2ND-SITE MUTATIONS IN CYCLIC AMP-SENSITIVE REVERTANTS OF A K-A MUTANTOF S49 MOUSE LYMPHOMA-CELLS REDUCE THE AFFINITY OF REGULATORY SUBUNITOF CYCLIC-AMP-DEPENDENT PROTEIN-KINASE FOR CATALYTIC SUBUNIT
Rd. Cauthron et al., 2ND-SITE MUTATIONS IN CYCLIC AMP-SENSITIVE REVERTANTS OF A K-A MUTANTOF S49 MOUSE LYMPHOMA-CELLS REDUCE THE AFFINITY OF REGULATORY SUBUNITOF CYCLIC-AMP-DEPENDENT PROTEIN-KINASE FOR CATALYTIC SUBUNIT, Journal of cellular physiology, 165(2), 1995, pp. 376-385
K-a mutants of S49 mouse lymphoma cells are generally heterozygous for
expression of wild-type and mutant regulatory (R) subunits of type 1(
a) cyclic AMP-(cAMP)-dependent protein kinase, where the mutant R subu
nit has a defect in cAMP-binding to one of two intrachain cAMP-binding
sites. Several cAMP-sensitive revertants of such a K-a mutant were fo
und previously to harbor second-site mutations in the mutant allele, a
nd we have now identified three such mutations by sequence analysis of
PCR-amplified cDNAs. The resulting amino acid changes were Ala98 to T
hr, Gly179 to Arp, or Gly224 to Asp. The K-a mutation in these strains
(Glu201 to Lys) eliminated cAMP-binding to the more aminoterminal cAM
P-binding site (site A). None of the second-site mutations restored th
is activity in bacterially expressed recombinant R subunit. On the oth
er hand, all three second-site mutations reduced the apparent affinity
of the mutant R subunit for catalytic (C) subunit with the effects of
the substitutions at Ala98 and Gly179 substantially greater than the
effect of the substitution at Gly224. Patterns of phosphorylation and
turnover of wild-type and mutant R subunits in intact revertant cells
were consistent with reduced association of the doubly mutant subunits
with C subunit but the free mutant subunits apparently were more stab
le than free wild-type subunits. Differences in metabolic turnover of
mutant and wild-type subunits did not correlate with the sensitivities
of the isolated proteins to proteolytic cleavage. (C) 1995 Wiley-Liss
, Inc.