- Research Article
Calreticulin affects cell adhesiveness through differential phosphorylation of insulin receptor substrate-1
Cellular & Molecular Biology Letters volume 19, pages 77–97 (2014)
Cellular adhesion to the underlying substratum is regulated through numerous signaling pathways. It has been suggested that insulin receptor substrate 1 (IRS-1) is involved in some of these pathways, via association with and activation of transmembrane integrins. Calreticulin, as an important endoplasmic reticulum-resident, calcium-binding protein with a chaperone function, plays an obvious role in proteomic expression. Our previous work showed that calreticulin mediates cell adhesion not only by affecting protein expression but also by affecting the state of regulatory protein phosphorylation, such as that of c-src. Here, we demonstrate that calreticulin affects the abundance of IRS-1 such that the absence of calreticulin is paralleled by a decrease in IRS-1 levels and the unregulated overexpression of calreticulin is accompanied by an increase in IRS-1 levels. These changes in the abundance of calreticulin and IRS-1 are accompanied by changes in cell-substratum adhesiveness and phosphorylation, such that increases in the expression of calreticulin and IRS-1 are paralleled by an increase in focal contact-based cellsubstratum adhesiveness, and a decrease in the expression of these proteins brings about a decrease in cell-substratum adhesiveness. Wild type and calreticulin-null mouse embryonic fibroblasts (MEFs) were cultured and the IRS-1 isoform profile was assessed. Differences in morphology and motility were also quantified. While no substantial differences in the speed of locomotion were found, the directionality of cell movement was greatly promoted by the presence of calreticulin. Calreticulin expression was also found to have a dramatic effect on the phosphorylation state of serine 636 of IRS-1, such that phosphorylation of IRS-1 on serine 636 increased radically in the absence of calreticulin. Most importantly, treatment of cells with the RhoA/ROCK inhibitor, Y-27632, which among its many effects also inhibited serine 636 phosphorylation of IRS-1, had profound effects on cell-substratum adhesion, in that it suppressed focal contacts, induced extensive close contacts, and increased the strength of adhesion. The latter effect, while counterintuitive, can be explained by the close contacts comprising labile bonds but in large numbers. In addition, the lability of bonds in close contacts would permit fast locomotion. An interesting and novel finding is that Y-27632 treatment of MEFs releases them from contact inhibition of locomotion, as evidenced by the invasion of a cell’s underside by the thin lamellae and filopodia of a cell in close apposition.
focal adhesion kinase
insulin receptor substrate 1
mouse embryonic fibroblast
Burridge, K., Fath, K., Kelly, T., Nuckolls, G. and Turner, C. Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu. Rev. Cell Biol. 4 (1988) 487–525.
McClay, D.R. and Ettensohn, C.A. Cell adhesion in morphogernesis. Annu. Rev. Cell Biol. 3 (1987) 319–345.
Strohmeier, R. and Bereiter-Hahn, J. Control of cell shape and locomotion by external calcium. Exp. Cell Res. 154 (1984) 412–420.
Hinrichsen, R.D. Calcium and calmodulin in the control of cellular behavior and motility. Biochim. Biophys. Acta 1155 (1993) 277–293.
Huttenlocher, A., Palecek, S.P., Lu, Q., Zhang, W.L., Mellgren, R.L., Lauffenburger, D.A., Ginsberg, M.H. and Horwitz, A.F. Regulation of cell migration by the calcium-dependent protease calpain. J. Biol. Chem. 272 (1997) 32719–32722.
Bolsover, S.R. Calcium signaling in growth cone migration. Cell Calcium 37 (2005) 395–402.
Bedard, K., Szabo, E., Michalak, M. and Opas, M. Cellular functions of endoplasmic reticulum chaperones calreticulin, calnexin, and ERp57. Int. Rev. Cytol. 245 (2005) 91–121.
Villagomez, M., Szabo, E., Podchenko, A., Feng, T., Papp, S. and Opas, M. Calreticulin and focal contact-dependent adhesion. Biochem. Cell Biol. 87 (2009) 545–556.
