Protein phosphorlyation in human peripheral blood lymphocytes. Phosphorylation of endogenous plasma membrane and cytoplasmic proteins.
Phosphorylation of endogenous proteins in subcellular fractions of human peripheral-blood lymphocytes was studied by one- and two-dimensional polyacrylamide-gel electrophoresis. Studies using extensively purified subcellular fractions indicated that the endogenous phosphorylating activity in the particulate fractions was derived primarily from the plasma membrane. Electrophoresis of (32)P-labelled subcellular fractions in two dimensions [O'Farrell (1975) J. Biol. Chem.250, 4007-4021] provided much greater resolution of the endogenous phosphoproteins than electrophoresis in one dimension, facilitating their excision from gels for quantification of (32)P content. More than 100 cytoplasmic and 20 plasma-membrane phosphorylated species were observed. Phosphorylation of more than 10 cytoplasmic proteins was absolutely dependent on cyclic AMP. In the plasma membrane, cyclic AMP-dependent phosphoproteins were observed with mol.wts. of 42000, 42000, 80000 and 90000 and pI values of 6.1, 6.3, 6.25 and 6.5 respectively. Phosphorylation of endogenous cytoplasmic and plasma-membrane proteins was rapid with t((1/2))=5-12s at 25 degrees C. Between 40 and 70% of the (32)P was recovered as phosphoserine and phosphothreonine when acid hydrolysates of isolated plasma-membrane phosphoproteins were analysed by high-voltage paper electrophoresis. The presence of cyclic AMP-dependent protein kinase and endogenous phosphate-acceptor proteins in the plasma membranes of lymphocytes provides a mechanism by which these cells might respond to plasma-membrane pools of cyclic AMP generated in response to stimulation by mitogens or physiological modulators of lymphocyte function.
D001797:Blood Protein Electrophoresis; D001798:Blood Proteins / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D066298:In Vitro Techniques; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000097:blood; D010750:Phosphoproteins / Q000097:blood; D010766:Phosphorylation; D013347:Subcellular Fractions / Q000378:metabolism
D001798:Blood Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D000242:Cyclic AMP; D002118:Calcium