Abstract
The catalytic subunit of cAMP-dependent protein kinase radiolabeled with [35S]methionine in wild-type S49 mouse lymphoma cells was degraded with half-lives of approximately 9.2 h in unstimulated cells and approximately 4.5 h in cells stimulated with a membrane-permeable cAMP analog. Turnover in kinase-negative mutant cells was about three times faster than in stimulated wild-type cells and appeared to involve a unique 47-kDa intermediate. Levels of catalytic subunit protein revealed by Western immunoblotting were consistent with the measured differences in turnover, but whereas the protein was mostly soluble in wild-type cell extracts, it was almost entirely insoluble in the mutant cell extracts. A substantial fraction of the catalytic subunit labeled in a 5-min pulse was soluble in kinase-negative cell extracts, but most of this material was rendered insoluble by incubating the cells for an additional 30 min before extraction. Degradation of the catalytic subunit in kinase-negative, but not in wild-type, cells was inhibited strongly by two specific peptide aldehyde inhibitors of the proteasomal chymotrypsin-like activity. An inhibitor of the proteasomal protease that prefers branched-chain amino acids had less of an effect on catalytic subunit degradation in the mutant cells.
Highlights
Kinase-negative1 mutants of S49 mouse lymphoma cells are defective in post-translational maturation and/or accumulation of the catalytic (C) subunit of cAMP-dependent protein kinase with the result that the cells have no detectable C subunit activity and little or no C subunit protein [1,2,3,4]
This difference in turnover rates accounts quite well for the 2–3-fold reductions in C subunit activity and/or protein reported in previous studies on a number of different cell systems treated chronically with either agents that elevate intracellular cAMP or cAMP analogs (6 –9)
C subunit turnover in kinϪ cells was three times faster than that in wild-type cells activated with CPT-cAMP and about six times faster than that in unstimulated wild-type cells
Summary
Kinase-negative (kinϪ) mutants of S49 mouse lymphoma cells are defective in post-translational maturation and/or accumulation of the catalytic (C) subunit of cAMP-dependent protein kinase with the result that the cells have no detectable C subunit activity and little or no C subunit protein [1,2,3,4]. As noted by Orellana and McKnight [3], the normal rate of C subunit production in kinϪ cells implies that the C subunit deficiency results from enhanced C subunit degradation Such accelerated turnover could be either the immediate cause of the mutant phenotype or a consequence of the production of defective C subunit protein. In kinϪ cells, the majority of newly synthesized C subunit remains insoluble [4] From this observation, we have hypothesized that the primary defect in kinϪ cells involves a failure to fold properly the newly synthesized C subunit protein [4]. We have hypothesized that the primary defect in kinϪ cells involves a failure to fold properly the newly synthesized C subunit protein [4] In this view, enhanced C subunit turnover might reflect the activity of a pathway specific for clearance of aberrant cell proteins. In kinϪ cells, is C subunit turnover faster than in wild-type cells, and the pathway for C subunit turnover appears to involve a unique intermediate
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