Geiger, B., Volk, T. and Volberg, T. Molecular heterogeneity of adherens junctions. J. Cell Biol. 101 (1985) 1523–1531.
Opas, M., Szewczenko-Pawlikowski, M., Jass, G.K., Mesaeli, N. and Michalak, M. Calreticulin modulates cell adhesiveness via regulation of vinculin expression. J. Cell Biol. 135 (1996) 1913–1923.
Fadel, M.P., Dziak, E., Lo, C.M., Ferrier, J., Mesaeli, N., Michalak, M. and Opas, M. Calreticulin affects focal contact-dependent but not close contactdependent cell-substratum adhesion. J. Biol. Chem. 274 (1999) 15085–15094.
Opas, M. and Fadel, M.P. Partial reversal of transformed fusiform phenotype by overexpression of calreticulin. Cell. Mol. Biol. Lett. 12 (2007) 294–307.
Papp, S., Fadel, M.P. and Opas, M. Dissecting focal adhesions in cells differentially expressing calreticulin — a microscopical study. Biol. Cell 99 (2007) 389–402.
Papp, S., Fadel, M.P., Kim, H., McCulloch, C.A. and Opas, M. Calreticulin affects fibronectin-based cell-substratum adhesion via the regulation of c-src activity. J. Biol. Chem. 282 (2007) 16585–16598.
Szabo, E., Papp, S. and Opas, M. Differential calreticulin expression affects focal contacts via the calmodulin/Camk II pathway. J. Cell. Physiol. 213 (2007) 269–277.
Burridge, K. and Chrzanowska-Wodnicka, M. Focal adhesions, contractility, and signaling. Annu. Rev. Cell Dev. Biol. 12 (1996) 463–518.
Burridge, K., Chrzanowska-Wodnicka, M. and Zhong, C.L. Focal adhesion assembly. Trends Cell Biol. 7 (1997) 342–347.
Jockusch, B.M., Bubeck, P., Giehl, K., Kroemker, M., Moscher, J., Rothkegel, M., Rüdiger, M., Schlüter, K., Stanke, G. and Winkler, J. The molecular architecture of focal adhesions. Annu. Rev. Cell Dev. Biol. 11 (1995) 379–416.
Vuori, K. and Ruoslahti, E. Association of insulin receptor substrate-1 with integrins. Science 266 (1994) 1576–1578.
Goel, H.L., Fornaro, M., Moro, L., Teider, N., Rhim, J.S., King, M. and Languino, L.R. Selective modulation of type 1 insulin-like growth factor receptor signaling and functions by beta1 integrins. J. Cell Biol. 166 (2004) 407–418.
Lebrun, P., Mothe-Satney, I., Delahaye, L., Van Obberghen, E. and Baron, V. Insulin receptor substrate-1 as a signaling molecule for focal adhesion kinase pp125(FAK) and pp60(src). J. Biol. Chem. 273 (1998) 32244–32253.
Lebrun, P., Baron, V., Hauck, C.R., Schlaepfer, D.D. and Van Obberghen, E. Cell adhesion and focal adhesion kinase regulate insulin receptor substrate-1 expression. J. Biol. Chem. 275 (2000) 38371–38377.
El Annabi, S., Gautier, N. and Baron, V. Focal adhesion kinase and Src mediate integrin regulation of insulin receptor phosphorylation. FEBS Lett. 507 (2001) 247–252.
Lee, Y.J., Hsu, T.C., Du, J.Y., Valentijn, A.J., Wu, T.Y., Cheng, C.F., Yang, Z. and Streuli, C.H. Extracellular matrix controls insulin signaling in mammary epithelial cells through the RhoA/Rok pathway. J. Cell. Physiol. 220 (2009) 476–484.
Nakamura, K., Bossy-Wetzel, E., Burns, K., Fadel, M.P., Lozyk, M., Goping, I.S., Opas, M., Bleackley, R.C., Green, D.R. and Michalak, M. Changes in endoplasmic reticulum luminal environment affect cell sensitivity to apoptosis. J. Cell Biol. 150 (2000) 731–740.
