The regulatory subunit of neural cAMP-dependent protein kinase II represents a unique gene product.
Although the major form of soluble cAMP-dependent protein kinase in bovine cerebral cortex can be classified as a type II kinase, the regulatory subunit (RII) can be distinguished from RII found in other tissues such as heart. Heart and brain RII were distinguished qualitatively by autophosphorylation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mobility of dephosphorylated heart RII shifted from an apparent Mr of 55,000 to 57,000 following autophosphorylation. In contrast, when RII purified from brain was autophosphorylated with [gamma-32P]ATP, two radiolabeled bands were visualized, a minor band (less than or equal to 20%) which migrated with an Mr of 57,000 similar to the heart protein and a band with Mr = 55,000 which did not shift its mobility in response to autophosphorylation. Brain RII was further distinguished from heart RII on the basis of cAMP binding. Millipore filtration and equilibrium dialysis indicated that 2 mol of cAMP bound/mol of RII in contrast to 4 mol/mol with heart RII. Immunological differences were also apparent. Radioimmunoassays using monoclonal antibodies to RII showed that the brain protein had less than 4% of the cross-reactivity of heart RII. Both immunoblotting and immunoprecipitation using monoclonal as well as serum antibodies established that the cross-reactivity in phosphorylated brain RII was associated exclusively with the 57,000 component that behaved like heart RII. The lack of cross-reactivity of neural RII with two different monoclonal antibodies targeted the hinge region of RII as an area where structural differences might be anticipated, and comparative sequence analysis of this region definitively established that the major form of RII in brain is a unique gene product from the RII expressed in heart.
Weldon
S L
SL
Mumby
M C
MC
Taylor
S S
SS
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002352:Carrier Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D002417:Cattle; D002540:Cerebral Cortex / Q000032:analysis; D003429:Cross Reactions; D000242:Cyclic AMP / Q000378:metabolism; D005796:Genes; D047908:Intracellular Signaling Peptides and Proteins; D008970:Molecular Weight; D009206:Myocardium / Q000032:analysis; D010766:Phosphorylation; D014018:Tissue Distribution
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C015482:protein kinase modulator; D000242:Cyclic AMP