Regulation of the two forms of glycogen phosphorylase by cAMP and its analogs in Dictyostelium discoideum

Abstract

We have recently reported the existence of two forms of glycogen phosphorylase (1,4-alpha-D-glucan: orthophosphate-alpha-glucosyltransferase; EC 2.4.1.1) in Dictyostelium discoideum. During development the activity of the glycogen phosphorylase b form decreased as the activity of the a form increased. The total phosphorylase activity remained constant. The physical and kinetic properties of the Dictyostelium enzyme were similar to those of the mammalian enzyme. In mammals, cAMP regulates the conversion of the two forms by a cAMP dependent protein kinase (cAMPdPK). We report here that if cAMP is added to a single cell suspension, the Dictyostelium phosphorylase activity becomes independent of 5'AMP and a 104 kd peptide appears. We also show the effect of several cAMP analogs on the phosphorylase activity in these single-cell suspensions. The cAMP analogs were selected on the basis of their affinities for the membrane-bound cAMP receptor or the cytoplasmic cAMPdPK. We found that relatively low levels, 100 microM, of cAMP or 2'd-cAMP added to aggregation-competent cells in shaking culture caused a loss of phosphorylase b activity and the appearance of phosphorylase a activity. The analog, 2'd-cAMP, has a high affinity for the cAMP receptor but a low affinity for the cAMPdPK. Two other analogs, Bt2-cAMP and 8-Br-cAMP, which have low affinities for the cAMP receptor but high affinities for the cAMPdPK, required high levels (500 microM) for 'b' to 'a' conversion. cDNAs to three cAMP-regulated genes--PL3, D11, and D3--were used as controls in the above experiments. In order to determine if intracellular levels of cAMP were involved in the regulation of phosphorylase activity, both the phosphorylase and the PL3, D11 and D3 mRNA levels were examined in cells suspended in a glucose/albumin mixture--a medium in which adenylate cyclase is inhibited. Under these conditions, neither gene regulation nor a change in the phosphorylase b to a activity occurred in response to added extra cellular cAMP. The results suggest that an intracellular increase in cAMP is involved in the regulation of the two forms of glycogen phosphorylase in Dictyostelium.

Regulation of the Dictyostelium glycogen phosphorylase 2 gene by cyclic AMP

“…The presence of exogenous cAMP induces the gp-2 mRNA, a result that is consistent with the observed cAMP induction of gp-2 activity and the gp-2 protein [Brickey et al, 1990;Rutherford et al, 19921. It seems likely that cAMP regulation occurs at the level of transcription, given previous studies on the regulation of gp-2 during Dictyostelium development [Firtel and Bonner, 1972;Thomas and Wright, 1976;Higgins and Dahmus, 19821.…”

Developmental and cAMP-mediated regulation of glycogen phosphorylase 1 in Dictyostelium discoideum

“…We have shown previously that exogenous cAMP causes a dramatic decrease in gp-1 enzyme activity (Brickey et al, 1990). Fig.…”

Disruption of glycogen phosphorylase gene expression in Dictyostelium: Evidence for altered glycogen metabolism and developmental coregulation of the gene products

“…The gpl enzyme is dependent on 5' AMP in the reaction mixture for activity, and the gpl protein can be phosphorylated in vivo and in vitro [31]. In log-phase cells, gpl is active, but as development proceeds, the activity of gpl decreases [4]. The exogenous addition of CAMP to shaking cultures of aggregationcompetent Dictyosrelium cells also results in a loss of gpl activity.…”