Specific Residues within the α2 Integrin Subunit Cytoplasmic Domain Regulate Migration and Cell Cycle Progression via Distinct MAPK Pathways
2001; Elsevier BV; Volume: 276; Issue: 34 Linguagem: Inglês
10.1074/jbc.m101921200
ISSN1083-351X
AutoresPaul Klekotka, Samuel A. Santoro, Haochuan Wang, Mary M. Zutter,
Tópico(s)HER2/EGFR in Cancer Research
ResumoThe α2 integrin subunit cytoplasmic domain is necessary for epidermal growth factor (EGF)-stimulated chemotactic migration and insulin-dependent entry into S-phase of mammary epithelial cells adherent to type I collagen. Truncation mutants revealed that the seven amino acids, KYEKMTK, in addition to the GFFKR motif were sufficient for these functions. Mutation of tyrosine 1134 to alanine inhibited the ability of the cells to phosphorylate p38 MAPK and to migrate in response to EGF but had only a modest effect on the ability of the cells to induce sustained phosphorylation of the ERK MAPK, to up-regulate cyclin E and cdk2 expression, and to enter S-phase when adherent to type I collagen. Conversely, mutation of the lysine 1136 inhibited the ability of the cells to increase cyclin E and cdk2 expression, to maintain long term phosphorylation of the ERK MAPK, and to enter S-phase but had no effect on the ability of the cells to phosphorylate the p38 MAPK or to migrate on type I collagen in response to EGF. Methionine 1137 was essential for both migration and entry into S-phase. Thus, distinctly different structural elements of the α2 integrin cytoplasmic domain are required to engage the signaling pathways leading to cell migration or cell cycle progression. The α2 integrin subunit cytoplasmic domain is necessary for epidermal growth factor (EGF)-stimulated chemotactic migration and insulin-dependent entry into S-phase of mammary epithelial cells adherent to type I collagen. Truncation mutants revealed that the seven amino acids, KYEKMTK, in addition to the GFFKR motif were sufficient for these functions. Mutation of tyrosine 1134 to alanine inhibited the ability of the cells to phosphorylate p38 MAPK and to migrate in response to EGF but had only a modest effect on the ability of the cells to induce sustained phosphorylation of the ERK MAPK, to up-regulate cyclin E and cdk2 expression, and to enter S-phase when adherent to type I collagen. Conversely, mutation of the lysine 1136 inhibited the ability of the cells to increase cyclin E and cdk2 expression, to maintain long term phosphorylation of the ERK MAPK, and to enter S-phase but had no effect on the ability of the cells to phosphorylate the p38 MAPK or to migrate on type I collagen in response to EGF. Methionine 1137 was essential for both migration and entry into S-phase. Thus, distinctly different structural elements of the α2 integrin cytoplasmic domain are required to engage the signaling pathways leading to cell migration or cell cycle progression. extracellular signal-regulated kinase mitogen-activated protein kinase epidermal growth factor cyclin-dependent kinase 2 bromodeoxyuridine Dulbecco's modified Eagle's medium polymerase chain reaction c-Jun NH2-terminal kinase mitogen-activated protein kinase The integrin family of heterodimeric cell surface adhesion receptors not only mediates adhesion to the extracellular matrix and other cells, but also serves to integrate signals from the outside to the inside of the cell (1Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2809) Google Scholar, 2Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Biol. 1995; 11: 549-599Crossref Scopus (1460) Google Scholar, 3Yamada K.M. Miyamoto S. Curr. Opin. Cell Biol. 1995; 7: 681-689Crossref PubMed Scopus (586) Google Scholar, 4Howe A. Aplin A.E. Alahari S.K. Juliano R.L. Curr. Opin. Cell Biol. 1998; 10: 220-231Crossref PubMed Scopus (582) Google Scholar, 5Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3785) Google Scholar). Signals from growth factor receptors acting in combination with signals from integrins are necessary to regulate complex cellular processes such as migration and proliferation. Adhesion to the extracellular matrix via integrins is required for growth factors to induce robust activation of both the extracellular signal-regulated kinase (ERK)1 