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Mutations that prevent cyclic nucleotide binding to binding sites A or B of type I cyclic AMP-dependent protein
D Ogreid1, S O Døskeland, K B Gorman
1Cell Biology Research Group, University of Bergen, Norway.
Abstract:
Cyclic nucleotide binding and activation properties of cAMP-dependent protein kinases from five independent mutants of S49 mouse lymphoma cells were studied. These mutants were all hemizygous for expression of mutant regulatory (R) subunits of the type I kinase with lesions that altered the electrostatic charge of R subunit: lesions in three of the mutants mapped to cAMP-binding site A, and those in two of the mutants mapped to cAMP-binding site B. A nucleotide mismatch assay using 32P-labeled cRNA and ribonuclease A confirmed and refined localization of the mutations to single amino acid residues implicated in cAMP binding. R subunits from all mutants retained the ability to bind cAMP, but binding behaved as if it were entirely to nonmutated sites: 1) relative affinities of 11 adenine-modified derivatives of cAMP for mutant enzymes were identical to their relative affinities for the site of wild-type kinase that corresponded to the nonmutated site of the mutant; 2) the potencies of these analogs as activators of mutant kinases were strictly correlated with their binding affinities (for wild-type enzyme activation potencies were correlated with mean affinities of the analogs for cAMP-binding sites A and B); 3) combinations of analogs with strong preferences for opposite cAMP-binding sites in wild-type kinase showed no synergism in activating mutant kinases; 4) dissociation of cAMP from mutant kinases was monophasic; and 5) high salt accelerated dissociation of cAMP from kinases with site B lesions but retarded dissociation from those with site A lesions.
Insights
Mutant cAMP-dependent protein kinases show altered cAMP binding site properties. These findings help understand kinase regulation and drug development for cyclic nucleotide-binding proteins.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Cyclic adenosine monophosphate (cAMP)-dependent protein kinases (CA-PKs) are crucial for cellular signaling.
- Mutations in regulatory subunits can alter kinase activity and cAMP binding.
- S49 mouse lymphoma cell mutants provide a model for studying CA-PK function.
Purpose of the Study:
- To investigate the cAMP binding and activation properties of CA-PKs from S49 mouse lymphoma cell mutants.
- To characterize the effects of specific lesions in cAMP-binding sites A and B of the regulatory subunit.
Main Methods:
- Utilized five independent S49 mouse lymphoma cell mutants with altered regulatory (R) subunits.
- Employed a nucleotide mismatch assay with 32P-labeled cRNA and ribonuclease A to localize mutations.
- Assessed cAMP analog binding affinities and activation potencies for mutant and wild-type kinases.
Main Results:
- Mutant R subunits retained cAMP binding but exclusively to non-mutated sites.
- Analog potencies correlated with binding affinities at the non-mutated site.
- No synergistic activation observed with analogs targeting opposite sites.
- cAMP dissociation was monophasic and salt-dependent, differing between site A and B lesions.
Conclusions:
- Mutations significantly alter cAMP binding site characteristics, impacting kinase activation.
- Findings elucidate the distinct roles of cAMP-binding sites A and B.
- Provides insights into CA-PK regulation and potential therapeutic targets.