A kinase-negative mutant of S49 mouse lymphoma cells is defective in posttranslational maturation of catalytic subunit of cyclic AMP-dependent protein kinase.
Kinase-negative mutants of S49 mouse lymphoma cells, which lack detectable catalytic (C) subunit of cyclic AMP-dependent protein kinase, nevertheless contain cytoplasmic mRNAs for the two major forms of C subunit, C alpha and C beta. Investigation of the metabolism of C subunits in wild-type and mutant cells was undertaken to identify the step(s) at which C subunit expression was defective in kinase-negative cells. [35S]methionine-labeled C subunits from cytosolic fractions of wild-type S49 cells or C subunit-overexpressing cell lines were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after purification by either affinity chromatography using a peptide inhibitor of C subunit as the ligand or immunoadsorption with an anti-C subunit antiserum. Immunoadsorption revealed electrophoretic forms of C alpha and C beta subunits that migrated faster than those detected in affinity-purified samples; this unexpected heterogeneity suggested that functional activation of C subunit may require posttranslational modification. Immunoadsorption of cytosolic fractions from wild-type cells labeled for various times with [35S]methionine revealed an additional posttranslational maturation step. The bulk of immunoadsorbable C subunit label in cells pulse-labeled for 5 min or less was in an insoluble fraction from which it could be solubilized with a detergent-containing buffer; solubilization of the newly synthesized material proceeded over an incubation period of about 10 min. The primary defect in kinase-negative cells appeared to be in this solubilization step, since about equal C subunit radioactivity was found in detergent extracts of wild-type and kinase-negative cells but very little was found in mutant cytosols. I speculate that an accessory factor required for proper folding of newly synthesized C subunit in defective in the kinase-negative cells.
Steinberg
R A
RA
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D002846:Chromatography, Affinity; D008223:Lymphoma; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000138:chemical synthesis; D011061:Poly A / Q000235:genetics; D011494:Protein Kinases / Q000235:genetics* / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification
D046911:Macromolecular Substances; D010455:Peptides; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D011061:Poly A; D012313:RNA; D011494:Protein Kinases