and c-Jun NH2-terminal kinase mitogen-activated protein kinase (JNK) cascades, which leads to progression through the G1 phase of the cell cycle (6Miyamoto S. Teramoto H. Coso O.A. Gutkind J.S. Burbelo P.D. Akiyama S.K. Yamada K.M. J. Cell Biol. 1995; 131: 791-805Crossref PubMed Scopus (1101) Google Scholar, 7Aplin 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, 8Oktay M. Wary K.K. Dans M. Birge R.B. Giancotti F.G. J. Cell Biol. 1999; 145: 1461-1469Crossref PubMed Scopus (247) Google Scholar, 9Wary K.K. Mainiero F. Isakoff S.J. Marcantonio E.E. Giancotti F.G. Cell. 1996; 87: 733-743Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar, 10Wary K.K. Mariotti A. Zurzolo C. Giancotti F.G. Cell. 1998; 94: 625-634Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar, 11Mainiero F. Murgia C. Wary K.K. Curatola A.M. Pepe A. Blumemberg M. Westwick J.K. Der C.J. Giancotti F.G. EMBO J. 1997; 16: 2365-2375Crossref PubMed Scopus (305) Google Scholar). During chemotactic migration integrin ligation not only provides the adhesion necessary for traction on the extracellular matrix but also activates intracellular signaling molecules such as Rac, Cdc42, and p38 MAPK (12Keely P.J. Westwick J.K. Whitehead I.P. Der C.J. Parise L.V. Nature. 1997; 390: 632-636Crossref PubMed Scopus (649) Google Scholar, 13Klekotka P.A. Santoro S.A. Zutter M.M. J. Biol. Chem. 2001; 276: 9503-9511Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar).The α1β1 and α2β1 integrins are expressed on the surface of many cell types where they serve as receptors for collagens and/or laminins (14Hall D.E. Reichardt L.F. Crowley E. Holley B. Moezzi H. Sonnenberg A. Damsky C.H. J. Cell Biol. 1990; 110: 2175-2184Crossref PubMed Scopus (301) Google Scholar, 15Ignatius M.J. Large T.H. Houde M. Tawil J.W. Barton A. Esch F. Carbonetto S. Reichardt L.F. J. Cell Biol. 1990; 111: 709-720Crossref PubMed Scopus (135) Google Scholar, 16Santoro S.A. Cell. 1986; 46: 913-920Abstract Full Text PDF PubMed Scopus (289) Google Scholar, 17Staatz W.D. Walsh J.J. Pexton T. Santoro S.A. J. Biol. Chem. 1990; 265: 4778-4781Abstract Full Text PDF PubMed Google Scholar). Studies by several groups, including our own, have shown that although the α1β1 and α2β1 integrins have similarities in ligand binding, they mediate different functions and are not redundant adhesive receptors (9Wary K.K. Mainiero F. Isakoff S.J. Marcantonio E.E. Giancotti F.G. Cell. 1996; 87: 733-743Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar, 10Wary K.K. Mariotti A. Zurzolo C. Giancotti F.G. Cell. 1998; 94: 625-634Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar, 18Langholz O. Rockel D. Mauch C. Kozlowska E. Bank I. Krieg T. Eckes B. J. Cell Biol. 1995; 131: 1903-1915Crossref PubMed Scopus (378) Google Scholar, 19Zutter M.M. Santoro S.A. Staatz W.D. Tsung Y.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7411-7415Crossref PubMed Scopus (198) Google Scholar, 20Riikonen T. Westermarck J. Koivisto L. Broberg A. Kahari V.M. Heino J. J. Biol. Chem. 1995; 270: 13548-13552Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 21Gotwals P.J. Chi-Rosso G. Lindner V. Yang J. Ling L. Fawell S.E. Koteliansky V.E. J. Clin. Invest. 1996; 97: 2469-2477Crossref PubMed Scopus (114) Google Scholar, 22Zutter M.M. Santoro S.A. Wu J.E. Wakatsuki T. Dickeson S.K. Elson E.L. Am. J. Pathol. 1999; 155: 927-940Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 23Ivaska J. Reunanen H. Westermarck J. Koivisto L. Kahari V.M. Heino J. J. Cell Biol. 1999; 147: 401-416Crossref PubMed Scopus (191) Google Scholar). Recent studies employing wild type, truncated, and chimeric α2 integrin subunits indicate that the unique phenotypic influences of the α2β1 integrin on cell proliferation and migration on collagenous substrates are mediated through the cytoplasmic domain of the α2 subunit (13Klekotka P.A. Santoro S.A. Zutter M.M. J. Biol. Chem. 2001; 276: 9503-9511Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 24Klekotka, P. A., Santoro, S. A., Ho, A., Dowdy, S. F., and Zutter, M. M. (2001) Am. J. Pathol., in press.Google Scholar). These findings suggested that certain residues present in the α2 integrin cytoplasmic domain not present in the α1 cytoplasmic