P.A. Nature. 2002; 417: 949-954Crossref PubMed Scopus (8397) Google Scholar). Cell Adhesion Assays and Preparation of Cell Lysates—Cells were briefly serum-starved before detachment with trypsin. Cells were pelleted in 153 medium (melanocytes) or MEM (SK-MEL-28 and SK-MEL-24 cells) containing 0.5% (w/v) BSA (Sigma) and 1 mg/ml soybean trypsin inhibitor (Invitrogen, Rockville, MD). Cells were washed once and then incubated non-adherently in the appropriate medium containing 0.5% BSA at 37 °C on a rotator. After 45 min, cells were plated onto ECM-coated dishes or maintained in suspension for between 3 and 9 h. The ECM components used were human fibronectin and vitronectin (BD Biosciences, San Diego, CA), both at a concentration of 10 μg/ml. In melanocyte experiments, some cells were stimulated with 20 ng/ml basic FGF (BD Biosciences) for 15 min. Cell lysates were prepared as previously described (28Aplin A.E. Juliano R.L. J. Cell Sci. 1999; 112: 695-706Crossref PubMed Google Scholar). Briefly, cells were washed in PBS and lysed in a modified radioimmune precipitation assay buffer containing 50 mm Hepes, pH 7.5, 1% Nonidet P-40, 0.5% sodium deoxycholate, 150 mm NaCl, 50 mm NaF, 1 mm sodium vanadate, 1 mm nitrophenyl phosphate, 0.2 μm calyculin A, 1 mm 4-(2-aminoethyl) benzenesulfonylfluoride HCl, and 10 μg/ml aprotinin. Cell lysates were cleared by centrifugation at 16,000 × g prior to protein analysis. Luciferase-based Elk-1 Reporter Assays—For Elk-1 transactivation assays, cells were transfected with three plasmids, pFA2-Elk-1, pFRluc, and pRL-CMV. pFA2-Elk-1 (Stratagene, La Jolla, CA) encodes a fusion between the GAL4 DNA binding domain and the trans-activation domain of Elk-1. pFR-luc (Stratagene) is a GAL4-based reporter plasmid controlling the transcription of Firefly luciferase. pRL-CMV-luc (Promega, Madison, WI) encodes Renilla luciferase under the control of the CMV promoter and was included for normalization. Luciferase levels in adherent and non-adherent cells were determined using the dual luciferase assay kit (Promega). Cells were extracted in passive lysis buffer and 20 μl of lysate was incubated with 100 μl of luciferin reagent. Firefly luminescence was measured for 10 s in a Turner Designs 20/20 luminometer. Subsequently, 100 μl of Stop and Glo® reagent was added, and the specific luminescence from Renilla luciferase was recorded for an additional 10 s. Within each sample, Firefly activity was normalized to Renilla luciferase activity. SDS-PAGE and Western Blotting—Cell lysates were separated by SDS-PAGE under reducing conditions, and proteins were transferred electrophoretically onto Immobilon P membranes (Millipore Corp., Bedford, MA). Membranes were blocked with PBS containing 1% BSA and 0.1% Tween 20 for 1 h and subsequently incubated with primary antibody overnight at 4 °C. Primary antibodies that recognize phospho-ERK (Cell Signaling Technology, Beverly, MA), total ERK1/2 (clone K-23, Santa Cruz Biotechnologies, Santa Cruz, CA), B-Raf (clone C-19, Santa Cruz Biotechnologies), and MKP-2 (clone S-18, Santa Cruz Biotechnologies) were used. Membranes were washed in PBS containing 0.1% Tween and incubated with goat anti-mouse or goat anti-rabbit IgG peroxidase conjugates (Calbiochem, San Diego, CA) for 1 h at room temperature. Western blots were developed using a SuperSignal chemiluminescent substrate (Pierce, Rockford, IL). Immunoreactivity was detected and quantified using a Fluor-S MultiImager and Quantity-One software (Bio-Rad, Hercules, CA). Immunofluorescence—Cells were either plated onto fibronectincoated coverslips for 2 h at 37 °C or, for non-adherent conditions, maintained in suspension for 1 h and 55 min and then attached to polylysine-coated coverslips for 5 min. No cell spreading was observed on polylysine under these