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Cell Anchorage Permits Efficient Signal Transduction Between Ras and Its Downstream Kinases

Cell Anchorage Permits Efficient Signal Transduction Between Ras and Its Downstream Kinases THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 14, Issue of April 4, pp. 8849 –8852, 1997 Communication © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. can trigger activation of MAP kinase (14 –16) and of other Cell Anchorage Permits protein kinases (17) that are part of the consensus signaling Efficient Signal Transduction pathway leading from receptor tyrosine kinases to Ras and then to a cytoplasmic kinase cascade comprising Raf, MEK1, Between Ras and Its MEK2, and MAP kinases (18). Downstream Kinases* Because integrins directly activate elements of the MAP kinase cascade, it is of interest to ask whether integrin-medi- (Received for publication, December 17, 1996, and in revised ated cell anchorage can also regulate the action of soluble form, January 31, 1997) mitogens on this cascade. If this were so, it would have impor- tant ramifications for understanding the anchorage depend- Tsung H. Lin‡, Qiming Chen§, Alan Howe, and R. L. Juliano¶ ence of cell cycle traverse. Previous studies of possible collab- oration between peptide mitogens and cell anchorage have led From the Department of Pharmacology, School of Medicine, University of North Carolina, to differing results. In some cases an enhancement of mitogen Chapel Hill, North Carolina 27599 signaling was observed in anchored cells as compared with their counterparts maintained in suspension, whereas in other Cell anchorage strongly affects the signal transduc- cases no such effect was observed (19 –22). In the present tion cascade initiated by peptide mitogens. For both investigation we have studied the collaboration between mito- epidermal growth factor and platelet-derived growth gens and anchorage in NIH 3T3 cells, a cell type that has been factor, activation of the consensus mitogen-activated widely used in signal transduction studies. We have examined protein kinase cascade is impaired when cells are held several steps in the signal transduction pathway leading from in suspension as compared with cells anchored to a fi- bronectin substratum. Upstream events in the signaling receptor tyrosine kinases to Ras and then to the downstream cascade, including tyrosine phosphorylation of the mi- kinases. We find that peptide mitogen activation of receptor togen receptor and GTP loading of Ras, are similar in tyrosine kinases and subsequent activation of Ras are inde- anchored and suspended cells. However, propagation of pendent of anchorage. However, signal transduction between the signal to Raf and subsequently to the downstream Ras and Raf is markedly attenuated in nonadherent cells, kinases MEK and mitogen-activated protein kinase is leading to reduced activation of Raf, MEK, and MAP kinase. markedly attenuated in suspended cells. Thus, there EXPERIMENTAL PROCEDURES seems to be a distinct anchorage-dependent step be- NIH 3T3 cells were maintained in Dulbecco’s minimal essential tween Ras and Raf in the signaling cascade initiated by medium containing 10% bovine calf serum and antibiotics. Confluent peptide mitogens. These observations may have impor- cells were serum-starved for 16 h before detachment by 0.05% trypsin tant implications for understanding the anchorage de- and 0.33 mM EDTA; trypsin activity was neutralized by 1 mg/ml soy- pendence of cell growth. bean trypsin inhibitor. Cells were suspended in Dulbecco’s minimal essential medium with 2% bovine serum albumin and incubated in suspension at 37 °C for 45 min in a rotator to allow kinases become quiescent. Cells were then either maintained in suspension or plated Cell anchorage to the proteins of the extracellular matrix is onto dishes coated with fibronectin (20 mg/ml) or with poly-L-lysine (20 known to have profound effects on cell differentiation (1, 2), cell mg/ml) and incubated at 37 °C for the indicated times. In some cases the growth (3), and apoptosis (4). A particularly important example suspended or adherent cells were stimulated with either EGF or PDGF of this concerns the recently described effects of anchorage on (Upstate Biotechnologies Inc.). Cell lysates were prepared and tested the expression and activity of components of the cell cycle for the activity of Raf, MEK, and MAP kinase using specific in vitro machinery, including cyclin D1-CDK4,6 complexes and cyclin kinase assays as described previously (17). The phosphorylation status of the EGF receptor and PDGF receptor were evaluated by immuno- E-CDK2 complexes (5, 6). These observations are clearly rele- precipitation of the receptor using antibodies obtained from H. S. Earp vant to the question of why both soluble mitogens and cell (EGF-R) or from Santa Cruz Biotechnology (PDGF-R) followed by West- anchorage are required for the growth of normal cells, whereas ern blotting with an anti-phosphotyrosine antibody and detection by the anchorage requirement is abrogated in transformed cells enhanced chemiluminescence (17). For studies of GTP loading of the (7). Many aspects of cell to extracellular matrix interactions Ras protein, cells were radiolabeled with [ P]orthophosphate, and the involve the integrin family of cell surface heterodimeric adhe- [ P]GTP and GDP bound to immunoprecipitated Ras were quantitated by thin layer chromatography and PhosphorImager analysis as de- sion proteins (8). Recently, it has become clear that integrins scribed (17). are signal transducing receptors (9, 10) capable of influencing a number of intracellular biochemical activities including protein RESULTS AND DISCUSSION tyrosine kinases (11), serine/threonine kinases (12), and ionic Because elements of the MAP kinase cascade are directly but transients (13). In particular, integrin-mediated cell adhesion transiently activated by integrin-mediated cell adhesion (17), we initially examined the kinetics of this process to find a time * This work was supported by National Institutes of Health Grants point when we could examine anchorage effects on mitogen- GM26165 and HL45100 (to R. L. J.). The costs of publication of this driven activation of MAP kinase without a direct contribution article were defrayed in part by the payment of page charges. This from integrin-mediated MAP kinase activation. As seen in Fig. article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1(A and B), when NIH 3T3 cells were held in suspension, EGF ‡ Present address: Pharmacopeia, 101 College Rd. East, Princeton, caused a robust tyrosine phosphorylation of EGF-R but had NJ 08540. § Present address: Hoffman LaRoche, 340 Kingsland St., Nutley, NJ 07110. The abbreviations used are: MAP, mitogen-activated protein; EGF, To whom correspondence should be addressed. Tel.: 919-966-4383; epidermal growth factor; PDGF, platelet-derived growth factor; EGF-R, Fax: 919-966-5640; E-mail: [email protected]. EGF receptor; PDGF-R, PDGF receptor. This paper is available on line at http://www-jbc.stanford.edu/jbc/ 8849 This is an Open Access article under the CC BY license. 8850 Integrin-Mitogen Collaboration in Signal Transduction FIG.1. Cell anchorage to fibronectin modulates MAP kinase activation but not EGF receptor activation. Serum-starved NIH 3T3 cells were harvested and then either maintained in suspension or allowed to adhere to substrata coated with fibronectin; in some cases (1) the cells were treated with EGF for 5 min. Tyrosine phosphorylation of the EGF receptor was evaluated by immunoprecipitation (IP) with anti-EGF-R antibody followed by Western immunoblotting (IB) with an anti-phosphotyrosine antibody. MAP kinase activity was evaluated by a band shift assay and by immunoprecipitation of the enzyme followed by an in vitro kinase assay using myelin basic protein (MBP) as a substrate as described previously (17). A, EGF-R tyrosine phosphorylation as a function of time after plating cells on fibronectin. B, MAP kinase activity as a function of time after plating cells on fibronectin. (For A and B 20 ng/ml of EGF was used.) C, EGF-R tyrosine phosphorylation as a function of the dose of EGF. D, MAP kinase activation as a function of the dose of EGF. (For C and D the EGF dose is given in ng/ml.) NAD, nonadherent (suspended) cells; Fn, cells adherent to fibronectin-coated substrata. only a very modest effect on MAP kinase (Fig. 1B, lanes 1 and 2). After 10 min of cell adhesion to fibronectin-coated substrata, when the cells were fully attached but not spread, there was a strong adhesion-mediated activation of MAP kinase; EGF stim- ulation caused tyrosine phosphorylation of EGF-R and further stimulated MAP kinase (Fig. 1B, lanes 3 and 4). A qualitatively similar situation also prevailed after 30 min of cell adhesion when the cells were partially spread (Fig. 1B, lanes 5 and 6). By 180 min, when the cells were well spread, in the absence of EGF there was only a basal level of MAP kinase activity, whereas treatment with EGF caused tyrosine phosphorylation of EGF-R and resulted in a strong stimulation of MAP kinase (Fig. 1B, lanes 7 and 8). Thus, in serum-starved 3T3 cells, EGF activation of its receptor seems to be independent of cell an- chorage; however, the MAP kinase response is strongly influ- enced by anchorage. In nonadherent cells, EGF produces only a weak activation of MAP kinase. Shortly after the cells adhere to the fibronectin substratum, EGF and anchorage have ap- proximately additive effects on MAP kinase activity. At longer times, EGF strongly activates MAP kinase in anchored cells, FIG.2. Mitogen activation of the MAP kinase cascade in an- chored or suspended cells. Serum-starved NIH 3T3 cells were har- whereas the direct activation by cell adhesion has returned to vested and then either maintained in suspension or allowed to adhere basal levels. In Fig. 1 (C and D) we examined EGF concentra- to substrata coated with fibronectin for 180 min; in some cases (1) the tion-response relationships for EGF-R and MAP kinase in cells cells were treated with either EGF (20 ng/ml) or PDGF (20 ng/ml) for 5 that have either been maintained in suspension or anchored to min. The components of the signaling cascade were immunoprecipi- fibronectin for 180 min. As shown, the concentration-response tated (IP) using specific antibodies, and their activities were assayed as described previously (17). A, EGF responses. Top panel, EGF-R, West- profile for EGF-R tyrosine phosphorylation was essentially ern blot (IB) with