Analysis of the dominance of mutations in cAMP-binding sites of murine type I cAMP-dependent protein kinase in activation of kinase from heterozygous mutant lymphoma cells.
Structural lesions in cAMP-binding sites of regulatory (R) subunit of cAMP-dependent protein kinase caused identical increases in apparent constants for cyclic nucleotide-dependent kinase activation in preparations from cells that were hemizygous or heterozygous for mutant R1 subunit expression. No wild-type kinase activation was observed in extracts from heterozygous mutant cells. This "dominance" was investigated by characterizing expression of wild-type and mutant R1 subunits and properties of protein kinase from S49 mouse lymphoma cell mutants heterozygous for expression of wild-type R1 subunits and R1 subunits with a lesion (Glu200) that inactivates cAMP-binding site A. By both studies of cAMP dissociation and two-dimensional gel analysis, wild-type R subunits comprised about 35% of total R1 subunits in heterozygous mutants. Synthesis of wild-type and mutant R1 subunits was equivalent, but wild-type subunits were degraded preferentially. Hydroxylapatite chromatography revealed a novel R1 subunit-containing species from heterozygous mutant preparations whose elution behavior suggested a trimeric kinase consisting of an R1 subunit dimer and one catalytic (C) subunit. Wild-type R1 subunit was found only in dimer and "trimer" peaks; the tetrameric kinase peak contained only mutant R1 subunit. It is concluded that C subunit binds preferentially to mutant R1 subunit in heterozygous cells forming either tetrameric kinase with mutant R1 subunit homodimers or trimeric kinase with R1 subunit heterodimers. This preferential binding results both in suppression of wild-type kinase activation and differential stabilization of mutant R1 subunits.
Shuntoh
H
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Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
Steinberg
R A
RA
D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism*; D004789:Enzyme Activation; D006579:Heterozygote; D007700:Kinetics; D008223:Lymphoma / Q000201:enzymology* / Q000235:genetics; D046911:Macromolecular Substances; D051379:Mice; D009154:Mutation; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D046911:Macromolecular Substances; D000242:Cyclic AMP; D011494:Protein Kinases