Nakamura, K., Zuppini, A., Arnaudeau, S., Lynch, J., Ahsan, I., Krause, R., Papp, S., De Smedt, H., Parys, J.B., Muller-Esterl, W., Lew, D.P., Krause, K.H., Demaurex, N., Opas, M. and Michalak, M. Functional specialization of calreticulin domains. J. Cell Biol. 154 (2001) 961–972.
Zigler, J.S., Jr., Lepe-Zuniga, J.L., Vistica, B. and Gery, I. Analysis of the cytotoxic effects of light-exposed HEPES-containing culture medium. In Vitro Cell Dev. Biol. 21 (1985) 282–287.
Menssen, H.D., Herlyn, M., Rodeck, U. and Koprowski, H. Rapid dissociation of adherent human tumor cells by ultrasound. J. Immunol. Methods 104 (1987) 1–6.
Szabo, E., Papp, S. and Opas, M. Calreticulin and cellular adhesion/migration-specific signaling pathways. J. Appl. Biomed. 4 (2006) 45–52.
Zamir, E. and Geiger, B. Molecular complexity and dynamics of cell-matrix adhesions. J. Cell Sci. 114 (2001) 3583–3590.
Critchley, D.R. and Gingras, A.R. Talin at glance. J. Cell Sci. 121 (2008) 1345–1347.
Geiger, B. and Yamada, K.M. Molecular architecture and function of matrix adhesions. Cold Spring Harb. Perspect. Biol. 3 (2011) a005033.
Reiss, K., Wang, J.Y., Romano, G., Tu, X., Peruzzi, F. and Baserga, R. Mechanisms of regulation of cell adhesion and motility by insulin receptor substrate-1 in prostate cancer cells. Oncogene 20 (2001) 490–500.
Sun, X.J. and Liu, F. Phosphorylation of IRS proteins Yin-Yang regulation of insulin signaling. Vitam. Horm. 80 (2009) 351–387.
Furukawa, N., Ongusaha, P., Jahng, W.J., Araki, K., Choi, C.S., Kim, H.J., Lee, Y.H., Kaibuchi, K., Kahn, B.B., Masuzaki, H., Kim, J.K., Lee, S.W. and Kim, Y.B. Role of Rho-kinase in regulation of insulin action and glucose homeostasis. Cell Metab. 2 (1997) 119–120.
Begum, N., Sandu, O.A., Ito, M., Lohmann, S.M. and Smolenski, A. Active Rho kinase (ROK-alpha ) associates with insulin receptor substrate-1 and inhibits insulin signaling in vascular smooth muscle cells. J. Biol. Chem. 277 (2002) 6214–6222.
Farah, S., Agazie, Y., Ohan, N., Ngsee, J.K. and Liu, X.J. A rho-associated protein kinase, ROKalpha, binds insulin receptor substrate-1 and modulates insulin signaling. J. Biol. Chem. 273 (1998) 4740–4746.
Chrzanowska-Wodnicka, M. and Burridge, K. Rho-stimulated contractility drives the formation of stress fibers and focal adhesions. J. Cell Biol. 133 (1996) 1403–1415.
Rottner, K., Hall, A. and Small, J.V. Interplay between Rac and Rho in the control of substrate contact dynamics. Curr. Biol. 9 (1999) 640–648.
Narumiya, S., Ishizaki, T. and Uehata, M. Use and properties of ROCKspecific inhibitor Y-27632. Methods Enzymol. 325 (2000) 273–284.
Guilherme, A., Torres, K. and Czech, M.P. Cross-talk between insulin receptor and integrin à5á1 signaling pathways. J. Biol. Chem. 273 (1998) 22899–22903.
Papp, S., Szabo, E., Kim, H., McCulloch, C.A. and Opas, M. Kinasedependent adhesion to fibronectin: regulation by calreticulin. Exp. Cell Res. 314 (2008) 1313–1326.
Curtis, A.S.G. The mechanism of adhesion of cells to glass. A study by interference reflection microscopy. J. Cell Biol. 20 (1964) 199–215.
