The Journal of Cell Biology, Vol 97, 1072-1080, Copyright © 1983 by The Rockefeller University Press
Sites of phosphorylation and mutation in regulatory subunit of cyclic AMP-dependent protein kinase from S49 mouse lymphoma cells: mapping to structural domains
RA Steinberg
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.
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