Sites of phosphorylation and mutation in regulatory subunit of cyclic AMP-dependent protein kinase from S49 mouse lymphoma cells: mapping to structural domains.
A novel peptide mapping approach has been used to map sites of charge modification to major structural domains of regulatory subunit (R) of type I cAMP-dependent protein kinase from S49 mouse lymphoma cells. Proteolytic fragments of crude, radiolabeled R were purified by cAMP affinity chromatography and displayed by two-dimensional polyacrylamide gel electrophoresis. [35S]methionine-labeled peptides containing sites of mutation or phosphorylation exhibited charge heterogeneity attributable to the modification. Phosphate-containing fragments were also labeled with [32P]orthophosphate to confirm their phosphorylation. Major fragments from [35S]methionine-labeled S49 cell R corresponded in size to carboxyterminal cAMP-binding fragments reported from proteolysis of purified type I Rs from various mammalian species; additional fragments were also visualized. End-specific markers in Rs from some mutant S49 sublines confirmed that cAMP-binding fragments extended to the carboxyterminus of R. Aminoterminal endpoints of fragments could be deduced, therefore, from peptide molecular weights. Clustering of proteolytic cleavage sites within the "hinge-region" separating aminoterminal and carboxyterminal domains of R permitted high resolution mapping in this region: the endogenous phosphate and a "phenotypically-silent" electrophoretic marker mutation fell within a 2.5-kdalton interval at its aminoterminal end. On the other hand, Ka mutations that increase the apparent constant for activation of kinase by cAMP mapped within the large cAMP-binding region of R. A map of charge density distribution within the hinge-region of R was constructed to facilitate structural comparisons between Rs from S49 cells and from other mammalian sources.
D000818:Animals; D002352:Carrier Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D047908:Intracellular Signaling Peptides and Proteins; D008223:Lymphoma; D051379:Mice; D009154:Mutation; D010766:Phosphorylation; D011494:Protein Kinases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C015482:protein kinase modulator; D000242:Cyclic AMP; D011494:Protein Kinases