anti-phosphotyrosine antibody; second panel, EGF-R identical in suspended cells and cells anchored to fibronectin expression, Western blot with anti-EGF-R; third panel, Raf-B activa- substrata. However, at all EGF concentrations tested, an- tion, immunoprecipitation with anti-Raf-B followed by an in vitro chored cells displayed a 3– 4-fold greater activation of MAP linked kinase assay (17) with kinase-dead MAP kinase (K MAPK)asa substrate; fourth panel, MEK activation, immunoprecipitation with kinase than did suspended cells (for example, compare lanes 4 anti-MEK followed by an in vitro kinase using kinase-dead MAP kinase and 10 in Fig. 1D, both at 5 ng/ml EGF). as a substrate (17); fifth panel, MAP kinase activation, immunoprecipi- We have also investigated how anchorage modulates mito- tation with anti-MAP kinase followed by an in vitro kinase assay using gen actions on other components of the MAP kinase cascade. As MBP as a substrate (17). B, PDGF responses. The same assays were used as in A, except that the top two panels employed specific antibodies shown in Fig. 2A, EGF stimulated similar levels of tyrosine to PDGF-R (Santa Cruz), and the third panel employed an antibody to phosphorylation of EGF-R in suspended or anchored cells. Raf-1. NAD, nonadherent cells; Fn, cells adherent to fibronectin-coated However, EGF produced substantially stronger activations of substrata; MBP, myelin basic protein. Raf-B, MEK, and MAP kinase in cells anchored to fibronectin substrata as compared with nonanchored cells. Raf-1 was also activated (weakly) by EGF in anchored cells but not in sus- suspended cells. However, PDGF treatment resulted in mark- pended cells (not shown). We decided to also examine anchor- edly stronger activation of Raf-1 and MEK in anchored cells as age dependence of signaling events mediated by PDGF, an- compared with suspended cells, as well as a more modest but other peptide mitogen. Exposure of 3T3 cells to PDGF caused a significant difference in MAP kinase activation. Thus, for substantially greater increase in overall cellular protein tyro- PDGF, as for EGF, cell anchorage seems to control the effi- sine phosphorylation than was observed with EGF (not shown). ciency of signal transduction between initial activation of the As seen in Fig. 2B, PDGF caused equivalent robust tyrosine receptor tyrosine kinase and subsequent activation of down- phosphorylation of its cognate receptor in both anchored and stream kinases. Integrin-Mitogen Collaboration in Signal Transduction 8851 FIG.4. Attenuation of the mitogen signaling cascade between Ras and Raf in nonanchored cells. This figure summarizes several independent experiments for EGF (A) and PDGF (B) stimulation. The tyrosine phosphorylation of immunoprecipitated EGF-R and PDGF-R was quantitated by laser densitometry of enhanced chemiluminescence FIG.3. Mitogen activation of Ras GTP loading in anchored or Western blots. The activation of Raf, MEK, and MAP kinases, as well as suspended cells. Serum-starved NIH 3T3 cells were harvested and GTP loading of Ras, were quantitated using a PhosphorImager. The then either maintained in suspension or allowed to adhere to substrata parameter shown on the ordinate (Mitogen Activation NAD/Fn) repre- coated with fibronectin for 180 min; in some cases (1) the cells were sents the ratio of mitogen activation in nonadherent cells (NAD) to that treated with EGF (20 ng/ml) or PDGF (20 ng/ml) for 5 min. Ras GTP observed in cells anchored on fibronectin substrata (Fn). In each case it loading was measured by thin layer chromatography as described (17). was calculated by subtracting the basal value from the mitogen-stim- The data are presented as the percentage of bound GTP ((GTP/(1.5 ulated value; the differences (termed dNAD or dFn) were divided to get GDP1GTP)) 3 100). The results for PDGF represent the means and the ratio given on the ordinate (dNAD/dFn). Thus, if cell anchorage had standard errors for three independent experiments, whereas for EGF no effect on the mitogen activation of a certain component in the the means of two independent experiments are shown. pathway, the ratio on the ordinate for that component would be 1; numbers less than 1 indicate that the mitogen-mediated activation is attenuated in nonadherent cells. The results represent the means and Because the GTP-bound form of Ras is a key transducer in standard errors for three independent experiments (two for EGF effects the mitogen signaling pathway (18), we decided to examine Ras on Ras). GTP loading in anchored or suspended 3T3 cells treated with either PDGF or EGF. As seen in Fig. 3, the basal GTP/GDP ratio was somewhat higher in suspension cells than in adher- activation of MAP kinase is 2–3-fold greater in cells plated on ent cells. Treatment with peptide mitogen resulted in a strong a fibronectin substratum, as opposed to a poly-lysine substra- increase in Ras GTP loading in both suspended cells and an- tum (data not shown). This suggests that the anchorage effects chored cells, with PDGF producing a somewhat greater effect on signaling that we have observed may be mediated by inte- than EGF. Thus, Ras GTP loading in response to mitogens grins; however, several additional lines of investigation will be occurred in suspended cells at