Characterization of multiple cyclic -AMP phosphodiesterase isoforms and differential regulation by isoflavones and tyrphostins
Cyclic-AMP phosphodiesterase (PDE) plays a critical role in the regulation of cAMP levels by catalyzing the hydrolysis of this ubiquitous and crucial signaling molecule. The implication of PDE in many signaling processes has increased its significance as a therapeutic regulatory target. Three distinct PDE isoforms were isolated and pharmacologically characterized from the HT4.7 neural cell line (PDE4) and bovine heart crude complex (PDE1 and PDE3). Two well known protein tyrosine kinase (PTK) inhibitors, genistein (isoflavone) and tyrphostin 51, were found to significantly inhibit the three PDE isoforms. Isozyme-selective differences in potency by the two inhibitors were observed. A series of isoflavone and tyrphostin compounds containing small structural changes demonstrated substantially different effects on the degradation of cyclic-AMP by multiple isoforms of cyclic nucleotide phosphodiesterase (PDE). Not only did the structural components of each isoflavone and tyrphostin effect their inhibitory potency of PDE1, PDE3 and PDE4, but striking differences were noted between PDE isoforms. Since these compounds are well known tyrosine kinase inhibitors, the three PDE isoforms were purified away from protein tyrosine kinase (PTK) activity by ion-exchange chromatography and immunoprecipitation to preclude the involvement of those enzymes. Insights were gained into the molecular determinants of isoflavones and tyrphostins that are important in PDE isoform-selective inhibition. These data were used to construct a model of isoflavone binding to the PDE active site. The results obtained in this research project may help to explain some of the therapeutic and intracellular signaling effects of isoflavones and tyrphostins. Additional information on the regulation of PDE was obtained by the discovery that pretreatment of HT4.7 cells with herbimycin A resulted in inhibition of PDE4 activity. The data suggested the mechanism of PDE inhibition by herbimycin was indirect and may be mediated by another protein. Furthermore, incubation of the cytoplasmic tyrosine kinase, Syk, with PDE1 and PDE4 isoforms altered their activity. In both cases PDE activity was lowered in the presence of Syk. The modulation of PDE activity by herbimycin A and Syk PTK provide opportunities for future research into unique PDE regulatory possibilities.
Morimoto, Purdue University.
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