A study of phosphodiesterase in the fission yeast Schizosaccharomyces pombe

Angelyn La Marra-Phillips, Boston College

Abstract

The phosphodiesterase of S. pombe has been purified and characterized. The phosphodiesterase has a molecular weight of approximately 66 kDa as determined from gel filtration chromatography and can be resolved into four bands by native polyacrylamide gel electrophoresis. Three of these four forms have been purified to apparent homogeneity and were shown to hydrolyze both cAMP and cGMP. On SDS polyacrylamide electrophoresis each of these forms separated into two polypeptides with molecular weights of 40 $\pm$ 1 kDa and 25 $\pm$ 1 kDa. The data indicated that the phosphodiesterase exists as a dimer and the different forms could be due to post-translational modifications. Effector studies demonstrated that S. pombe phosphodiesterases could not be classified according to the mammalian classification system. In synchronized cells a series of spikes of cAMP were found to coincide with increases in cAMP phosphodiesterase activity. An initial spike correlated with daughter cell separation. The increase in cAMP phosphodiesterase activity was also observed in the presence of protein synthesis inhibitors. The data suggested that phosphodiesterase activity is regulated by cAMP through post-translational modification. S. pombe cAMP phosphodiesterase activity was inhibited by a tyrosine specific phosphatase and a phosphotyrosine specific antibody while cGMP phosphodiesterase activity was stimulated by the presence of protein kinase and ATP. Competition analysis of cAMP and cGMP indicated that the two substrates were hydrolyzed at the same catalytic site. This suggested that tyrosine phosphorylation plays a role in regulating S. pombe phosphodiesterase activity and that different residues are involved in the binding of cAMP and cGMP. In the presence of 10 uM caffeine, S. pombe cells exhibited an abnormal elongated phenotype and slower growth rate. These effects were initially attributed to increased concentrations of cAMP that presumably resulted from caffeine inhibition of phosphodiesterase. Studies with an adenyl cyclase null mutant demonstrated that the abnormal phenotype was not due to increased cAMP levels. Caffeine was shown, however, to inhibit phosphodiesterase activity in purified cell extracts which suggested that caffeine had a pleiotropic effect.

Recommended Citation

Angelyn La Marra-Phillips, "A study of phosphodiesterase in the fission yeast Schizosaccharomyces pombe" (January 1, 1997). Boston College Dissertations and Theses. Paper AAI9818617.
http://escholarship.bc.edu/dissertations/AAI9818617