conditions. Cells were fixed and incubated with phospho-ERK1/2 (BIOSOURCE, Camarillo, CA) primary and Alexa Fluor 488 goat anti-rabbit secondary (Molecular Probes) antibodies. Nuclei were stained with Hoechst reagent 33342 (Molecular Probes). Fluorescent staining was viewed on an Olympus BX60 microscope equipped for epifluorescence. Images were captured using a Spot charge-coupled device camera and processed using deconvolution Slide Book software. Adhesion to Fibronectin Regulates Growth Factor Activation of ERK1/2 in Primary Melanocytes—Adhesion to the ECM regulates signaling events that promote cell proliferation and survival (3Assoian R.K. Schwartz M.A. Curr. Opin. Genet. Dev. 2001; 11: 48-53Crossref PubMed Scopus (270) Google Scholar). Initially, we analyzed whether adhesion controls growth factor-induced activation of ERK1/2 in primary human melanocytes, a cell type that has not been well characterized for adhesion control of signaling events. In melanocytes, basic FGF is known to activate ERK1/2 and enhance cell growth (29Nesbit M. Nesbit H.K. Bennett J. Andl T. Hsu M.Y. Dejesus E. McBrian M. Gupta A.R. Eck S.L. Herlyn M. Oncogene. 1999; 18: 6469-6476Crossref PubMed Scopus (98) Google Scholar, 30Tada A. Pereira E. Beitner-Johnson D. Kavanagh R. Abdel-Malek Z.A. J. Invest. Dermatol. 2002; 118: 316-322Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Serum-deprived primary melanocytes were either maintained in suspension or allowed to adhere to fibronectin before stimulation with FGF. Basal ERK1/2 activities were low in serum-free conditions in both suspended and adherent cells, as determined by Western blot analysis of cell lysates with phospho-ERK1/2 antibodies (Fig. 1A). FGF treatment resulted in efficient ERK1/2 activation in cells adherent to fibronectin but weak activation in suspended melanocytes. Low level ERK1/2 activation was also observed in FGF-treated cells that were attached non-specifically to polylysine-coated dishes (data not shown). Consistent with anti-phospho ERK1/2 analysis, a mobility shift that is indicative of phosphorylation was detected only in adherent FGF-stimulated cells upon Western blotting with an antibody to total ERK1/2. Quantification of Western blot data showed that FGF-mediated ERK1/2 activity was ∼3-fold higher in fibronectin-adherent melanocytes compared with suspended cells (Fig. 1B). These findings demonstrate that adhesion of primary melanocytes to the ECM is permissive for FGF activation of ERK1/2. Constitutive Activation of ERK1/2 in V599E B-Raf-expressing Melanoma Cells—It was reported recently that individual point mutations in B-Raf that lead to its constitutive activation are frequently found in melanoma cells (25Davies H. Bignell G.R. Cox C. Stephens P. Edkins S. Clegg S. Teague J. Woffendin H. Garnett M.J. Bottomley W. Davis N. Dicks E. Ewing R. Floyd Y. Gray K. Hall S. Hawes R. Hughes J. Kosmidou V. Menzies A. Mould C. Parker A. Stevens C. Watt S. Hooper S. Wilson R. Jayatilake H. Gusterson B.A. Cooper C. Shipley J. Hargrave D. Pritchard-Jones K. Maitland N. Chenevix-Trench G. Riggins G.J. Bigner D.D. Palmieri G. Cossu A. Flanagan A. Nicholson A. Ho J.W. Leung S.Y. Yuen S.T. Weber B.L. Seigler H.F. Darrow T.L. Paterson H. Marais R. Marshall C.J. Wooster R. Stratton M.R. Futreal P.A. Nature. 2002; 417: 949-954Crossref PubMed Scopus (8397) Google Scholar). Because B-Raf is an upstream component in the MEK to ERK1/2 kinase module, we analyzed whether ERK1/2 were activated in human melanoma cell lines. Two melanoma lines were tested, SK-MEL-28 and SK-MEL-24, that carry the most common activating B-Raf mutation (V599E) (25Davies H. Bignell G.R. Cox C. Stephens P. Edkins S. Clegg S. Teague J. Woffendin H. Garnett M.J. Bottomley W. Davis N. Dicks E. Ewing R. Floyd Y. Gray K. Hall S. Hawes R. Hughes J. Kosmidou V. Menzies A. Mould C. Parker A. Stevens C. Watt S. Hooper S. Wilson R. Jayatilake H. Gusterson B.A. Cooper C. Shipley J. Hargrave D. Pritchard-Jones K. Maitland N. Chenevix-Trench G. Riggins G.J. Bigner D.D. Palmieri G. Cossu A. Flanagan A. Nicholson A. Ho J.W. Leung S.Y. Yuen S.T. Weber B.L. Seigler H.F. Darrow T.L. Paterson H. Marais R. Marshall C.J. Wooster R. Stratton M.R. Futreal P.A. Nature. 