Izzard, C.S. and Lochner, L.R. Cell-to-substrate contacts in living fibroblasts: an interference reflexion study with an evaluation of the technique. J. Cell Sci. 21 (1976) 129–159.
Bereiter-Hahn, J., Fox, C.H. and Thorell, B. Quantitative reflection contrast microscopy of living cells. J. Cell Biol. 82 (1979) 767–779.
Gingell, D. and Todd, I. Interference reflection microscopy. A quantitative theory for image interpretation and its application to cell-substratum separation measurement. Biophys. J. 26 (1979) 507–526.
Omelchenko, T., Vasiliev, J.M., Gelfand, I.M., Feder, H.H. and Bonder, E.M. Mechanisms of polarization of the shape of fibroblasts and epitheliocytes: Separation of the roles of microtubules and Rho-dependent actin-myosin contractility. Proc. Natl. Acad. Sci. USA 99 (2002) 10452–10457.
Worthylake, R.A. and Burridge, K. RhoA and ROCK promote migration by limiting membrane protrusions. J. Biol. Chem. 278 (2003) 13578–13584.
Vega, F.M., Fruhwirth, G., Ng, T. and Ridley, A.J. RhoA and RhoC have distinct roles in migration and invasion by acting through different targets. J. Cell Biol. 193 (2011) 655–665.
Opas, M. Adhesion of cells to protein carpets: do cells’ feet have to be black? Cell Motil. Cytoskeleton 11 (1988) 178–181.
Opas, M. and Kalnins, V.I. Microfilament distribution and adhesion patterns in cultured cells after glutaraldehyde-formaldehyde fixation. Eur. J. Cell Biol. 33 (1984) 60–65.
Weber, I. Reflection interference contrast microscopy. Methods Enzymol. 361 (2003) 34–47.
Wang, J.Y., Gualco, E., Peruzzi, F., Sawaya, B.E., Passiatore, G., Marcinkiewicz, C., Staniszewska, I., Ferrante, P., Amini, S., Khalili, K. and Reiss, K. Interaction between serine phosphorylated IRS-1 and beta1-integrin affects the stability of neuronal processes. J. Neurosci. Res. 85 (2007) 2360–2373.
Wang, Q., Bilan, P.J. and Klip, A. Opposite effects of insulin on focal adhesion proteins in 3T3-L1 adipocytes and in cells overexpressing the insulin receptor. Mol. Biol. Cell 9 (1998) 3057–3069.
Gail, M.H. and Boone, C.W. The locomotion of mouse fibroblasts in tissue culture. Biophys. J. 10 (1970) 980–993.
Couchman, J.R. and Rees, D.A. Actomyosin organization for adhesion, spreading, growth and movement in chick fibroblasts. Cell Biol. Int. Rep. 3 (1979) 431–439.
Couchman, J.R. and Rees, D.A. The behaviour of fibroblasts migrating from chick heart explants: Changes in adhesion, locomotion and growth, and in the distribution of actomyosin and fibronectin. J. Cell Sci. 39 (1979) 149–165.
Kolega, J., Shure, M.S., Chen, W.T. and Young, N.D. Rapid cellular translocation is related to close contacts formed between various cultured cells and their substrata. J. Cell Sci. 54 (1982) 23–34.
Pouyssegur, J. and Pastan, I. The directionality of locomotion of mouse fibroblasts. Role of cell adhesiveness. Exp. Cell Res. 121 (1979) 373–382.
Rid, R., Schiefermeier, N., Grigoriev, I., Small, J.V. and Kaverina, I. The last but not the least: the origin and significance of trailing adhesions in fibroblastic cells. Cell Motil. Cytoskeleton 61 (2005) 161–171.
Abercrombie, M. and Dunn, G.A. Adhesions of fibroblasts to substratum during contact inhibition observed by interference reflection microscopy. Exp. Cell Res. 92 (1975) 57–62.
Heath, J.P. and Dunn, G.A. Cell to substratum contacts of chick fibroblasts and their relation to the microfilament system. A correlated interference-reflexion and high-voltage electron-microscope study. J. Cell Sci. 29 (1978) 197–212.
