MOLECULAR-CLONING OF THE P45 SUBUNIT OF PYRUVATE-DEHYDROGENASE KINASE

Purified preparations of rat heart pyruvate dehydrogenase kinase have two polypeptides with molecular weights of 48,000 (p48) and 45,000 (p4 5). Recently, we reported the primary structure of p48 (Popov, K. M., Kedishvili, N. Y., Zhao, Y., Shimomura, Y., Crabb, D. W., and Harris, R. A. (1993) J. Biol. Chem. 268, 26602-26606) and presented evidence t hat (i) it exhibits kinase activity for pyruvate dehydrogenase and (ii ) it belongs to a family of mitochondrial protein kinases unique from other eukaryotic protein kinases. Here, we report the molecular clonin g and deduced amino acid sequence of p45. The protein sequence of p45 has 70% identity to the protein sequence of p48. Minor differences exi st throughout the protein sequences with the greatest difference occur ring at the amino termini. Recombinant p45 protein, expressed in Esche richia coli and purified to homogeneity, catalyzed the phosphorylation and inactivation of kinase-depleted pyruvate dehydrogenase complex, i ndicating that p45 and p48 correspond to different isoforms of pyruvat e dehydrogenase kinase. Northern blot analysis revealed a single hybri dizing species of 2.5 kilobases. The highest level of p45 message expr ession was found in heart and skeletal muscle and the lowest in spleen and lung. Liver, kidney, brain, and testis express intermediate amoun ts of p45 RNA. In contrast, p48 mRNA is predominantly expressed in hea rt, with other tissues expressing only a modest amount of this message . Tissue-specific expression of isoforms of pyruvate dehydrogenase kin ase may indicate the existence of tissue-specific mechanisms for the r egulation of pyruvate dehydrogenase activity.