least as well as in anchored cells; needed to fully confirm this possibility. in fact, suspended cells usually showed higher levels of GTP Anchorage dependence of cell growth is one of the most loading than anchored cells. This observation, along with those fundamental differences between normal and transformed cells of Fig. 2, suggests that peptide mitogen signaling pathways are (24). Our observations indicate that cell anchorage can influ- intact and operate efficiently in both suspended and anchored ence the efficiency of signal tranduction in mitogenic pathways. cells up to the level of Ras but are attenuated between Ras and This suggests the possibility that adhesion effects on early the Raf kinases in the nonanchored cells. signaling events may play an important role in anchorage Quantitation of anchorage effects on activation of several dependence of cell growth, although other factors may also be components of the EGF- and PDGF-triggered pathways is involved. For both EGF and PDGF, the upstream events of the shown in Fig. 4. The data are expressed as the ratio of the mitogen signaling pathway were independent of anchorage. mitogen activation in suspended cells versus anchored cells. Thus, receptor tyrosine kinase activation and GTP loading of The downstream kinases (Rafs, MEKs, and MAP kinases) dis- Ras were robust in both anchored and suspended cells. For both play substantial reductions in activity in nonanchored cells, mitogens, however, cell adhesion had a profound effect on the but Ras and the receptor tyrosine kinases do not. Although the activation of the MEK-kinases Raf-B and Raf-1 and clear-cut exact magnitudes of the anchorage effects on activation of the effects of lesser magnitude on MEK and MAP kinase. The individual components of the pathway differ somewhat be- nonlinearity of the effects we have observed on anchorage tween EGF and PDGF, the trend is similar. The observation regulation of signal transduction may be due to the extensive that EGF and PDGF produce qualitatively similar but quanti- branching and cross-talk that is known to occur in the MAP tatively distinct effects on the consensus MAP kinase cascade is kinase cascade (25). Our observations suggest that in sus- not surprising, because individual receptor tyrosine kinases pended cells, there is a rather sharp break in the signaling are known to have distinct effects on cell growth and differen- cascade between Ras and the Raf kinases. Because a major role tiation (23). of Ras in signal transduction is to recruit Raf to the plasma Although cell anchorage to a fibronectin-coated substratum membrane (26), our findings suggest that cell anchorage con- clearly has a substantial impact on the MAP kinase cascade, it tributes to this process. One plausible model is that integrin- is not yet certain that this is purely an integrin-mediated dependent focal contacts formed during cell adhesion partici- phenomenon. Preliminary experiments have shown that EGF pate in the recruitment and subsequent activation of Raf. 8852 Integrin-Mitogen Collaboration in Signal Transduction 7. Clarke, G. D., Stoker, M. G., Ludlow, A. & Thornton, M. (1970) Nature 227, Cell anchorage to fibronectin, a process primarily mediated 798 – 801 by integrins, resulted in 2–3-fold greater activation of MAP 8. Hynes, R. O. (1992) Cell 69, 11–25 kinases by peptide mitogens as compared with suspended cells. 9. Rosales, C., O’Brien, V., Kornberg, L. & Juliano, R. L. (1995) Biochim. Biophys. Acta 1242, 77–98 At this point it is unclear whether a change in MAP kinase 10. Clark, E. A. & Brugge, J. S. (1995) Science 286, 233–235 activation of this magnitude would account for the strong effect 11. Richardson, A. & Parsons, J. T. (1996) Nature 380, 538 –540 that anchorage has on cell growth. It is important to note, 12. Hannigan, G. E., Leung-Hagensteijn, C., Fitz-Gibbon, L., Coppolino, M. G., Radeva, G., Filmus, J., Bell, J. C., and Dedhar, S. (1996) Nature 379, 91–96 however, that the effects of anchorage on Raf-1 or Raf-B acti- 13. Schwartz, M. A. (1993) J. Cell Biol. 120, 1003–1010 vation were much greater (8 –20-fold). Raf family kinases are 14. Chen, Q., Kinch, M. S., Lin, T. H., Burridge, K. & Juliano, R. L. (1994) J. Biol. thought to have downstream targets other than the MAP ki- Chem. 269, 26602 -26605 15. Zhu, X. & Assoian, R. K. (1995) Mol. Biol. Cell 6, 273–282 nase pathway (27, 28). Thus, it seems possible that the anchor- 16. Schlaepfer, D. D., Hanks, S., Hunter, T. & van der Geer, P. (1994) Nature 372, age modulation of mitogen signaling reported here, particu- 786 –791 larly the dramatic effect on Raf family kinases, may be 17. Chen, Q., Lin, T. H., Der, C. J. & Juliano, R. L. (1996) J. Biol Chem. 271, 18122–18127 important aspects of cell growth control. 18. Egan, S. E. & Weinberg, R. A. (1993) Science 365, 781–783 19. McNamee, H. P., Ingber, D. E. & Schwartz, M. A. (1993) J. Cell. Biol. 121, Acknowledgments—We thank Andrew Aplin and Channing Der for 673– 678 valuable comments on the manuscript. 