Protein Phosphorylation in Endothelial Cell Membranes: Relationship to Growth Control
The activation of various kinase systems during the proliferative response of fibroblasts and certain tumor cell lines is well documented. The intracellular signalling pathway involved in the triggering of endothelial cell (EC) mitogenesis, however, has received little attention. We have employed cultured porcine aortic EC to examine the possible role of protein phosphorylation events in EC growth control. Triton-solubilized membranes from proliferating (sparse) and quiescent (confluent) EC were incubated under standard kinase assay conditions and radiolabeled membrane phosphoproteins were separated by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. The extent of phosphorylation was dramatically elevated in sparse over confluent samples for four phosphoproteins including two serine phosphoproteins (180 kDa and 100 kDa), a 97 kDa protein phosphorylated on both serine and threonine and a 55 kDa protein which exhibited a specific and dramatic increase in tyrosine phosphorylation. Cofactor studies revealed that Mn2+ is the optimum cofactor for the EC membrane kinase(s) and that Mg2+ can replace Mn2+ only for the kinase acting on the 100 kDa protein. The kinases, acting on all four phosphoproteins, were not dependent on Ca2+, cAMP, or cGMP. The observed membrane phosphorylation patterns were similar in porcine and bovine aortic EC but were different from human fibroblasts or 3T3 cells. Sparse, quiescent EC exhibited a phosphoprotein pattern similar to that of confluent cultures, suggesting a growth dependence for the enhanced kinase activity that was observed in membranes from proliferating cells.
KeywordsPhosphorylation Event Mitogenic Response Kinase System Multiple Intracellular Signalling Pathway Bovine Capillary Endothelial Cell
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Bormann, B-J, Huang, C-K, Lam, G.F. and Jaffe, E.A. Thrombin-induced vimentin phosphorylation in cultured human umbilical vein endothelial cells. J. Biol. Chem.
261:10471–10474, 1986.PubMedGoogle Scholar
Cohen, P. The role of protein phosphorylation in neural and hormonal control of cellular activity. Nature.
296:613–620, 1982.PubMedCrossRefGoogle Scholar
Cohen, S., Carpenter, G. and King, L., Jr. Epidermal growth factor-receptor-protein kinase interactions. Co-purification of receptor and epidermal growth-factor-enhanced phosphorylation activity. J. Biol. Chem.
255:4834–4842, 1980.PubMedGoogle Scholar
Cooper, J.A. and Hunter, T. Changes in protein phosphorylation in Rous sarcoma virus-transformed chick embryo cells. Mol. Cell Biol.
1:165–178, 1981.PubMedGoogle Scholar
Cooper, J.A., Bowen-Pope, D.F., Raines, E., Ross, R. and Hunter, T. Similar effects of platelet-derived growth factor and epidermal growth factor on the phosphorylation of tyrosine in cellular proteins. Cell.
31:263–273, 1982.PubMedCrossRefGoogle Scholar
Daniel, T.O., Gibbs, V.C., Milfay, D.F. and Williams, L.T. Agents that increase cAMP accumulation block endothelial c-sis induction by thrombin and transforming growth factor-ß. J. Biol. Chem.
262:11893–11896, 1987.PubMedGoogle Scholar
Davies, P.F. and Ross, R. Mediation of pinocytosis in cultured arterial smooth muscle cells and endothelial cells by platelet-derived growth factor. J. Cell. Biol.
79:663–671, 1978.PubMedCrossRefGoogle Scholar
Doctrow, S.R. and Folkman, J. Protein kinase C activators suppress stimulation of capillary endothelial cell growth by angiogenic endothelial mitogens. J. Cell. Biol.
104:679–687, 1987.PubMedCrossRefGoogle Scholar
Ek, B., Westermark, B., Wasteson, A. and Heldin, C.H. Stimulation of tyrosine-specific phosphorylation by platelet-derived growth factor. Nature.
295:419–420, 1982.PubMedCrossRefGoogle Scholar
Huang, S.S., and Huang, J.S. Association of bovine brain-derived growth factor receptor with protein tyrosine kinase activity. J. Biol. Chem.
261:9568–9571, 1986.PubMedGoogle Scholar
Kazlauskas, A. and DiCorleto, P.E. Comparison of the phosphorylation events in membranes from proliferating vs. quiescent endothelial cells. J. Cell. Physiol.
130:228–244, 1987.PubMedCrossRefGoogle Scholar
Mackie, K., Lai, Y., Nairn, A.C., Greengard, P., Pitt, B.R. and Laze, J.S. Protein phosphorylation in cultured endothelial cells. J. Cell. Physiol.
128:367–374, 1986.PubMedCrossRefGoogle Scholar
Nishizuka, Y. The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature.
308:693–698, 1984.PubMedCrossRefGoogle Scholar
Nishizuka, Y. Studies and prospectives of protein kinase C. Science.
Schwartz, S.M. Selection and characterization of bovine aortic endothelial cells. In Vitro
. 14:966–980, 1978.PubMedCrossRefGoogle Scholar
Thomas, G., Martin-Perez, J., Siegman, M. and Otto, A.M. The effect of serum, EGF, PGF and insulin of S6 phosphorylation and the initiation of protein and DNA synthesis. Cell.
30:235–242, 1982.PubMedCrossRefGoogle Scholar
Uratsuji, Y. and DiCorleto, P.E. Growth-dependent subcellular redistribution of protein kinase C in cultured porcine aortic endothelial cells. J. Cell. Physiol.
136:431–438, 1988.PubMedCrossRefGoogle Scholar
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