Wehland, J., Osborn, M. and Weber, K. Cell-to-substratum contacts in living cells: a direct correlation between interference-reflexion and indirect-immunofluorescence microscopy using antibodies against actin and alphaactinin. J. Cell Sci. 37 (1979) 257–273.
Izzard, C.S. and Lochner, L.R. Formation of cell-to-substrate contacts during fibroblast motility: an interference-reflexion study. J. Cell Sci. 42 (1980) 81–116.
Yates, J.R. and Izzard, C.S. Cell-to-substrate contacts in an adhesiondefective mutant of Balb/c3T3 cells. J. Cell Sci. 52 (1981) 183–196.
Arthur, W.T. and Burridge, K. RhoA inactivation by p190RhoGAP regulates cell spreading and migration by promoting membrane protrusion and polarity. Mol. Biol. Cell 12 (2001) 2711–2720.
Patla, I., Volberg, T., Elad, N., Hirschfeld-Warneken, V., Grashoff, C., Fassler, R., Spatz, J.P., Geiger, B. and Medalia, O. Dissecting the molecular architecture of integrin adhesion sites by cryo-electron tomography. Nat. Cell Biol. 12 (2010) 909–915.
Sinnett-Smith, J., Lunn, J.A., Leopoldt, D. and Rozengurt, E. Y-27632, an inhibitor of Rho-associated kinases, prevents tyrosine phosphorylation of focal adhesion kinase and paxillin induced by bombesin: dissociation from tyrosine phosphorylation of p130(CAS). Exp. Cell Res. 266 (2001) 292–302.
Ward, M.D. and Hammer, D.A. A theoretical analysis for the effect of focal contact formation on cell-substrate attachment strength. Biophys. J. 64 (1993) 936–959.
Gallant, N.D., Michael, K.E. and Garcia, A.J. Cell adhesion strengthening: contributions of adhesive area, integrin binding, and focal adhesion assembly. Mol. Biol. Cell 16 (2005) 4329–4340.
Stricker, J., Aratyn-Schaus, Y., Oakes, P.W. and Gardel, M.L. Spatiotemporal constraints on the force-dependent growth of focal adhesions. Biophys. J. 100 (2011) 2883–2893.
Coyer, S.R., Singh, A., Dumbauld, D.W., Calderwood, D.A., Craig, S.W., Delamarche, E. and Garcia, A.J. Nanopatterning reveals an ECM area threshold for focal adhesion assembly and force transmission that is regulated by integrin activation and cytoskeleton tension. J. Cell Sci. 125 (2012) 5110–5123.
Reinhart-King, C.A., Dembo, M. and Hammer, D.A. The dynamics and mechanics of endothelial cell spreading. Biophys. J. 89 (2005) 676–689.
Schindl, M., Wallraff, E., Deubzer, B., Witke, W., Gerisch, G. and Sackmann, E. Cell-substrate interactions and locomotion of Dictyostelium wild-type and mutants defective in three cytoskeletal proteins: A study using quantitative reflection interference contrast microscopy. Biophys. J. 68 (1995) 1177–1190.
Loomis, W.F., Fuller, D., Gutierrez, E., Groisman, A. and Rappel, W.J. Innate non-specific cell substratum adhesion. PLoS ONE 7 (2012) e42033.
Bereiter-Hahn, J., Strohmeier, R., Kunzenbacher, I., Beck, K. and V”th, M. Locomotion of Xenopus epidermis cells in primary culture. J. Cell Sci. 52 (1981) 289–311.
Lee, J. and Jacobson, K. The composition and dynamics of cell-substratum adhesions in locomoting fish keratocytes. J. Cell Sci. 110 (1997) 2833–2844.
Renkawitz, J., Schumann, K., Weber, M., Lammermann, T., Pflicke, H., Piel, M., Polleux, J., Spatz, J.P. and Sixt, M. Adaptive force transmission in amoeboid cell migration. Nat. Cell Biol. 11 (2009) 1438–1443.
Weiss, L. and Harlos, J.P. Short-term interactions between cell surfaces. Progr. Surf. Sci. 1 (1972) 355–405.