20. Hotchin, N. A. & Hall, A. (1995) J. Cell. Biol. 131, 1857–1865 21. Bohmer, R. M., Scharf, E. & Assoian, R. K. (1996) Mol Biol. Cell 7, 101–111 REFERENCES 22. Vuori, K. & Ruoslahti, E. (1994) Science 266, 1576 –1578 1. Streuli, C. H., Edwards, G. M., Delcommenne, M., Whitelaw, C. B. A., Burdon, 23. Marshall, C. J. (1995) Cell 80, 179 –185 T. G., Schindler, C. & Watson, C. J. (1995) J. Biol. Chem. 270, 21639 –21644 24. Ruddon, R. (1995) in Cancer Biology, p. 101, Oxford University Press, 2. Juliano, R. L. & Haskill, S. (1993) J. Cell Biol. 120, 577–585 New York 3. Varner, J. A., Emerson, D. A. & Juliano, R. L. (1995) Mol. Biol. Cell. 6, 725–740 25. Vojtek, A. B. & Cooper, J. A. (1995) Cell 82, 527–529 4. Ruoslahti, E. & Reed, J. C. (1994) Cell 77, 477– 478 26. Leevers, S. J., Paterson, H. F. & Marshall, C. J. (1994) Nature 369, 411– 414 5. Fang, F., Orend, G., Watanabe, N., Hunter, T. & Ruoslahti, E. (1996) Science 27. Avruch, J., Zhang, X. F. & Kyriakis, J. M. (1994) Trends Biochem. Sci. 19, 271, 499 –502 279–83 6. Zhu, X., Ohtsubo, M., Bohmer, R. M., Roberts, J. M. & Assoian, R. K. (1996) J. Cell Biol. 133, 391– 403 28. Galaktionov, K., Jessus, C. & Beach, D. (1995) Genes & Dev. 9, 1046 –58 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Cell Anchorage Permits Efficient Signal Transduction Between Ras and Its Downstream Kinases

Lin, Tsung H.; Chen, Qiming; Howe, Alan; Juliano, R.L.
Journal of Biological Chemistry – Apr 1, 1997
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 14, Issue of April 4, pp. 8849 –8852, 1997 Communication © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. can trigger activation of MAP kinase (14 –16) and of other Cell Anchorage Permits protein kinases (17) that are part of the consensus signaling Efficient Signal Transduction pathway leading from receptor tyrosine kinases to Ras and then to a cytoplasmic kinase cascade comprising Raf, MEK1, Between Ras and Its MEK2, and MAP kinases (18). Downstream Kinases* Because integrins directly activate elements of the MAP kinase cascade, it is of interest to ask whether integrin-medi- (Received for publication, December 17, 1996, and in revised ated cell anchorage can also regulate the action of soluble form, January 31, 1997) mitogens on this cascade. If this were so, it would have impor- tant ramifications for understanding the anchorage depend- Tsung H. Lin‡, Qiming Chen§, Alan Howe, and R. L. Juliano¶ ence of cell cycle traverse. Previous studies of possible collab- oration between peptide mitogens and cell anchorage have led From the Department of Pharmacology, School of Medicine, University of North Carolina, to differing results. In some cases an enhancement of mitogen Chapel Hill, North Carolina 27599 signaling was observed in anchored cells as compared with their counterparts maintained in suspension, whereas in other Cell anchorage strongly affects the signal transduc- cases no such effect was observed (19 –22). In the present tion cascade initiated by peptide mitogens. For both investigation we have studied the collaboration between mito- epidermal growth factor and platelet-derived growth gens and anchorage in NIH 3T3 cells, a cell type that has been factor, activation of the consensus mitogen-activated widely used in signal transduction studies. We have examined protein kinase cascade is impaired when cells are held several steps in the signal transduction pathway leading from in suspension as compared with cells anchored to a fi- bronectin substratum. Upstream events in the signaling receptor tyrosine kinases to Ras and then to the downstream cascade, including tyrosine phosphorylation of the mi- kinases. We find that peptide mitogen activation of receptor togen receptor and GTP loading of Ras, are similar in tyrosine kinases and subsequent activation of Ras are inde- anchored and suspended cells. However, propagation of pendent of anchorage. However, signal transduction between the signal to Raf and subsequently to the downstream Ras and Raf is markedly attenuated in nonadherent cells, kinases MEK and mitogen-activated protein kinase is leading to reduced activation of Raf, MEK, and MAP kinase. markedly attenuated in suspended cells. Thus, there EXPERIMENTAL PROCEDURES seems to be a distinct anchorage-dependent step be- NIH 3T3 cells were maintained in Dulbecco’s minimal essential tween Ras and Raf in the signaling cascade initiated by medium containing 10% bovine calf serum and antibiotics. Confluent peptide mitogens. These observations may have impor- cells were serum-starved for 16 h before detachment by 0.05% trypsin tant implications for understanding the anchorage de- and 0.33 mM EDTA; trypsin activity was neutralized by 1 mg/ml soy- pendence of cell growth. bean trypsin inhibitor. Cells were suspended in Dulbecco’s minimal essential medium with 2% bovine serum albumin and incubated in suspension at 37 °C for 45 min in a rotator to allow kinases become quiescent. Cells were then either maintained in suspension or plated Cell anchorage to the proteins of the extracellular matrix is onto dishes coated with fibronectin (20 mg/ml) or with poly-L-lysine (20 known to have profound effects on cell differentiation (1, 2), cell mg/ml) and incubated at 37 °C for the indicated times. In some cases the growth (3), and apoptosis (4). A particularly important example suspended or adherent cells were stimulated with either EGF or PDGF of this concerns the recently described effects of anchorage on (Upstate Biotechnologies Inc.). Cell lysates were prepared