J. Biol. Chem., Vol. 263, Issue 33, 17397-17404, Nov, 1988
Mutations that prevent cyclic nucleotide binding to binding sites A or B of type I cyclic AMP-dependent protein kinase
D Ogreid, SO Doskeland, KB Gorman and RA Steinberg
Cell Biology Research Group, University of Bergen, Norway.
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.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