domain might be responsible for mediating the differing phenotypes.To begin to understand these differences we have undertaken a mutagenesis study to identify the residues within the α2integrin cytoplasmic domain that are required to mediate chemotactic migration in response to epidermal growth factor (EGF) and entry into the cell cycle on type I collagen matrices in response to insulin. We find that the seven amino acids following the conserved GFFKR sequence are sufficient to recapitulate the phenotype of the full-length α2 integrin cytoplasmic domain in mammary epithelial cells. Mutation of the methionine at position 1137 to alanine inhibited both chemotactic migration and entry into S-phase. Mutation of the tyrosine at position 1134 to alanine inhibited migration, whereas it had only a modest effect on the ability of the cells to enter S-phase in the presence of insulin when adherent to type I collagen. Mutation of the lysine at position 1136 to alanine severely inhibited the ability of the mammary epithelial cells to enter S-phase, whereas it had no effect on the ability of the cells to migrate in response to EGF on a type I collagen matrix. Furthermore, these results confirmed our previous findings that phosphorylation of p38 MAPK downstream of the α2 integrin cytoplasmic domain was required for EGF-stimulated chemotactic migration and that up-regulation of cyclin E and cyclin dependent kinase 2 (cdk2) was required for entry into S-phase.RESULTSOur previous studies demonstrated that the α2, but not the α1, integrin subunit was essential for mediating EGF-stimulated chemotactic migration on type I collagen matrices (21Gotwals P.J. Chi-Rosso G. Lindner V. Yang J. Ling L. Fawell S.E. Koteliansky V.E. J. Clin. Invest. 1996; 97: 2469-2477Crossref PubMed Scopus (114) Google Scholar,13Klekotka P.A. Santoro S.A. Zutter M.M. J. Biol. Chem. 2001; 276: 9503-9511Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In agreement with recent studies (24Klekotka, P. A., Santoro, S. A., Ho, A., Dowdy, S. F., and Zutter, M. M. (2001) Am. J. Pathol., in press.Google Scholar) and as shown in Fig.1A, the α2integrin cytoplasmic domain was required to support insulin-dependent entry into S-phase of mammary epithelial cells cultured on type I collagen in a minimal media containing only insulin. The α1 cytoplasmic domain did not support entry into S-phase, as also shown in Fig. 1 A. To identify the regions of the α2 integrin cytoplasmic domain that were important for mediating EGF-stimulated chemotactic migration and insulin-dependent entry into S-phase, we constructed a series of truncated α2 integrin cytoplasmic domains containing 14 and 7 amino acids beyond the conserved GFFKR motif (1146 and 1139, respectively) (Fig. 1 B).The 1146 and 1139 constructs were transfected into the NMuMG-3 cell line, and stable cell lines were selected for in G418 (850 µg/ml). Surface expression of the human α2 integrin subunit was evaluated by flow cytometry, and clones that expressed the α2 integrin extracellular domain at levels similar to the X2C2 (wild type) transfectants were utilized (data not shown). Clones of both the 1146 and 1139 transfectants adhered to and spread on type I collagen in a manner similar to the X2C2 transfectants (data not shown). As shown in Fig. 2A, clones of the α2 integrin cytoplasmic domain truncations, 1146 and 1139, migrated in response to EGF on type I collagen matrices in a manner comparable to cells expressing the full-length α2 integrin cytoplasmic domain (X2C2). Similarly, both the 1146 and 1139 transfectants were able to enter S-phase, as measured by bromodeoxyuridine (BrdUrd) incorporation, following adhesion to type I collagen in the presence of insulin as effectively as the X2C2 transfectants (Fig. 2 B). These results indicate that the seven amino acids following the GFFKR motif of the α2integrin cytoplasmic domain were sufficient to support EGF-stimulated chemotactic migration as well as insulin-dependent entry into S-phase in mammary epithelial cells adherent to type I collagen. In addition, since the 1139 construct encoded a truncated α2 integrin cytoplasmic domain that was one amino acid shorter than the α1 integrin cytoplasmic domain, the different