Alternative ribosomal initiation gives rise to chicken brain-type creatine kinase isoproteins with heterogeneous amino termini.
In higher eukaryotes three different types of creatine kinases (CK) are expressed: the muscle-specific M-CK, the ubiquitous cytoplasmic B-CKs, and the mitochondrial Mi-CKs. They fulfill multiple tasks in cells with an intensive energy metabolism. Isolated chicken B-CK can be resolved by two-dimensional gel electrophoresis into a major acidic Ba-CK and a major basic Bb-CK protein species which are very likely produced from the unique chicken B-CK gene (Wirz, T., Hossle, J. P., Soldati, T., and Perriard, J.-C. (1989) Experientia (Basel) 45, 32 (abstr.]. However, close inspection of the gels indicates the presence of additional B-CK species. This additional heterogeneity is generated by two distinct post-transcriptional processes. Post-translational phosphorylation was shown to contribute to heterogeneity of both Ba- and Bb-CK isoproteins and appears to modulate their enzymatic activity (A. F. Q. Quest, H. M. Eppenberger, and T. Wallimann, manuscript in preparation). Alternative ribosomal initiation of Bb-CK synthesis at multiple sites was shown to occur in cell free systems as well as in vivo, resulting in proteins differing in the length of their amino termini. Using site-directed mutagenesis to "switch off" each of the first four methionine codons of a full length Bb-CK cDNA, we were able to correlate each protein product with one distinct translational start site. An additional protein species appears to be produced by initiation at a noncanonical start codon. We propose that a leucine codon may be used as a translational start site. Evidence is presented to support the role of these amino-terminal truncated subunits in the regulation of the enzyme.
Soldati
T
T
Institute for Cell Biology, Swiss Federal Institute of Technology, Zürich.
Schäfer
B W
BW
Perriard
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D002645:Chickens / Q000235:genetics*; D003001:Cloning, Molecular; D003062:Codon; D003402:Creatine Kinase / Q000235:genetics* / Q000302:isolation & purification; D004247:DNA / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D007527:Isoenzymes; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism*; D014158:Transcription, Genetic
D003062:Codon; D007527:Isoenzymes; D046911:Macromolecular Substances; D012333:RNA, Messenger; D004247:DNA; D003402:Creatine Kinase