Identification of a novel PP2C-type mitochondrial phosphatase - PubMed (original) (raw)

Identification of a novel PP2C-type mitochondrial phosphatase

Mandar Joshi et al. Biochem Biophys Res Commun. 2007.

Erratum in

Abstract

A novel phosphatase has been cloned and partially characterized. It has a mitochondrial leader sequence and its amino acid sequence places it in the PP2C family like two known mitochondrial phosphatases. Western blot analysis of subcellular fractions and confocal microscopy of 3T3L1 preadipocytes expressing the GFP-tagged protein confirm its mitochondrial localization. Western blot analysis indicates that the protein is expressed in several mouse tissues, with highest expression in brain, heart, liver, and kidney. The recombinant protein exhibits Mn(2+)-dependent phosphoserine phosphatase activity against the branched-chain alpha-keto acid dehydrogenase complex, suggesting the enzyme may play a role in regulation of branched chain amino acid catabolism. Whether there are other mitochondrial substrates for the enzyme is not known.

PubMed Disclaimer

Figures

Fig. 1

Fig. 1

Sequence alignment of PTMP with human PDP1, PP2C-α and PP2C-β (GenBank accession nos. Q9P0J1, P35813, O75688 respectively). The catalytically important amino acids characteristic of PP2C phosphatases are indicated by inverted red triangles. Conserved sequence motifs are indicated by red amino acid residues and blue arrows.

Fig. 2

Fig. 2

Subcellular localization of the novel PP2C. (A) Mitochondrial leader sequence of PTMP. Conserved ser at -5, leu at -8, and arg at -10 positions corresponding to the putative cleavage site between the 29th and 30th amino acid are indicated in red. (B) Confocal microscopy image showing localization of the PTMP-GFP fusion protein in mitochondria of 3T3-L1 preadipocytes. (C) Western blot analysis of subcellular fractions.

Fig. 3

Fig. 3

(A) Phosphatase activity of PTMP with _p_-nitrophenyl phosphate. Lineweaver-Burk plot is given in the insert. (B) Phosphatase activity of PTMP towards BCKDC (○) and PDC (●).

Fig. 4

Fig. 4

(A) Western blot analysis with both pre-immune serum and serum obtained after immunization with the peptide. PTMP runs at apparent molecular weight of 41 kDa on SDS-PAGE. No corresponding band was observed at this molecular weight with pre-immune serum. Samples from left: whole liver extract, liver mitochondrial extract, whole kidney extract, and kidney mitochondrial extract. Signal corresponding to PTMP is indicated by the arrow. (B) Detection of PTMP in heart using affinity purified antibody. To test the specificity of the antibody, protein blot was incubated with antibody diluted 50 times in blocking buffer [% milk powder in 50 mM Tris-HCl, 150 mM NaCl, 0.1% (v/v) Tween 20)] with and without 2 mg/ml peptide. The signal corresponding to PTMP was blocked by the peptide. (C) Tissue specific expression of PTMP in mice. Samples from left: recombinant hPTMP (runs slower because of his tag), brain, heart, liver, kidney, muscle, testis, spleen, lung, adipose.

References

    1. Bowker-Kinley MM, Davis WI, Wu P, Harris RA, Popov KM. Evidence for existence of tissue-specific regulation of the mammalian pyruvate dehydrogenase complex. Biochem J. 1998;329( Pt 1):191–6. - PMC - PubMed
    1. Popov KM, Zhao Y, Shimomura Y, Kuntz MJ, Harris RA. Branched-chain alpha-ketoacid dehydrogenase kinase. Molecular cloning, expression, and sequence similarity with histidine protein kinases. J Biol Chem. 1992;267:13127–30. - PubMed
    1. Huang B, Gudi R, Wu P, Harris RA, Hamilton J, Popov KM. Isoenzymes of pyruvate dehydrogenase phosphatase. DNA-derived amino acid sequences, expression, and regulation. J Biol Chem. 1998;273:17680–8. - PubMed
    1. Damuni Z, Merryfield ML, Humphreys JS, Reed LJ. Purification and properties of branched-chain alpha-keto acid dehydrogenase phosphatase from bovine kidney. Proc Natl Acad Sci U S A. 1984;81:4335–8. - PMC - PubMed
    1. Damuni Z, Reed LJ. Purification and properties of the catalytic subunit of the branched-chain alpha-keto acid dehydrogenase phosphatase from bovine kidney mitochondria. J Biol Chem. 1987;262:5129–32. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources