Cyclic AMP-resistant mutants of S49 mouse lymphoma cells hemizygous for expression of regulatory subunit of type I cyclic AMP-dependent protein kinase.
For use in studies of the functional organization of regulatory (R) subunit of type I cAMP-dependent protein kinase, 84 independent cyclic AMP-resistant mutants were isolated from sublines of S49 mouse lymphoma cells that are hemizygous for expression of the R subunit. Mutants were characterized by two-dimensional gel analysis of the R subunits, assays of kinase activation, and assays of cAMP-binding. All but eight of the mutants had kinases with increased apparent Kas for cAMP-dependent activation, and studies with site-selective cAMP analogs revealed considerable phenotypic diversity among these mutants. Forty-nine of the mutants had "charge-shift" lesions that mapped to regions of the R subunit polypeptide implicated in cAMP-binding. Twenty-five of the "charge-shift mutants" expressed only mutant R subunits, and the lesions in most of these isolates inhibited binding of cAMP to mutated cAMP-binding sites. The remainder of the charge-shift mutants expressed both mutant R subunit and R subunit with wild-type gel mobilities. The origin of these "heterozygous" mutants from parental "hemizygous" cells remains a puzzle.
Steinberg
R A
RA
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
Murphy
C S
CS
Russell
J L
JL
Gorman
K B
KB
D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology*; D004351:Drug Resistance / Q000235:genetics; D006579:Heterozygote; D007700:Kinetics; D008223:Lymphoma / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D009154:Mutation; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism
D000242:Cyclic AMP; D011494:Protein Kinases