and tested the expression and activity of components of the cell cycle for the activity of Raf, MEK, and MAP kinase using specific in vitro machinery, including cyclin D1-CDK4,6 complexes and cyclin kinase assays as described previously (17). The phosphorylation status of the EGF receptor and PDGF receptor were evaluated by immuno- E-CDK2 complexes (5, 6). These observations are clearly rele- precipitation of the receptor using antibodies obtained from H. S. Earp vant to the question of why both soluble mitogens and cell (EGF-R) or from Santa Cruz Biotechnology (PDGF-R) followed by West- anchorage are required for the growth of normal cells, whereas ern blotting with an anti-phosphotyrosine antibody and detection by the anchorage requirement is abrogated in transformed cells enhanced chemiluminescence (17). For studies of GTP loading of the (7). Many aspects of cell to extracellular matrix interactions Ras protein, cells were radiolabeled with [ P]orthophosphate, and the involve the integrin family of cell surface heterodimeric adhe- [ P]GTP and GDP bound to immunoprecipitated Ras were quantitated by thin layer chromatography and PhosphorImager analysis as de- sion proteins (8). Recently, it has become clear that integrins scribed (17). are signal transducing receptors (9, 10) capable of influencing a number of intracellular biochemical activities including protein RESULTS AND DISCUSSION tyrosine kinases (11), serine/threonine kinases (12), and ionic Because elements of the MAP kinase cascade are directly but transients (13). In particular, integrin-mediated cell adhesion transiently activated by integrin-mediated cell adhesion (17), we initially examined the kinetics of this process to find a time * This work was supported by National Institutes of Health Grants point when we could examine anchorage effects on mitogen- GM26165 and HL45100 (to R. L. J.). The costs of publication of this driven activation of MAP kinase without a direct contribution article were defrayed in part by the payment of page charges. This from integrin-mediated MAP kinase activation. As seen in Fig. article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1(A and B), when NIH 3T3 cells were held in suspension, EGF ‡ Present address: Pharmacopeia, 101 College Rd. East, Princeton, caused a robust tyrosine phosphorylation of EGF-R but had NJ 08540. § Present address: Hoffman LaRoche, 340 Kingsland St., Nutley, NJ 07110. The abbreviations used are: MAP, mitogen-activated protein; EGF, To whom correspondence should be addressed. Tel.: 919-966-4383; epidermal growth factor; PDGF, platelet-derived growth factor; EGF-R, Fax: 919-966-5640; E-mail: [email protected]. EGF receptor; PDGF-R, PDGF receptor. This paper is available on line at http://www-jbc.stanford.edu/jbc/ 8849 This is an Open Access article under the CC BY license. 8850 Integrin-Mitogen Collaboration in Signal Transduction FIG.1. Cell anchorage to fibronectin modulates MAP kinase activation but not EGF receptor activation. Serum-starved NIH 3T3 cells were harvested and then either maintained in suspension or allowed to adhere to substrata coated with fibronectin; in some cases (1) the cells were treated with EGF for 5 min. Tyrosine phosphorylation of the EGF receptor was evaluated by immunoprecipitation (IP) with anti-EGF-R antibody followed by Western immunoblotting (IB) with an anti-phosphotyrosine antibody. MAP kinase activity was evaluated by a band shift assay and by immunoprecipitation of the enzyme followed by an in vitro kinase assay using myelin basic protein (MBP) as a substrate as described previously (17). A, EGF-R tyrosine phosphorylation as a function of time after plating cells on fibronectin. B, MAP kinase activity as a function of time after plating cells on fibronectin. (For A and B 20 ng/ml of EGF was used.) C, EGF-R tyrosine phosphorylation as a function of the dose of EGF. D, MAP kinase activation as a function of the dose of EGF. (For C and D the EGF dose is given in ng/ml.) NAD, nonadherent (suspended) cells; Fn, cells adherent to fibronectin-coated substrata. only a very modest effect on MAP kinase (Fig. 1B, lanes 1 and 2). After 10 min of cell adhesion to fibronectin-coated substrata, when the cells were fully attached but not spread, there was a strong adhesion-mediated activation of MAP kinase; EGF stim- ulation caused tyrosine phosphorylation of EGF-R and further stimulated MAP kinase (Fig. 1B, lanes 3 and 4). A qualitatively similar situation also prevailed after 30 min of cell adhesion when the cells were partially spread (Fig. 1B, lanes 5 and 6). By 180 min, when the cells were well spread, in the absence of EGF there was only a basal level of MAP kinase activity, whereas treatment with EGF caused tyrosine phosphorylation of EGF-R and resulted in a strong stimulation of MAP kinase (Fig. 1B, lanes 7 and 8). Thus, in serum-starved 3T3 cells, EGF activation of its receptor seems to be independent of cell an- chorage; however, the MAP kinase response is strongly influ- enced by anchorage. In nonadherent cells, EGF produces only a weak activation of MAP kinase. Shortly after the cells adhere to the fibronectin substratum, EGF and anchorage have ap- proximately additive effects on MAP kinase activity. At longer times, EGF strongly activates