phenotypes supported by the α1 and α2 cytoplasmic domains fused to the α2integrin extracellular and transmembrane domains were a consequence of specific sequences and not a function of the length of the cytoplasmic domain per se.Figure 2α2 cytoplasmic domain-dependent insulin-stimulated entry into S-phase and EGF-stimulated chemotactic migration on type I collagen. A, X2C2, X2C1, 1146, and 1139 transfectants were serum-starved for 24 h and plated on the upper surface of a transwell filter coated with type I collagen (25 µg/ml). Migration through the pores of the filter was either unstimulated (Control) or stimulated with EGF (10 ng/ml) (EGF). Cell migration proceeded for 5 h in a 5% CO2 humidified chamber at 37 °C. The number of cells attached to the lower surface of the transwell filter was quantitated microscopically. Results are presented as the mean ± S.E. of at least three separate experiments. B, entry into S-phase by clonal cell lines of the X2C2, X2C1, 1146, and 1139 transfectants was determined 24 h after the serum-starved cells were plated on type I collagen in DMEM plus insulin (5 µg/ml). The percentage of cells that incorporated BrdUrd during the final 2 h of incubation was determined. Results shown represent the mean ± S.E. of at least three separate experiments. C, X2C2, X2C1, 1135, and X2C0 transfectants were serum-starved for 24 h and plated on the upper surface of a transwell filter coated with type I collagen (25 µg/ml). Migration through the pores of the filter was either unstimulated (Control) or stimulated with EGF (10 ng/ml) (EGF). Cell migration proceeded for 5 h in a 5% CO2 humidified chamber at 37 °C. The number of cells attached to the lower surface of the transwell filter was quantitated microscopically. Results are presented as the mean ± S.E. of at least three separate experiments. D, entry into S-phase by clonal cell lines of the X2C2, X2C1, 1135, and X2C0 transfectants was determined 24 h after the serum-starved cells were plated on type I collagen in DMEM plus insulin (5 µg/ml). The percentage of cells that incorporated BrdUrd during the final 2 h of incubation was determined. Results shown represent the mean ± S.E. of at least three separate experiments.View Large Image Figure ViewerDownload (PPT)We have shown previously that the α2 integrin subunit with a cytoplasmic domain truncated after the GFFKR motif (X2C0) was not able to support either entry into S-phase in the presence of insulin or EGF-stimulated chemotactic migration on type I collagen (22Zutter M.M. Santoro S.A. Wu J.E. Wakatsuki T. Dickeson S.K. Elson E.L. Am. J. Pathol. 1999; 155: 927-940Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar,13Klekotka P.A. Santoro S.A. Zutter M.M. J. Biol. Chem. 2001; 276: 9503-9511Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Since the data in Fig. 2, A and B,demonstrated that the seven amino acids following the GFFKR motif were sufficient to replicate the phenotype of the full-length α2 integrin cytoplasmic domain, we made an additional cytoplasmic domain truncation by deleting the carboxyl-terminal 17 amino acids of the α2 integrin cytoplasmic domain. As shown in Fig. 1 B, this deletion created a construct that encoded a cytoplasmic domain that consisted of the GFFKR motif followed by the three amino acids of the α2integrin cytoplasmic domain (1135). The 1135 construct was transfected into the NMuMG-3 cell line, and clonal cell lines that expressed the α2 integrin extracellular domain at levels comparable to the X2C2, 1146, and 1139 transfectants were established, as described above (data not shown). Clones of the 1135 transfectants adhered to and spread on type I collagen in a manner similar to the X2C2 transfectants (data not shown). As shown in Fig. 2 C, the ability of the 1135 transfectants to migrate in response to a chemotactic gradient of EGF was significantly reduced as compared with either the X2C2 or 1139 transfectants. Deletion of the four amino acids from the 1139 construct to create the 1135 construct also abrogated the ability of the 1135 transfectants to enter S-phase when plated on type I collagen in the presence of insulin (Fig. 2 D). These results indicate that one or more of the four amino acids deleted