Curtis, A.S.G. and Büültjens, T.E.J. Cell adhesion and locomotion. Ciba Found. Symp. 4 (1973) 172–186.
Leader, W.M., Stopak, D. and Harris, A.K. Increased contractile strength and tightened adhesions to the substratum result from reverse transformation of CHO cells by dibutyryl cyclic adenosine monophosphate. J. Cell Sci. 64 (1983) 1–11.
Ward, M.D. and Hammer, D.A. Morphology of cell-substratum adhesion. Influence of receptor heterogeneity and nonspecific forces. Cell Biophys. 20 (1992) 177–222.
Abercrombie, M., Heaysman, J.E.M. and Pegrum, S.M. The locomotion of fibroblasts in culture. IV. Electron microscopy of the leading lamella. Exp. Cell Res. 67 (1971) 359–367.
Pegrum, S.M. Contact relationships between chicke embryo cells growing in monolayer culture after infection with Rous sarcoma virus. Exp. Cell Res. 138 (1982) 147–157.
Selhuber-Unkel, C., Erdmann, T., Lopez-Garcia, M., Kessler, H., Schwarz, U.S. and Spatz, J.P. Cell adhesion strength is controlled by intermolecular spacing of adhesion receptors. Biophys. J. 98 (2010) 543–551.
Rees, D.A., Couchman, J.R., Smith, C.G., Woods, A. and Wilson, G. Cellsubstratum interactions in the adhesion and locomotion of fibroblasts. Philos. Trans. R. Soc. Lond. B Biol. Sci. 299 (1982) 169–176.
Woods, A., Smith, C.G., Rees, D.A. and Wilson, G. Stages in specialization of fibroblast adhesion and deposition of extracellular matrix. Eur. J. Cell Biol. 32 (1983) 108–116.
Duband, J.L., Nuckolls, G.H., Ishihara, A., Hasegawa, T., Yamada, K.M., Thiery, J.P. and Jacobson, K. Fibronectin receptor exhibits high lateral mobility in embryonic locomoting cells but is immobile in focal contacts and fibrillar streaks in stationary cells. J. Cell Biol. 107 (1988) 1385–1396.
Bell, G.I., Dembo, M. and Bongrand, P. Cell adhesion: Competition between nonspecific repulsion and specific bonding. Biophys. J. 45 (1984) 1051–1064.
Abercrombie, M. and Heaysman, J.E.M. Observations on the social behaviour of cells in tissue culture: I. Speed of movement of chick heart fibroblasts in relation to their mutual contacts. Exp. Cell Res. 5 (1953) 111–131.
Abercrombie, M. and Heaysman, J.E.M. Observations on the social behaviour of cells in tissue culture. II. “Monolayering” of fibroblasts. Exp. Cell Res. 6 (1954) 293–306.
Nelson, C.M., Pirone, D.M., Tan, J.L. and Chen, C.S. Vascular endothelialcadherin regulates cytoskeletal tension, cell spreading and focal adhesions by stimulating RhoA. Mol. Biol. Cell 15 (2004) 2943–2953.
Kadir, S., Astin, J.W., Tahtamouni, L., Martin, P. and Nobes, C.D. Microtubule remodelling is required for the front-rear polarity switch during contact inhibition of locomotion. J. Cell Sci. 124 (2011) 2642–2653.
Anear, E. and Parish, R.W. The effects of modifying RhoA and Rac1 activities on heterotypic contact inhibition of locomotion. FEBS Lett. 586 (2012) 1330–1335.
Mayor, R. and Carmona-Fontaine, C. Keeping in touch with contact inhibition of locomotion. Trends Cell Biol. 20 (2010) 319–328.
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Czarnowski, A., Papp, S., Szaraz, P. et al. Calreticulin affects cell adhesiveness through differential phosphorylation of insulin receptor substrate-1. Cell Mol Biol Lett 19, 77–97 (2014). https://doi.org/10.2478/s11658-014-0181-9
- Insulin receptor substrate-1
- Focal contacts
- Close contacts