2002; 417: 949-954Crossref PubMed Scopus (8397) Google Scholar). Under serum-free conditions, ERK1/2 activities were high in the SK-MEL-28 and SK-MEL-24 cells, as determined by Western blot analysis with phospho-ERK antibodies (Fig. 2). Treatment with the MEK inhibitor compound, U0126, inhibited phosphorylation of ERK1/2, and serum failed to enhance ERK1/2 phosphorylation in both cell lines. These results indicate that the ERK1/2 pathway is constitutively active downstream of mutant B-Raf in the SK-MEL-28 and SK-MEL-24 cells. Activation of ERK1/2 Is Adhesion-independent in Melanoma Cells—We have previously shown that expression of an activated form of Raf-1, known as 22W Raf, is able to recover ERK1 activity in suspended NIH 3T3 cells (14Aplin A.E. Stewart S.A. Assoian R.K. Juliano R.L. J. Cell Biol. 2001; 153: 273-282Crossref PubMed Scopus (223) Google Scholar). In addition to Raf-1, B-Raf, and A-Raf act as MAP3K in the ERK1/2 cascade (6Chong H. Vikis H.G. Guan K.-L. Cell. Signaling. 2003; 15: 463-469Crossref PubMed Scopus (346) Google Scholar). We asked whether ERK1/2 activation is adhesion-independent in malignant melanoma cell lines harboring mutant V599E B-Raf. SK-MEL-24 cells maintained in suspension or adhered to ECM components in serum-free conditions were lysed and analyzed by Western blotting for phospho- and total ERK1/2. ERK1/2 phosphorylation was highly active in SK-MEL-24 cells that were either suspended or replated onto fibronectin over a 9-h time course (Fig. 3A). Mobility-shifted ERK1/2, corresponding to activation, is evident in the Western blot of SK-MEL-24 cell lysates with total ERK1/2 antibody. Similarly, phosphorylation of ERK1/2 was high in both non-adherent and adherent SK-MEL-28 cells over a 9-h time course (Fig. 3B). Comparable results were observed whether SK-MEL-28 cells were replated onto vitronectin or fibronectin-coated dishes (data not shown). Thus, ERK1/2 are activated regardless of adhesion in mutant B-Raf expressing melanoma cell lines. Activated ERK Localizes to the Nucleus in Adherent and Non-adherent Melanoma Cells—Our published findings in NIH 3T3 mouse fibroblasts show that adhesion regulates both growth factor activation and the nuclear accumulation of activated ERKs (14Aplin A.E. Stewart S.A. Assoian R.K. Juliano R.L. J. Cell Biol. 2001; 153: 273-282Crossref PubMed Scopus (223) Google Scholar). To determine in melanoma cells whether ERK1/2 are subject to adhesion regulation downstream of activation, we analyzed phospho-ERK accumulation in the nucleus of B-Raf V599E-expressing SK-MEL-28 cells. By deconvolution immunofluorescence, we observed phospho-ERK staining was highly localized to the nucleus in the majority of non-adherent and adherent cells (Fig. 4). As expected, treatment with U0126 reduced levels of phospho-ERK staining. These data show that the localization of activated ERK1/2 to the nucleus is not dependent upon adhesion in SK-MEL-28 cells. The Effect of Adhesion on GAL4-Elk-1 Transactivation and MKP-2 Levels in Melanoma Cells—In the nucleus, ERK1/2 phosphorylate transcription factors, including Elk-1 (31Gille H. Sharrocks A.D. Shaw P.E. Nature. 