title: Inhibition of protein phosphatase-1 by clavosines A and B. Novel members of the calyculin family of toxins.
abstract: Site-directed mutagenesis was used to investigate the mechanism of interaction between the catalytic subunit of human protein phosphatase-1 (PP-1cgamma) and members of the calyculin family of toxins. Clavosines A and B are related to calyculins but are glycosylated with a trimethoxy rhamnose group. We provide experimental evidence implicating Tyr-134 as an important residue in PP-1cgamma that mediates interactions with the calyculins. Mutation of Tyr-134 to Phe, to prevent hydrogen bond formation, resulted in a slight increase in sensitivity of PP-1cgamma to clavosines A and B and calyculin A. In contrast, a Y134A mutant was 10-fold less sensitive to inhibition by all three inhibitors. The greatest effect on inhibition was found by substituting an Asp for Tyr-134 in the phosphatase. Clavosine B inhibited PP-1cgamma Y134D with a 310-fold decrease in potency. Clavosine A and calyculin A were also markedly poorer inhibitors of this mutant. These results suggest that a hydrogen bond between Tyr-134 and the calyculins is unlikely to be essential for inhibitor binding to the phosphatase. The clavosines and calyculin A were tested for their ability to inhibit other mutants of PP-1cgamma (including Ile-133, Val-223, and Cys-291). Our mutagenesis studies provide an experimental basis for assessing models of calyculin binding found in the literature (Lindvall, M. K., Pihko, P. M., and Koskinen, A. M. (1997) J. Biol. Chem. 272, 23312-23316; Gupta, V., Ogawa, A. K., Du, X., Houk, K. N., and Armstrong, R. W. (1997) J. Med. Chem. 40, 3199-3206; Gauss, C. M., Sheppeck, I. J., Nairn, A. C., and Chamberlain, R. (1997) Bioorg. Med. Chem. 5, 1751-1773). A new model for clavosine and calyculin A binding to PP-1c is presented that is consistent with previous structure-function experiments and which accommodates key structural features of the clavosines, including the novel rhamnose moiety.
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. tara_mccready@darwin.biochem.ualberta.ca
authors.forename: T L
authors.initials: TL
authors.lastname: McCready
authors.forename: B F
authors.initials: BF
authors.lastname: Islam
authors.forename: F J
authors.initials: FJ
authors.lastname: Schmitz
authors.forename: H A
authors.initials: HA
authors.lastname: Luu
authors.forename: J F
authors.initials: JF
authors.lastname: Dawson
authors.forename: C F
authors.initials: CF
authors.lastname: Holmes
mesh_terms: D000818:Animals; D002417:Cattle; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli; D006801:Humans; D006860:Hydrogen Bonding; D008387:Marine Toxins; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000201:enzymology; D010755:Organophosphates / Q000737:chemistry* / Q000494:pharmacology; D010080:Oxazoles / Q000737:chemistry* / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000235:genetics; D011485:Protein Binding; D054645:Protein Phosphatase 1; D011994:Recombinant Proteins; D014443:Tyrosine / Q000235:genetics
chemical_list: D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010755:Organophosphates; D010080:Oxazoles; D011994:Recombinant Proteins; C404954:clavosine A; C404955:clavosine B; D014443:Tyrosine; C059041:calyculin A; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1