Comparison of the phosphorylation events in membranes from proliferating vs. quiescent endothelial cells
Authors: Kazlauskas Andrius , Dicorleto Paul E. 
Published: February 2005

In an attempt to elucidate the intracellular events regulating the proliferation of endothelial cells (EC), we have compared the phosphorylation events in membranes prapared from proliferating (sparse) and quiescent (confluent) EC. Triton‐solubilized membranes from sparse and confluent EC were incubated at pH 6.5 in the presence of divalent cations and [32P]ATP. Membrane proteins were then separated by SDS‐PAGE and the radiolabeled phosphoproteins visulaized by autoradiography. The overall kinase activity per milligram protein was 1.7 ± 0.2‐fold greater in membranes prepared from proliferating than from quiescent cells. The extent of phosphorylation was dramatically elevated in sparse over confluent samples for four phosphoproteins having the following approximate molecular masses: 180, 100, 97, and 55 kDa. The 180 and 100 kDa phosphoproteins exhibited 3.6‐ and 7.4‐fold higher labeling, respectively, in sparse than in confluent membranes and both were phosphorylated on serine residues exclusively. The 97 kDa phosphoprotein was 11.6‐fold higher in sparse membranes and contained both phosphoserine (p‐ser) and phosphotheronine (p‐thr), the latter comprising 61% of the radioactivity. The 55 kDA phosphoprotein contained 62% p‐ser, 16% p‐thr, and 22% phosphotyrosine (p‐tyr) and was 2.3‐fold higher in sparse membranes. Of these four phosphoproteins, only the 55 kDa protein was phosphorylated in confluent samples to an appreciable degree. Whereas the p‐ser and p‐thr content of the 55 kDa band increased moderately in sparse vs. confluent sample (1.8‐fod increase), the tyrosine residues of this protein iin sparse membranes were radiolabeled to a much greater extent relative to confluent membranes (5.4‐fold increase). Analysis of the cofactor requirements of the FC membrane kinase(s) revealed that Mn2+ is the optimum cofactor and that Mg2+ can replace Mn2+ only for the kinase acting on the 100 kDa band. This suggests the presence of multiple EC membrane kinases. In the presence of both cofactors, the phosphorylation pattern is similar to the pattern obtained with Mn2+ alone. The kinase activity acting on all four phosphoproteins was independent of Ca2+, cAMP, cGMP, and phorbol 12‐myristate 13‐acetate. The mechanism responsible for the difference in kinase activity of proliferating vs. quiescent cells was not due to an inhibitor or enhanced phosphatase activity in confluent cells; the phosphorylation patterns obtained with sparse solubilized membranes and a mixture of sparse and confluent solubilized membranes were similar. The observed differences in phosphorylation events between sparse and confluent membranes occurred in multiple strains of two types of EC—pig aortic and bovine aortic—but were not apparent in membranes prepared from proliferating and quiescent human foreskin fibroblasts or 3T3 cells. Sparse endothelial cells made quiescent by serum deprivation exhibited reduced kinase activity with a phosphoprotein pattern similar to that of confluent cells; therefore, the enhanced kinase activity in sparse membranes may be growth‐dependent.