1992; 358: 414-417Crossref PubMed Scopus (816) Google Scholar). We next examined ERK1/2-induced activation of a reporter driven by Elk-1. Cells were transfected with GAL4 Elk-1 fusion and firefly luciferase reporter constructs and either maintained in suspension or replated onto fibronectin for 3 or 6 h. GAL4-Elk-1-driven luciferase activity was constitutively high and was unaffected by loss of adhesion in both SK-MEL-24 and SK-MEL-28 cells (Fig. 5A). In both melanoma cells lines, GAL4-Elk-1 activity was inhibited dramatically upon treatment with the MEK inhibitor, U0126. We also analyzed the expression level of MAP kinase phosphatase-2 (MKP2) that is regulated at the transcriptional level by ERK1/2 (32Brondello J.M. Brunet A. Pouyssegur J. McKenzie F.R. J. Biol. Chem. 1997; 272: 1368-1376Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). MKP2 levels were readily detectable by Western blot analysis of lysates from adherent SK-MEL-24 and SK-MEL-28 cells under serum-free conditions (Fig. 5B). Expression of MKP2 was maintained in cells upon loss of adhesion over the 9-h time period but was dramatically down-regulated upon inhibition of MEK. Together, these data indicate that activated ERK1/2 in mutant V599E B-Raf cells are able to promote transcription events in the absence of cell adhesion. Expression of Mutant B-Raf Initiates Adhesion-independent ERK1/2 Signaling in Melanocytes—To test whether mutations in B-Raf are causative for adhesion-independent ERK1/2 signaling, we utilized a construct encoding V599E B-Raf that contains the most common B-Raf mutation found in melanomas. Transfection of this construct into COS-7 cells resulted in the expression of a 95-kDa protein that was detected with anti-B-Raf antibodies but was not present in control transfected cell lysates (Fig. 6A). Next, normal human melanocytes were transfected with mutant V599E B-Raf or control vector, in addition to GAL4 Elk-1 fusion and firefly luciferase reporter constructs. Cells were either maintained in suspension or readhered to fibronectin for 6 h before analysis of GAL4-Elk-1-driven luciferase activity. GAL4-Elk-1 transactivation was low in control transfected cells in both non-adherent and adherent conditions (Fig. 6B). Notably, V599E B-Raf transfection initiated efficient GAL4-Elk-1 transactivation in both suspended and adherent melanocytes. Despite marked GAL4-Elk-1-driven luciferase activity, we were unable to detect expression of exogenous B-Raf in melanocyte cell lysates by Western analysis due to lower transfection efficiency than COS-7 cells. Nevertheless, because ERK1/2 regulates Elk-1 activity, these results indicate that V599E B-Raf is sufficient to activate ERK1/2 signaling in an adhesion independent manner in normal melanocytes. Integrin-mediated adhesion plays an important role in cell proliferation, survival, and migration (3Assoian R.K. Schwartz M.A. Curr. Opin. Genet. Dev. 2001; 11: 48-53Crossref PubMed Scopus (270) Google Scholar, 5Howe A.K. Aplin A.E. Juliano R.L. Curr. Opin. Genet. Dev. 2002; 12: 30-35Crossref PubMed Scopus (249) Google Scholar). To understand how integrins control these processes, we have studied adhesion-dependent regulation of growth factor signaling through the ERK1/2 cascade. Our finding that adhesion regulates FGF activation of ERK1/2 in normal primary melanocytes is, to our knowledge, the first demonstration of adhesion control of a growth factor-activated kinase cascade in these cells. Our observations are consistent with published results in fibroblasts and endothelial cells, in which adhesion and growth factor-mediated signals have been found to converge at the level of activation of the growth factor receptor, Raf, and MEK (33Miyamoto S. Teramoto H. Gutkind J.S. Yamada K.M. J. Cell Biol. 1996; 135: 1633-1642Crossref PubMed Scopus (680) Google Scholar, 34Schneller M. Vuori K. Ruoslahti E. EMBO J. 1997; 16: 5600-5607Crossref PubMed Scopus (427) Google Scholar, 35Soldi R. Mitola S. Strasly M. Defilippi P. Tarone G. Bussolino F. EMBO J. 1999; 18: 882-892Crossref