Regulation of bovine kidney branched-chain 2-oxoacid dehydrogenase complex by reversible phosphorylation

Abstract

Bovine kidney mitochondria1 branched-chain 2-oxoacid dehydrogenase complex is inactivated by covalent phosphorylation catalysed by a specific protein kinase intrinsic to the complex. It has been shown previously [Cook, K. C., Lawson, R. and Yeaman, S. J. (1983) FEBS Lett. 157,59-621 that tryptic digestion ofphosphorylated complex releases three phosphopeptides, indicative of multisite phosphorylation.In this communication we report several findings. The mitochondria1 branched-chain 2-oxoacid dehydrogenase complex catalyses a rate-limiting step in the oxidation of the essential branched-chain amino acids, namely leucine. isoleucine and valine [l]. This complex is analogous to the well-characterised pyruvate and 2-oxoglutarate component ( E2) around which are arranged the oxoacid dehydrogenase (E 1) and dihydrolipoyl dehydrogenase (E3) components. As with the mammalian pyruvate dehydrogenase complex the E l component of the branched-chain 2-oxoacid dehydrogenase consists of two types of subunit, termed x and /j [2]. The activity of the branched-chain 2-oxacid dehydrogenase complex is regulated by phosphorylation of the x subunit of the E l component by a protein kinase intrinsic to the complex [3 -51.I t has been shown previously by ourselves [6] and others [7] that more than one site on the s1 subunit can be phosphorylated by the kinase. The same sites are also phosphorylated within intact mitochondria 183. Inactivation of the complex in i9itr.o apparently correlates with phosphorylation of one site [6] and the amino acid sequence surrounding that site has been determined [9].Previously we have shown that tryptic digestion of phosphor-ylated complex yields three phosphopeptides termed T1, T3 and T3 [6], the major inactivating site being contained in peptitlc T1. In this communication we show that these three phosphopcptides contain only two sites of phosphorylation.The sequence around the second site has been determined. Both sites can be dephosphorylated in vitro by the cytosolic protein phosphatases 2A and 2C. Re-activation of the complex correlates with dephosphorylation of the site contained in tryptic phosphopeptide TI. MATEKlALS AND METHODSAll materials were of the highest available purity and were IS described in [5]. EnzymesBranched-chain 2-oxoacid dehydrogenase was purified from bovine kidney as described in [ 5 ] except that the final preparation was resuspended in 50 mM Tris/HCl pH 7.0 in place of 30 mM sodium phosphate. Phosphorylated branched-chain 2-oxoacid dehydrogenase was prepared as in [5]. The catalytic subunits of protein phosphatases 1A and 2A were purified to homogeneity from rabbit skeletal muscle by Mr H. Y. Lim-Tung [lo]. Protein phosphatase 2B was purified from rabbit skeletal muscle up to and including the affi-gel blue chromatography step by Dr A. A. Stewart [I I]. Protein phosphatase 2C was partially purified from rat liver by Professor Philip Cohen, using chromatography on DEAEcellulose and Sephadex GI00 superfine [12]. Trypsin (treated with L-I -tosyl-amido-2-phenylethyl chlorome...

Structure of rat BCKD kinase: Nucleotide-induced domain communication in a mitochondrial protein kinase

“…The activity of the BCKD complex is regulated by a reversible phosphorylation͞dephosphorylation cycle in response to dietary and hormonal stimuli (7). Phosphorylation of Ser-292 and Ser-302 in the ␣-subunit of E1 by BCK results in complete inactivation of E1 and, therefore, the BCKD complex (8).…”

Cloning and sequencing of a cluster of genes encoding branched-chain alpha-keto acid dehydrogenase from Streptomyces avermitilis and the production of a functional E1 [alpha beta] component in Escherichia coli

“…A computer homology search of translated nucleotide and peptide sequence databases by using the deduced amino acid sequence of the S. avermitilis ORF1 and ORF2 sequences showed the best scores with E1␣ and E1␤ subunits of several BCDH and PDH complexes from different species (Table 1). A multiple amino acid sequence alignment of the S. avermitilis ORF1 product with those of the E1␣ BCDH and E1␣ PDH subunits in Table 1 showed similarity to structural motifs found in all E1␣ subunits of ␣-keto acid dehydrogenase complexes examined so far, such as the TPPbinding motif (25), a putative subunit interaction site (65), and the phosphorylation sites (I and II) of the E1␣ chains of the mammalian BCDH and PDH complexes (12,47,70) (Fig. 2).…”

Purification of Active E1α2β2 of Pseudomonas Putida Branched‐Chain‐Oxoacid Dehydrogenase

“…This is confirmed by the higher value for M , than for the associating Mutagenesis of homologous phosphorylation sites of Ela from P putidu. The activity of mammalian branched-chain-oxoacid dehydrogenase is regulated by phosphorylation [20,54,551 which inactivates the complex. Phosphorylation occurs at Ser293 and Ser313 of rat liver branched-chain-oxoacid dehydrogenase [20, 221, a region which is highly conserved in P. putida branched-chain-oxoacid dehydrogenase (Fig.…”