Inhibition of protein phosphatase-1 by clavosines A and B - Novel members of the calyculin family of toxins

Site-directed mutagenesis was used to investigate the mechanism of interact ion between the catalytic subunit of human protein phosphatase-l (PP-1c gam ma) and members of the calyculin family of toxins. Clavosines A and B are r elated to calyculins but are glycosylated with a trimethoxy rhamnose group. We provide experimental evidence implicating Tyr-134 as an important resid ue in PP-1c gamma 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-1c gamma 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 subst ituting an Asp for Tyr-134 in the phosphatase. Clavosine B inhibited PP-1c gamma Y134D with a 310-fold decrease in potency. Clavosine A and calyculin A were also markedly poorer inhibitors of this mutant. These results sugges t that a hydrogen bond between Tyr-134 and the calyculins is unlikely to be essential for inhibitor binding to the phosphatase. The clavosines and cal yculin A were tested for their ability to inhibit other mutants of PP-1c ga mma (including Ile-133, Val-223, and Cys-291). Our mutagenesis studies prov ide an experimental basis for assessing models of calyculin binding found i n 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. Che m. 5, 1751-1773). A new model for clavosine and calyculin A binding to PP-l c is presented that is consistent with previous structure-function experime nts and which accommodates key structural features of the clavosines, inclu ding the novel rhamnose moiety.