PubMed Scopus (534) Google Scholar, 36Moro L. Venturino M. Bozzo C. Silengo L. Altruda F. Beguinot L. Tarone G. Defilippi P. EMBO J. 1998; 17: 6622-6632Crossref PubMed Scopus (506) Google Scholar, 37Lin T.H. Chen Q. Howe A. Juliano R.L. J. Biol. Chem. 1997; 272: 8849-8852Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 38Renshaw M.W. Ren X.D. Schwartz M.A. EMBO J. 1997; 16: 5592-5599Crossref PubMed Scopus (270) Google Scholar, 39Aplin A.E. Short S.M. Juliano R.L. J. Biol. Chem. 1999; 274: 31223-31228Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). In melanocytes, it is likely that the point of convergence occurs at or above B-Raf activation, because ERK1/2 signaling is adhesion-independent in V599E B-Raf-expressing melanocytes and melanoma cells. We are presently performing further experiments to determine the exact adhesion regulated step leading to ERK1/2 activation in melanocytes. In addition to regulation of growth factor activation, adhesion regulates trafficking of ERK1/2 to the nucleus in fibroblasts and epithelial cells (14Aplin A.E. Stewart S.A. Assoian R.K. Juliano R.L. J. Cell Biol. 2001; 153: 273-282Crossref PubMed Scopus (223) Google Scholar, 21Danilkovitch A. Donley S. Skeel A. Leonard E.J. Mol. Cell. Biol. 2000; 20: 2218-2227Crossref PubMed Scopus (99) Google Scholar, 22Lai J.-M. Wu S. Huang D.-Y. Chang Z.-F. Mol. Cell. Biol. 2002; 22: 7581-7592Crossref PubMed Scopus (41) Google Scholar); an effect that is likely mediated by the cytoplasmic tail of the integrin beta subunit (40Hirsch E. Barberis L. Brancaccio M. Azzolino O. Xu D. Kyriakis J.M. Silengo L. Giancotti F.G. Tarone G. Fassler R. Altruda F. J. Cell Biol. 2002; 157: 481-492Crossref PubMed Scopus (76) Google Scholar). Our initial data indicate that this step is not strongly adhesion-dependent in melanocytes, because V599E B-Raf-initiated Elk-1 transactivation is comparable in suspended and adherent cells. However, V599E B-Raf is highly active and persistently activates ERK1/2, hence further experiments are required to determine whether under alternative conditions, for example transient ERK1/2 activation or expression of other Raf isoforms, ERK1/2 trafficking to the nucleus is regulated by adhesion in melanocytes. Adhesion regulation of ERK1/2 is likely critical for melanocyte proliferation and survival. Basic FGF that is secreted from keratinocytes is a known factor that promotes melanocyte proliferation (41Halaban R. Langdon R. Birchall N. Cuono C. Baird A. Scott G. Moellmann G. McGuire J. J. Cell Biol. 1988; 107: 1611-1619Crossref PubMed Scopus (424) Google Scholar, 42Halaban R. Pigment Cell Res. 2000; 13: 4-14Crossref PubMed Scopus (146) Google Scholar). Similarly, the ERK1/2 pathway is known to regulate G1 cell cycle progression in normal human melanocytes and melanoma cell lines (25Davies H. Bignell G.R. Cox C. Stephens P. Edkins S. Clegg S. Teague J. Woffendin H. Garnett M.J. Bottomley W. Davis N. Dicks E. Ewing R. Floyd Y. Gray K. Hall S. Hawes R. Hughes J. Kosmidou V. Menzies A. Mould C. Parker A. Stevens C. Watt S. Hooper S. Wilson R. Jayatilake H. Gusterson B.A. Cooper C. Shipley J. Hargrave D. Pritchard-Jones K. Maitland N. Chenevix-Trench G. Riggins G.J. Bigner D.D. Palmieri G. Cossu A. Flanagan A. Nicholson A. Ho J.W. Leung S.Y. Yuen S.T. Weber B.L. Seigler H.F. Darrow T.L. Paterson H. Marais R. Marshall C.J. Wooster R. Stratton M.R. Futreal P.A. Nature. 2002; 417: 949-954Crossref PubMed Scopus (8397) Google Scholar, 43Koo H.-M. VanBrocklin M. McWilliams M.J. Leppla S.H. Duesbery N.S. Woude G.F.V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3052-3057Crossref PubMed Scopus (138) Google Scholar), at least in part through down-regulation of the CDK inhibitor, p27 kip1 (44Kortylewski M. Heinrich P.C. Kauffmann M.E. Bohm M. MacKiewicz A. Behrmann I. Biochem. J. 2001; 357: 297-303Crossref PubMed Scopus (73) Google Scholar). Melanocyte survival is also dependent upon adhesion (27Scott G. Cassidy L. Busacco A. J. Invest. Dermatol. 