MAP kinase in anchored cells, FIG.2. Mitogen activation of the MAP kinase cascade in an- chored or suspended cells. Serum-starved NIH 3T3 cells were har- whereas the direct activation by cell adhesion has returned to vested and then either maintained in suspension or allowed to adhere basal levels. In Fig. 1 (C and D) we examined EGF concentra- to substrata coated with fibronectin for 180 min; in some cases (1) the tion-response relationships for EGF-R and MAP kinase in cells cells were treated with either EGF (20 ng/ml) or PDGF (20 ng/ml) for 5 that have either been maintained in suspension or anchored to min. The components of the signaling cascade were immunoprecipi- fibronectin for 180 min. As shown, the concentration-response tated (IP) using specific antibodies, and their activities were assayed as described previously (17). A, EGF responses. Top panel, EGF-R, West- profile for EGF-R tyrosine phosphorylation was essentially ern blot (IB) with anti-phosphotyrosine antibody; second panel, EGF-R identical in suspended cells and cells anchored to fibronectin expression, Western blot with anti-EGF-R; third panel, Raf-B activa- substrata. However, at all EGF concentrations tested, an- tion, immunoprecipitation with anti-Raf-B followed by an in vitro chored cells displayed a 3– 4-fold greater activation of MAP linked kinase assay (17) with kinase-dead MAP kinase (K MAPK)asa substrate; fourth panel, MEK activation, immunoprecipitation with kinase than did suspended cells (for example, compare lanes 4 anti-MEK followed by an in vitro kinase using kinase-dead MAP kinase and 10 in Fig. 1D, both at 5 ng/ml EGF). as a substrate (17); fifth panel, MAP kinase activation, immunoprecipi- We have also investigated how anchorage modulates mito- tation with anti-MAP kinase followed by an in vitro kinase assay using gen actions on other components of the MAP kinase cascade. As MBP as a substrate (17). B, PDGF responses. The same assays were used as in A, except that the top two panels employed specific antibodies shown in Fig. 2A, EGF stimulated similar levels of tyrosine to PDGF-R (Santa Cruz), and the third panel employed an antibody to phosphorylation of EGF-R in suspended or anchored cells. Raf-1. NAD, nonadherent cells; Fn, cells adherent to fibronectin-coated However, EGF produced substantially stronger activations of substrata; MBP, myelin basic protein. Raf-B, MEK, and MAP kinase in cells anchored to fibronectin substrata as compared with nonanchored cells. Raf-1 was also activated (weakly) by EGF in anchored cells but not in sus- suspended cells. However, PDGF treatment resulted in mark- pended cells (not shown). We decided to also examine anchor- edly stronger activation of Raf-1 and MEK in anchored cells as age dependence of signaling events mediated by PDGF, an- compared with suspended cells, as well as a more modest but other peptide mitogen. Exposure of 3T3 cells to PDGF caused a significant difference in MAP kinase activation. Thus, for substantially greater increase in overall cellular protein tyro- PDGF, as for EGF, cell anchorage seems to control the effi- sine phosphorylation than was observed with EGF (not shown). ciency of signal transduction between initial activation of the As seen in Fig. 2B, PDGF caused equivalent robust tyrosine receptor tyrosine kinase and subsequent activation of down- phosphorylation of its cognate receptor in both anchored and stream kinases. Integrin-Mitogen Collaboration in Signal Transduction 8851 FIG.4. Attenuation of the mitogen signaling cascade between Ras and Raf in nonanchored cells. This figure summarizes several independent experiments for EGF (A) and PDGF (B) stimulation. The tyrosine phosphorylation of immunoprecipitated EGF-R and PDGF-R was quantitated by laser densitometry of enhanced chemiluminescence FIG.3. Mitogen activation of Ras GTP loading in anchored or Western blots. The activation of Raf, MEK, and MAP kinases, as well as suspended cells. Serum-starved NIH 3T3 cells were harvested and GTP loading of Ras, were quantitated using a PhosphorImager. The then either maintained in suspension or allowed to adhere to substrata parameter shown on the ordinate (Mitogen Activation NAD/Fn) repre- coated with fibronectin for 180 min; in some cases (1) the cells were sents the ratio of mitogen activation in nonadherent cells (NAD) to that treated with EGF (20 ng/ml) or PDGF (20 ng/ml) for 5 min. Ras GTP observed in cells anchored on fibronectin substrata (Fn). In each case it loading was measured by thin layer chromatography as described (17). was calculated by subtracting the basal value from the mitogen-stim- The data are presented as the percentage of bound GTP ((GTP/(1.5 ulated value; the differences (termed dNAD or dFn) were divided to get GDP1GTP)) 3 100). The results for PDGF represent the means and the ratio given on the ordinate (dNAD/dFn). Thus, if cell anchorage had standard errors for three independent experiments, whereas for EGF no effect on the mitogen activation of a certain component in the the means of two independent experiments are shown. pathway, the ratio on the ordinate for that component would be 1; numbers less than 1 indicate that the mitogen-mediated activation is attenuated in nonadherent cells. The results represent the means