In vitro phosphorylation of the red blood cell cytoskeleton complex by cyclic AMP-dependent protein kinase from erythrocyte membrane.
Hydrosoluble proteins extracted from human erythrocyte ghosts by dialysis at low ionic strength and alkaline pH contain a cyclic AMP-dependent protein kinase which phosphorylates in vitro the cytoskeleton components in crude extracts. Spectrin components 1 and 2, actin and protein band 4.1 undergo this cyclic AMP-dependent endogenous phosphorylation together with low molecular weight peptides solubilized with the cytoskeleton in hydrosoluble extract. However, pure spectrin and purified erythrocyte G-actin were not phosphorylated by purified cyclic AMP-dependent protein kinase from erythrocyte membrane. Purified G-actin when added free to crude extract does not undergo phosphorylation by the cyclic AMP-dependent protein kinase present in this extract. In contrast, purified cyclic AMP-dependent protein kinase added either to crude extract or to the purified cytoskeleton complex (spectrin, actin and protein band 4.1), phosphorylates spectrin, actin and protein band 4.1. We can conclude that (1) cyclic AMP-dependent phosphorylation of red cell cytoskeleton occurs in vitro only when the cytoskeleton components are in a complexed form; (2) red cell actin, like other cellular actins, may be phosphorylated by cyclic AMP-dependent protein kinase but only in the oligomeric form and not in the G form.
D000199:Actins / Q000378:metabolism; D001798:Blood Proteins / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology; D003598:Cytoskeletal Proteins; D004910:Erythrocyte Membrane / Q000201:enzymology; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D009479:Neuropeptides; D010766:Phosphorylation; D011494:Protein Kinases / Q000097:blood*; D013049:Spectrin / Q000378:metabolism; D014461:Ultracentrifugation
D000199:Actins; D001798:Blood Proteins; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D009479:Neuropeptides; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1; D013049:Spectrin; D000242:Cyclic AMP; D011494:Protein Kinases