to create the 1135 construct was essential for mediating the functions of the α2 integrin cytoplasmic domain exhibited by the X2C2 and 1139 transfectants.Comparison of the seven amino acids distal to the GFFKR motif in the human α2 integrin cytoplasmic domain to those residues in the murine and bovine α2 integrin cytoplasmic domains revealed that only three of the amino acids are conserved across all three species (Fig. 3A). The three conserved residues include the tyrosine at position 1134, the lysine at position 1136, and the methionine at position 1137. Therefore, each of these three residues was individually mutated to an alanine in the 1139 cytoplasmic tail to create the 1139 Y1134A, 1139 K1136A, and 1139 M1137A constructs, respectively (Fig. 3 A). The tyrosine at position 1134 was also mutated to a phenylalanine (1139 Y1134F). Finally, although not conserved across all three species, the threonine at position 1138 of the human α2 subunit was mutated to alanine. These constructs were transfected into the NMuMG-3 cell line, and stable cell lines were established and selected as described above (data not shown). Individual clones of all four transfectants adhered to and spread on type I collagen in a manner similar to the X2C2 transfectants (data not shown). As shown in Fig.3 B, mutation of the lysine at position 1136 had no effect on the ability of the 1139 K1136A transfectants to migrate in response to EGF on type I collagen matrices. Substitution of the tyrosine at position 1134 with a phenylalanine had no effect on the ability of the 1139 Y1134F transfectants to migrate. However, mutation of either the tyrosine at position 1134 to alanine or the methionine at position 1137 to alanine severely reduced the ability of both the 1139 Y1134A and 1139 M1137A transfectants to migrate in response to EGF. Mutation of the threonine at position 1138 to alanine did not have a significant effect on chemotactic migration. These findings established the essential nature of the methionine at position 1137 and an aromatic amino acid at position 1134 for the ability of the α2integrin cytoplasmic domain to support EGF-dependent chemotactic migration by mammary epithelial cells on type I collagen matrices.Figure 3α2 cytoplasmic domain-dependent insulin-stimulated entry into S-phase and EGF-stimulated chemotactic migration on type I collagen is dependent on specific residues. A, species comparison of the membrane-proximal 14 amino acids of the α2 integrin cytoplasmic domains from the human, murine, and bovine sequences available in GenBankTM. The conserved residues distal to the GFFKR motif are underlined (upper panel). Diagram of the 1139 Y1134A, 1139 Y1134F, 1139 K1136A, and 1139 M1137A constructs illustrating the amino acid mutation (underlined) in the truncated human α2 integrin cytoplasmic domain (lower panel). B, X2C2, X2C1, 1139, 1139 Y1134A, 1139 Y1134F, 1139 K1136A, and 1139 M1137A transfectants were serum-starved for 24 h and plated on the upper surface of a transwell filter coated with type I collagen (25 µg/ml). Migration through the pores of the filter was either unstimulated (Control) or stimulated with EGF (10 ng/ml) (EGF). Cell migration proceeded for 5 h in a 5% CO2 humidified chamber at 37 °C. The number of cells attached to the lower surface of the transwell filter was quantitated microscopically. Results are presented as the mean ± S.E. of at least three separate experiments. C, entry into S-phase by clones of the X2C2, X2C1, 1139, 1139 Y1134A, 1139 Y1134F, 1139 K1136A, and 1139 M1137A transfectants was determined 24 h after the serum-starved cells were plated on type I collagen in DMEM plus insulin (5 µg/ml). The percentage of cells that incorporated BrdUrd during the final 2 h of incubation was determined. Results shown represent the mean ± S.E. of at least three separate experiments.View Large Image Figure ViewerDownload (PPT)Next, we evaluated the ability of the 1139 Y1134A, 1139 K1136A, and 1139 M1137A transfectants to enter S-phase in an insulin-dependent manner following adhesion to type I collagen. The data in Fig. 3 C demonstrate that mutation of either the lysine at position 1136 or the methionine at position 1137 abrogated the ability of the 