Identification and characterization of the human HCG V gene product as a novel inhibitor of protein phosphatase-1.
The catalytic subunit of mammalian protein phosphatase-1 (PP1) is known to bind to a number of regulatory subunits, whose functions include the targeting of the catalytic subunit to the molecular proximity of its substrate proteins. In addition, PP1 is potently inhibited by several inhibitory polypeptides that include inhibitor-1 and inhibitor-2. In this study the yeast two-hybrid system was used to screen a human cDNA library for putative PP1-binding proteins. Ten putative positive clones were identified, one of which was found to be a partial cDNA of the hemochromatosis candidate gene V (HCG V) whose function was previously unknown. The full-length protein of 126 amino acid residues was expressed in Escherichia coli as a glutathione S-transferase fusion protein and also as a nonfusion protein. The recombinant protein inhibited recombinant and rabbit muscle protein phosphatase-1 with IC50s of ca. 1 nM, but did not inhibit PP2A. The term inhibitor-3 is proposed for this novel inhibitor. It is extremely hydrophilic, is heat stable, and behaves anomalously on SDS-PAGE with an apparent molecular mass of 23 kDa and on gel filtration with a relative molecular weight of 55 000, in contrast to its calculated molecular mass of 14 kDa. These characteristics are shared by the previously described protein phosphatase-1 inhibitor-2 and inhibitor-1 proteins.
Zhang
J
J
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla 10595, USA.
Zhang
L
L
Zhao
S
S
Lee
E Y
EY
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004722:Endoribonucleases; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D005822:Genetic Vectors; D006432:Hemochromatosis / Q000235:genetics*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases; D010765:Phosphorylase Phosphatase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D054645:Protein Phosphatase 1; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D044767:Ubiquitin-Protein Ligases
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D016601:RNA-Binding Proteins; C021042:protein phosphatase inhibitor-1; C116196:PPP1R11 protein, human; D044767:Ubiquitin-Protein Ligases; D004722:Endoribonucleases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D010765:Phosphorylase Phosphatase; C097081:PPP1R8 protein, human