1997; 108: 147-153Abstract Full Text PDF PubMed Scopus (71) Google Scholar). Inhibition of MEK promotes apoptosis in human melanoma cells (17Jost M. Huggett T.M. Kari C. Rodeck U. Mol. Biol. Cell. 2001; 12: 1519-1527Crossref PubMed Scopus (93) Google Scholar, 18Le Gall M. Chambard J.C. Breittmayer J.P. Grall D. Pouyssegur J. Van Obberghen-Schilling E. Mol. Biol. Cell. 2000; 11: 1103-1112Crossref PubMed Scopus (156) Google Scholar, 43Koo H.-M. VanBrocklin M. McWilliams M.J. Leppla S.H. Duesbery N.S. Woude G.F.V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3052-3057Crossref PubMed Scopus (138) Google Scholar); survival may be mediated by ERK1/2-dependent up-regulation of the αvβ3 integrin that can interact with the dermal collagen matrix (45Woods D. Cherwinski H. Venetsanakos E. Bhat A. Gysin S. Humbert M. Bray P.F. Saylor V.L. McMahon M. Mol. Cell. Biol. 2001; 21: 3192-3205Crossref PubMed Scopus (111) Google Scholar, 46Montgomery A. Reisfeld R. Cheresh D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8856-8860Crossref PubMed Scopus (416) Google Scholar). Notably, levels of β3 integrin are increased during the vertical phase of melanoma growth (47Albelda S.M. Mette S.A. Elder D.E. Stewart R. Damjanovich L. Herlyn M. Buck C.A. Cancer Res. 1990; 50: 6757-6764PubMed Google Scholar, 48Van Belle P.A. Elenitsas R. Satyamoorthy K. Wolfe J.T. Guerry D. Schuchter L. Van Belle T.J. Albelda S. Tahin P. Herlyn M. Elder D.E. Hum. Pathol. 1999; 30: 562-567Crossref PubMed Scopus (112) Google Scholar). Thus, the loss of adhesion-dependent control of ERK1/2 signal transduction may be a critical step in neoplastic conversion. Although normal cells require both adhesion and soluble mitogens for growth, malignant cells are able to by-pass these needs (23Hanahan D. Weinberg R.A. Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (22628) Google Scholar). The identification of mutations in B-Raf that are associated with melanomas prompted us to address whether adhesion-dependent regulation of ERK1/2 signaling was altered in melanoma cells. Both ERK1 and -2 were constitutively active in SK-MEL-24 and SK-MEL-28 cells, consistent with recent reports on other melanoma cell lines and tissue sections (44Kortylewski M. Heinrich P.C. Kauffmann M.E. Bohm M. MacKiewicz A. Behrmann I. Biochem. J. 2001; 357: 297-303Crossref PubMed Scopus (73) Google Scholar, 49Cohen C. Zavala-Pompa A. Sequeira J.H. Shoji M. Sexton D.G. Cotsonis G. Cerimele F. Govindarajan B. Macaron N. Arbiser J.L. Clin. Cancer Res. 2002; 8: 3728-3733PubMed Google Scholar, 50Satyamoorthy K. Li G. Gerrero M.R. Brose M.S. Volpe P. Weber B.L. van Belle P. Elder D.E. Herlyn M. Cancer Res. 2003; 63: 756-759PubMed Google Scholar). Enhanced Raf/MEK/ERK signaling is known to promote transcription of genes linked to cell proliferation, survival, and invasion (16Schulze A. Lehmann K. Jefferies H.B.J. McMahon M. Downward J. Genes Dev. 2001; 15: 981-994Crossref PubMed Scopus (222) Google Scholar). Our data in melanoma cells show that ERK1/2 are efficiently activated, accumulate in the nucleus, and promote transcriptional events in an adhesion-independent manner. Both SK-MEK-28 and SK-MEL-24 cells display anchorage-independent properties (51Cirielli C. Riccioni T. Yang C. Pili R. Gloe T. Chang J. Inyaku K. Passaniti A. Capogrossi M.C. Int. J. Cancer. 1995; 63: 673-679Crossref PubMed Scopus (63) Google Scholar, 52Gasperi-Campani A. Musa A.R. Roncuzzi L. Melanoma Res. 1993; 3: 363-367Crossref PubMed Scopus (7) Google Scholar). 