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

✍ Scribed by Hisato Shuntoh; Robert A. Steinberg

• Publisher: John Wiley and Sons
• Year: 1991
• Tongue: English
• Weight: 876 KB
• Volume: 146
• Category: Article
• ISSN: 0021-9541
• DOI: 10.1002/jcp.1041460112

✦ Synopsis

Structural lesions in CAMP-binding sites of regulatory (R) subunit of CAMPdependent 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 R, 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 R, subunits and properties of protein kinase from S49 mouse lymphoma cell mutants heterozygous for expression of wild-type R, subunits and R, subunits with a lesion ( G I U ~~~) 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 R, subunits in heterozygous mutants. Synthesis of wild-type and mutant R, subunits was equivalent, but wild-type subunits were degraded preferentially. Hydroxylapatite chromatography revealed a novel R, subunit-containing species from heterozygous mutant preparations whose elution behavior suggested a trimeric kinase consisting of an R, subunit dimer and one catalytic (C) subunit. Wild-type R, subunit was found only in dimer and "trimer" peaks; the tetrameric kinase peak contained only mutant R, subunit. It is concluded that C subunit binds preferentially to mutant R, subunit in heterozygous cells forming either tetrameric kinase with mutant R, subunit homodirners or trimeric kinase with R, subunit heterodimers. This preferential binding results both in suppression of wild-type kinase activation and differential stabilization of mutant R, subunits.