Calcium depletion of Dictyostelium cells selectively inhibits cyclic nucleotide phosphodiesterase synthesis at a post-transcriptional step

Abstract

We reported previously that Ca2+ deprivation of Dictyostelium discoideum amoebae inhibits both the induction by cyclic AMP of soluble cyclic nucleotide phosphodiesterase (PD) activity in wild-type cells and the constitutive production of this activity in mutant HC35 cells. In this study, the mutant strain has been used to characterize this phenomenon further and to attempt to identify the Ca2+-dependent step(s) in PD production. Incubation of HC35 cells with 0.9 mM EGTA increased the rate of 45Ca2+ efflux from preloaded cells and partially inhibited PD production. The effect of EGTA on both processes was enhanced considerably by the presence of 1 ftM A23187. This concentration of ionophore alone had little effect on either Ca2+ efflux or PD production. Addition of 1μM free extracellular Ca2+ to amoebae incubated with EGTA and A23187 increased PD production two- to sixfold. Ca2+ depletion did not appreciably inhibit total protein synthesis or total protein glycosylation as determined by the incorporation of [3H]leucine or [3H]mannose, respectively. Also, the abundance of PD mRNAs was approximately the same in Ca2+-depleted and control cells. In contrast, immunoblot analysis of extracellular fluids and intracellular extracts of these cells revealed a dramatic reduction (up to 85%) in the levels of PD-related polypeptides associated with the Ca2+-depleted cells. These results suggest that Ca2+ deprivation selectively inhibits PD production by impairing a post-transcriptional process.