2S. R. Conner, G. Scott, and A. E. Aplin, unpublished observations. Thus, our findings indicate that one event leading to anchorage-independence is the ability to overcome integrin-dependent control of the ERK1/2 cascade. We were able to by-pass this control of ERK1/2 signaling in normal melanocytes by expression of V599E B-Raf. B-Raf is an activator of MEK in the ERK1/2 pathway in melanocytes (53Busca R. Abbe P. Mantoux F. Aberdam E. Peyssonnaux C. Eychene A. Ortonne J.-P. Ballotti R. EMBO J. 2000; 19: 2900-2910Crossref PubMed Scopus (295) Google Scholar), and the significance of control of ERK1/2 signaling for the malignant conversion of melanocytes is underscored by the knowledge that the BRAF gene is mutated in approximately two-thirds of melanomas (25Davies H. Bignell G.R. Cox C. Stephens P. Edkins S. Clegg S. Teague J. Woffendin H. Garnett M.J. Bottomley W. Davis N. Dicks E. Ewing R. Floyd Y. Gray K. Hall S. Hawes R. Hughes J. Kosmidou V. Menzies A. Mould C. Parker A. Stevens C. Watt S. Hooper S. Wilson R. Jayatilake H. Gusterson B.A. Cooper C. Shipley J. Hargrave D. Pritchard-Jones K. Maitland N. Chenevix-Trench G. Riggins G.J. Bigner D.D. Palmieri G. Cossu A. Flanagan A. Nicholson A. Ho J.W. Leung S.Y. Yuen S.T. Weber B.L. Seigler H.F. Darrow T.L. Paterson H. Marais R. Marshall C.J. Wooster R. Stratton M.R. Futreal P.A. Nature. 2002; 417: 949-954Crossref PubMed Scopus (8397) Google Scholar), ERK1/2 are constitutively active in melanoma tissue sections and cell lines (this study and Refs. 49Cohen C. Zavala-Pompa A. Sequeira J.H. Shoji M. Sexton D.G. Cotsonis G. Cerimele F. Govindarajan B. Macaron N. Arbiser J.L. Clin. Cancer Res. 2002; 8: 3728-3733PubMed Google Scholar and 50Satyamoorthy K. Li G. Gerrero M.R. Brose M.S. Volpe P. Weber B.L. van Belle P. Elder D.E. Herlyn M. Cancer Res. 2003; 63: 756-759PubMed Google Scholar), and introduction of activated MEK promotes transformation of an immortalized murine melanocyte cell line (54Govindarajan B. Bai X. Cohen C. Zhong H. Kilroy S. Louis G. Moses M. Arbiser J.L. J. Biol. Chem. 2003; 278: 9790-9795Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Thus, inhibition of B-Raf or downstream components in the ERK1/2 cascade has potential as therapeutic strategy to block melanoma growth. In summary, our findings provide a link between alterations in adhesion-dependent regulation of growth factor activation of ERK1/2 and the acquisition of malignant properties. Efficient ERK1/2 activation is dependent upon both adhesion and growth factors in normal melanocytes but is independent of these cues in melanoma cells (for model see Fig. 7). These findings highlight the importance of adhesion-dependent regulation of the ERK1/2 pathway for normal cell processes and the significance of alterations in this control for transformation and the acquisition of anchorage-independent properties. We thank Dr. Richard Marais (Cancer Research UK Centre for Cell and Molecular Biology, Institute of Cancer Research, London, UK) for kindly providing the mutant B-Raf construct. We are very grateful to Drs. Mike DiPersio and Kevin Pumiglia for critically reading this manuscript." @default.
• W2061261084 created "2016-06-24" @default.
• W2061261084 creator A5031804974 @default.
• W2061261084 creator A5062746446 @default.
• W2061261084 creator A5085366413 @default.
• W2061261084 date "2003-09-01" @default.
• W2061261084 modified "2023-09-26" @default.
• W2061261084 title "Adhesion-dependent Activation of the ERK1/2 Cascade Is By-passed in Melanoma Cells" @default.
• W2061261084 cites W1576773659 @default.
• W2061261084 cites W1965343041 @default.
• W2061261084 cites W1980855212 @default.
• W2061261084 cites W1984362855 @default.
• W2061261084 cites W1985035689 @default.
• W2061261084 cites W1987680808 @default.
• W2061261084 cites W1990105301 @default.
• W2061261084 cites W1999157780 @default.
• W2061261084 cites W2004638017 @default.
