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Exogenous substrate stimulates autodephosphorylation of cyclic-AMP-dependent protein kinase II
B T Gjertsen1, B Fauske, S O Døskeland
1Department of Anatomy and Cell Biology, University of Bergen, Norway.
Abstract:
The autophosphorylated regulatory subunit (32P-RII) of cyclic-AMP-dependent protein kinase II was efficiently dephosphorylated by its C subunit in the absence of added ADP, provided that Mg/ATP and a standard protein kinase peptide substrate were present. This raises the possibility that autodephosphorylation could be significant in the intact cell. Only the cyclic-AMP-complexed free form of 32P-RII was efficiently dephosphorylated, indicating that the autodephosphorylation was intermolecular. Autodephosphorylation of 32P-RII in the presence of MgATP and kemptide occurred with formation of [gamma-32P]ATP, suggesting transfer of 32P of phospho-RII to a transient C*(MgADP) complex formed during the forward kinase reaction with peptide as substrate. Autodephosphorylation promoted by phosphorylation of exogenous substrates could operate also for other kinases conforming to a mechanism where MgADP remains bound to the active site after the other product (phosphorylated substrate) has left the catalytic complex.
Insights
Cyclic-AMP-dependent protein kinase II undergoes autodephosphorylation, where the regulatory subunit (32P-RII) is dephosphorylated by its catalytic subunit. This intermolecular process, forming [gamma-32P]ATP, suggests significance within intact cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- Cyclic-AMP-dependent protein kinase II (PKAII) plays crucial roles in cellular signaling.
- The regulatory subunit (RII) of PKAII is known to autophosphorylate.
- Understanding the regulation of PKAII activity is vital for comprehending cellular processes.
Purpose of the Study:
- To investigate the mechanism and significance of PKAII autodephosphorylation.
- To determine the conditions under which autodephosphorylation occurs.
- To explore the potential implications of autodephosphorylation in vivo.
Main Methods:
- In vitro dephosphorylation assays using autophosphorylated RII (32P-RII) and the catalytic subunit (C) of PKAII.
- Analysis of reaction products, including the formation of [gamma-32P]ATP.
- Investigation of the role of Mg/ATP and peptide substrates in the dephosphorylation process.
Main Results:
- Autophosphorylated RII (32P-RII) was efficiently dephosphorylated by the C subunit without added ADP, requiring Mg/ATP and a peptide substrate.
- Dephosphorylation was intermolecular, occurring only with the cyclic-AMP-complexed free form of 32P-RII.
- Formation of [gamma-32P]ATP indicated a transfer of phosphate from RII to a transient catalytic complex.
Conclusions:
- PKAII autodephosphorylation is an intermolecular process facilitated by the catalytic subunit.
- The formation of [gamma-32P]ATP suggests a novel dephosphorylation mechanism involving a transient catalytic complex.
- Autodephosphorylation may be a significant regulatory mechanism for PKAII activity in intact cells.