Thrombin and histamine stimulate the phosphorylation of elongation factor 2 in human umbilical vein endothelial cells.
The effects of thrombin and histamine on protein phosphorylation in intact cultured human umbilical vein endothelial cells (HUVEC) prelabeled with 32PO4 were investigated. Incubation of HUVEC with either thrombin or histamine, agonists known to induce rapid transient increases in intracellular calcium levels in HUVEC, caused a rapid reversible increase in the phosphorylation of a protein with a Mr = 100,000 independent of the presence of extracellular calcium. Immunological and biochemical studies demonstrated that this Mr = 100,000 protein is elongation factor 2 (EF-2), a substrate previously shown to be phosphorylated by calcium/calmodulin-dependent protein kinase III (Nairn, A. C., and Palfrey, H. C. (1987) J. Biol. Chem. 262, 17299-17303). EF-2 is crucial for protein synthesis because it catalyzes the translocation of peptidyl-tRNA on the ribosome. Phosphoamino acid analysis of the EF-2 immunoprecipitated from HUVEC revealed that all of the thrombin-stimulated phosphorylation occurred on threonine. EF-2 was also phosphorylated when HUVEC were treated with the calcium ionophore, ionomycin. Phosphorylation of EF-2 was not increased by treatment with D-Phe-Pro-Arg-chloromethyl ketone thrombin, phorbol dibutyrate, forskolin, or 8-bromo-cGMP. The transient nature of the phosphorylation of EF-2 is consistent with it having a role in mediating some of the transient effects of thrombin and histamine on endothelial cell protein synthesis and functional capabilities.
Mackie
K P
KP
Department of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York 10021.
Nairn
A C
AC
Hampel
G
G
Lam
G
G
Jaffe
E A
EA
D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D006632:Histamine / Q000494:pharmacology*; D006801:Humans; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology*; D013997:Time Factors; D014471:Umbilical Veins / Q000187:drug effects / Q000378:metabolism
D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D006632:Histamine; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D013917:Thrombin