• W2061261084 cites W2006443734 @default.
• W2061261084 cites W2010489503 @default.
• W2061261084 cites W2011468496 @default.
• W2061261084 cites W2014252192 @default.
• W2061261084 cites W2014461843 @default.
• W2061261084 cites W2027923032 @default.
• W2061261084 cites W2031818280 @default.
• W2061261084 cites W2033060201 @default.
• W2061261084 cites W2034269086 @default.
• W2061261084 cites W2036614475 @default.
• W2061261084 cites W2038777362 @default.
• W2061261084 cites W2040283898 @default.
• W2061261084 cites W2042921323 @default.
• W2061261084 cites W2045000224 @default.
• W2061261084 cites W2053268382 @default.
• W2061261084 cites W2054566768 @default.
• W2061261084 cites W2059287983 @default.
• W2061261084 cites W2075082022 @default.
• W2061261084 cites W2078636606 @default.
• W2061261084 cites W2093208114 @default.
• W2061261084 cites W2102369096 @default.
• W2061261084 cites W2106465904 @default.
• W2061261084 cites W2108398407 @default.
• W2061261084 cites W2111563081 @default.
• W2061261084 cites W2114474216 @default.
• W2061261084 cites W2121627701 @default.
• W2061261084 cites W2127150495 @default.
• W2061261084 cites W2135143455 @default.
• W2061261084 cites W2140929616 @default.
• W2061261084 cites W2145569043 @default.
• W2061261084 cites W2153084734 @default.
• W2061261084 cites W2154169742 @default.
• W2061261084 cites W2156636963 @default.
• W2061261084 cites W2160771960 @default.
• W2061261084 cites W2160926271 @default.
• W2061261084 cites W2163188200 @default.
• W2061261084 cites W2164767056 @default.
• W2061261084 cites W2164965551 @default.
• W2061261084 cites W2317270770 @default.
• W2061261084 cites W2334880322 @default.
• W2061261084 cites W4213141530 @default.
• W2061261084 cites W4230897605 @default.
• W2061261084 doi "https://doi.org/10.1074/jbc.m305797200" @default.
• W2061261084 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12821662" @default.
• W2061261084 hasPublicationYear "2003" @default.
• W2061261084 type Work @default.
• W2061261084 sameAs 2061261084 @default.
• W2061261084 citedByCount "51" @default.
• W2061261084 countsByYear W20612610842012 @default.
• W2061261084 countsByYear W20612610842013 @default.
• W2061261084 countsByYear W20612610842014 @default.
• W2061261084 countsByYear W20612610842015 @default.
• W2061261084 countsByYear W20612610842016 @default.
• W2061261084 countsByYear W20612610842017 @default.
• W2061261084 countsByYear W20612610842018 @default.
• W2061261084 countsByYear W20612610842019 @default.
• W2061261084 countsByYear W20612610842020 @default.
• W2061261084 crossrefType "journal-article" @default.
• W2061261084 hasAuthorship W2061261084A5031804974 @default.
• W2061261084 hasAuthorship W2061261084A5062746446 @default.
• W2061261084 hasAuthorship W2061261084A5085366413 @default.
• W2061261084 hasBestOaLocation W20612610841 @default.
• W2061261084 hasConcept C12554922 @default.
• W2061261084 hasConcept C178790620 @default.
• W2061261084 hasConcept C185592680 @default.
• W2061261084 hasConcept C2777658100 @default.
• W2061261084 hasConcept C34146451 @default.
• W2061261084 hasConcept C43617362 @default.
• W2061261084 hasConcept C502942594 @default.
• W2061261084 hasConcept C84416704 @default.
• W2061261084 hasConcept C85789140 @default.
• W2061261084 hasConcept C86803240 @default.
• W2061261084 hasConcept C95444343 @default.
• W2061261084 hasConceptScore W2061261084C12554922 @default.
• W2061261084 hasConceptScore W2061261084C178790620 @default.
• W2061261084 hasConceptScore W2061261084C185592680 @default.
• W2061261084 hasConceptScore W2061261084C2777658100 @default.
• W2061261084 hasConceptScore W2061261084C34146451 @default.
• W2061261084 hasConceptScore W2061261084C43617362 @default.
• W2061261084 hasConceptScore W2061261084C502942594 @default.
• W2061261084 hasConceptScore W2061261084C84416704 @default.

• next