and Because the GTP-bound form of Ras is a key transducer in standard errors for three independent experiments (two for EGF effects the mitogen signaling pathway (18), we decided to examine Ras on Ras). GTP loading in anchored or suspended 3T3 cells treated with either PDGF or EGF. As seen in Fig. 3, the basal GTP/GDP ratio was somewhat higher in suspension cells than in adher- activation of MAP kinase is 2–3-fold greater in cells plated on ent cells. Treatment with peptide mitogen resulted in a strong a fibronectin substratum, as opposed to a poly-lysine substra- increase in Ras GTP loading in both suspended cells and an- tum (data not shown). This suggests that the anchorage effects chored cells, with PDGF producing a somewhat greater effect on signaling that we have observed may be mediated by inte- than EGF. Thus, Ras GTP loading in response to mitogens grins; however, several additional lines of investigation will be occurred in suspended cells at least as well as in anchored cells; needed to fully confirm this possibility. in fact, suspended cells usually showed higher levels of GTP Anchorage dependence of cell growth is one of the most loading than anchored cells. This observation, along with those fundamental differences between normal and transformed cells of Fig. 2, suggests that peptide mitogen signaling pathways are (24). Our observations indicate that cell anchorage can influ- intact and operate efficiently in both suspended and anchored ence the efficiency of signal tranduction in mitogenic pathways. cells up to the level of Ras but are attenuated between Ras and This suggests the possibility that adhesion effects on early the Raf kinases in the nonanchored cells. signaling events may play an important role in anchorage Quantitation of anchorage effects on activation of several dependence of cell growth, although other factors may also be components of the EGF- and PDGF-triggered pathways is involved. For both EGF and PDGF, the upstream events of the shown in Fig. 4. The data are expressed as the ratio of the mitogen signaling pathway were independent of anchorage. mitogen activation in suspended cells versus anchored cells. Thus, receptor tyrosine kinase activation and GTP loading of The downstream kinases (Rafs, MEKs, and MAP kinases) dis- Ras were robust in both anchored and suspended cells. For both play substantial reductions in activity in nonanchored cells, mitogens, however, cell adhesion had a profound effect on the but Ras and the receptor tyrosine kinases do not. Although the activation of the MEK-kinases Raf-B and Raf-1 and clear-cut exact magnitudes of the anchorage effects on activation of the effects of lesser magnitude on MEK and MAP kinase. The individual components of the pathway differ somewhat be- nonlinearity of the effects we have observed on anchorage tween EGF and PDGF, the trend is similar. The observation regulation of signal transduction may be due to the extensive that EGF and PDGF produce qualitatively similar but quanti- branching and cross-talk that is known to occur in the MAP tatively distinct effects on the consensus MAP kinase cascade is kinase cascade (25). Our observations suggest that in sus- not surprising, because individual receptor tyrosine kinases pended cells, there is a rather sharp break in the signaling are known to have distinct effects on cell growth and differen- cascade between Ras and the Raf kinases. Because a major role tiation (23). of Ras in signal transduction is to recruit Raf to the plasma Although cell anchorage to a fibronectin-coated substratum membrane (26), our findings suggest that cell anchorage con- clearly has a substantial impact on the MAP kinase cascade, it tributes to this process. One plausible model is that integrin- is not yet certain that this is purely an integrin-mediated dependent focal contacts formed during cell adhesion partici- phenomenon. Preliminary experiments have shown that EGF pate in the recruitment and subsequent activation of Raf. 8852 Integrin-Mitogen Collaboration in Signal Transduction 7. Clarke, G. D., Stoker, M. G., Ludlow, A. & Thornton, M. 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Raf family kinases are 14. Chen, Q., Kinch, M. S., Lin, T. H., Burridge, K. & Juliano, R. L. (1994) J. Biol. thought to have downstream targets other than the MAP ki- Chem. 269, 26602 -26605 15. Zhu, X. & Assoian, R. K. (1995) Mol. Biol. Cell 6, 273–282 nase pathway (27, 28). Thus, it seems possible that the anchor- 16. Schlaepfer, D. D., Hanks, S., Hunter, T. & van der Geer, P. (1994) Nature 372, age modulation of mitogen signaling reported here, particu- 786 –791 larly the dramatic effect on Raf family kinases, may be 17. Chen, Q., Lin, T. H., Der, C. J. & Juliano, R. L. (1996) J. Biol Chem. 271, 18122–18127 important aspects of cell growth control. 18. Egan, S. E. & Weinberg, R. A. (1993) Science 365, 781–783 19. McNamee, H. P., Ingber, D. E. & Schwartz, M. A. (1993) J. Cell. Biol. 121, Acknowledgments—We thank Andrew Aplin and Channing Der for 673– 678 valuable comments on the manuscript. 20. Hotchin, N. A. & Hall, A. (1995) J. Cell. Biol. 131, 1857–1865 21. Bohmer, R. 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Published: Apr 1, 1997

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