1139 K1136A and 1139 M1137A transfectants to enter S-phase on type I collagen in the presence of insulin. However, in contrast to its effect on chemotactic migration, mutation of the tyrosine at position 1134 to an alanine had only a modest effect on the ability of the 1139 Y1134A transfectants to enter S-phase in an insulin-dependent manner when adherent to type I collagen. Replacement of the tyrosine at position 1134 with phenylalanine had little to no effect on the ability of the 1139 Y1134F transfectants to enter S-phase in the presence of insulin. Mutation of the threonine residue at position 1138 to alanine was also without effect (data not shown). These data demonstrate that the methionine at position 1137 was required for both EGF-dependent chemotactic migration and insulin-dependent entry into S-phase, whereas the lysine at position 1136 was required for entry into S-phase but not for migration. Conversely, the tyrosine, or at least an aromatic amino acid, at position 1134 was required for migration but was not essential for supporting α2 integrin cytoplasmic domain-dependent entry into S-phase in the presence of insulin in mammary epithelial cells expressing a truncated α2 integrin cytoplasmic domain.Progression through the G1 phase of the cell cycle and into S-phase requires signals from both growth factor receptors and the extracellular matrix. Signals from growth factor receptors and integrins converge on several signaling pathways including the ERK MAPK pathway (7Aplin 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, 28Renshaw M.W. Ren X. Schwartz M.A. EMBO J. 1997; 16: 5592-5599Crossref PubMed Scopus (269) Google Scholar, 29Chen Q. Lin T.H. Der C.J. Juliano R.L. J. Biol. Chem. 1996; 271: 18122-18127Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 30Bottazzi M.E. Assoian R.K. Trends Cell Biol. 1997; 7: 348-352Abstract Full Text PDF PubMed Scopus (55) Google Scholar, 31Howe A.K. Juliano R.L. J. Biol. Chem. 1998; 273: 27268-27274Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 32Renshaw M.W. Price L.S. Schwartz M.A. J. Cell Biol. 1999; 147: 611-618Crossref PubMed Scopus (189) Google Scholar). We were interested in determining if the ERK MAPK pathway was differentially activated in the transfectants that enter S-phase (the X2C2, 1139, 1139 Y1134F, and 1139 Y1134A transfectants)versus those that do not enter S-phase (the X2C1, K1136A, and M1137A transfectants). The results in Fig.4A, using an anti-phospho-ERK-specific antibody to evaluate the phosphorylation status of the ERK MAPK demonstrated that the X2C2, 1139, 1139 Y1134F, and 1139 Y1134A transfectants exhibited sustained phosphorylation (up to 24 h) of the ERK MAPK following adhesion to collagen substrates. In contrast, the X2C1, 1139 K1136A, and 1139 M1137A transfectants phosphorylated the ERK MAPK for the first 4 h following adhesion to type I collagen in the presence of insulin and then the levels of phospho-ERK decreased to base-line levels (Fig.4 A). To determine if sustained activation of the ERK MAPK pathway was required for entry into S-phase, we utilized a pharmacologic inhibitor of MAPK kinase, PD98059 (33Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2584) Google Scholar). As shown in Fig.4 B, incubation of the X2C2 transfectants with PD98059 for the entire 24 h of the experiment significantly inhibited the number of cells entering S-phase, with a p < 0.002. When PD98059 was present for the first 6 h of the experiment and then removed and fresh media added, the inhibitor had no effect on the ability of the X2C2 transfectants to enter S-phase. Conversely, if the inhibitor was added after the first 6 h of the experiment, the compound inhibited the ability of the X2C2 transfectants to enter S-phase just as if it had been present for the entire experiment (Fig.4 B). Immunoblot analysis revealed that PD98059 inhibited the phosphorylation of ERK and that when PD98059 was removed the X2C2 transfectants were able to induce moderate levels of ERK phosphorylation again. Therefore, sustained phosphorylation (>6 h) of the ERK MAPK was required for mammary epithelial cells to enter S-phase following adhesion to type I collagen via the α2β1 integrin. Whereas adhesion to type I collagen of the X2C2, 1139, 1139 Y1134F, and 1139 Y1134A transfectants in the presence of insulin initiated a signal transduction cascade that induced the sustained phosphorylation of ERK required for entry into S-phase, the X2C1, 1139 K1136A, and 1139 M1137A transfectants were unable to induce sustained phosphorylation of ERK upon adhesion to collagen and were therefore unable to enter S-phase.Figure 4α2 cytoplasmic domain-dependent phosphorylation of ERK MAPK is dependent on specific residues. A, following serum starvation the X2C2, X2C1, 1139, 1139 K1136A, 1139 Y1134F, 1139 M1137A, and 1139 Y1134A transfectants were either lysed (time = 0) or plated on type I collagen in the presence of insulin (5 µg/ml). Following adhesion, cells were lysed at defined intervals including 1, 4, 8, 16, and 24 h. Immunoblot analysis of cell lysates was performed using the monoclonal anti-phospho-ERK MAPK antibody (P-ERK) followed by the polyclonal anti-total ERK MAPK antibody (Total ERK). B, entry into S-phase of the X2C2 transfectants was determined 24 h after the serum-starved cells were plated on type I collagen in DMEM plus insulin (5 µg/ml) (Control), in the presence of PD98059 (50 µm) (PD98059), in the presence of PD98059 for 6 h followed by DMEM plus insulin for the remaining 18 h (0–6 h + PD98059 6–24 h −PD98059), or in the absence of PD98059 for 6 h followed by PD98059 for the remaining 18 h (0–6 h −PD98059 6–24 h + PD98059). The percentage of cells that incorporated BrdUrd during the final 2 h of incubation was determined. Results shown represent the mean ± S.E. of at least three separate experiments (upper panel). Following serum starvation the X2C2 transfectants were lysed (time = 0) or plated on type I collagen in DMEM plus insulin (5 µg/ml) in the absence of PD98059 (X2C2), in the presence of PD98059 (50 µm) for 6 h followed by media without PD98059 for the remaining 18 h (0–6 h + PD98059 6–24 −PD98059), or in the absence of PD98059 for 6 h followed by the addition of PD98059 (50 µm) for the remaining 18 h (0–6 h −PD98059 6–24 h + PD98059). Following adhesion, cells were lysed at defined intervals including 1, 4, 8, 16, and 24 h. Immunoblot analysis of cell lysates was performed using the monoclonal anti-phospho-ERK MAPK antibody (P-ERK) followed by the polyclonal anti-total ERK MAPK antibody (Total ERK) (lower panel).View Large Image Figure ViewerDownload (PPT)Following adhesion to collagen the X2C2 transfectants up-regulated the levels of cyclin E and cdk2 as they progressed through the G1 phase of the cell cycle (Fig.5A). However, the X2C1 transfectants induced a transient increase in cyclin E levels but failed to up-regulate cdk2 following adhesion to type I collagen and were arrested in G1. To determine if the inability of the 1139 K1136A and 1139 M1137A transfectants to progress through G1 and into S-phase was due to a failure to induce cyclin E and/or cdk2 expression, the transfectants were serum-starved, plated on type I collagen in the presence of insulin, and lysed at defined time points. As shown in Fig. 5 A, transfectants that were able to enter S-phase (X2C2, 1139, 1139 Y1134F, and 1139 Y1134A) up-regulated the expression of both cyclin E and cdk2 following adhesion to type I collagen in an insulin-dependent manner, as expected. The X2C1 transfectants failed to increase substantially the expression of cdk2 or induce sustained up-regulation of cyclin E (Fig.5 A). The 1139 K1136A transfectants induced cyclin E expression but not up-regulation of cdk2, and the 1139 M1137A transfectants failed to substantially induce expression of cdk2 or sustained up-regulation of cyclin E (Fig. 5 A). These results suggested that α2 integrin cytoplasmic domain-dependent induction of cyclin E and cdk2 levels, and therefore entry into S-phase, following adhesion to type I collagen was dependent on the presence of a lysine at position 1136 and a methionine at position 1137.Figure 5Increased cyclin E and cdk2 expression is dependent on specific residues within the α2 integrin cytoplasmic domain. A, the levels of cyclin E, cdk2, and actin expressed by the X2C2, 1139, 1139 Y1134F, 1139 Y1134A, X2C1, 1139 K1136A, and 1139 M1137A transfectants following adhe
Referência(s)