An increase of cAMP-dependent protein kinase during development in Polysphondylium pallidum.

Polysphondylium pallidum is a cellular slime mold in which, unlike in Dictyostelium discoideum, cAMP is not the chemotactic agent. The occurrence of a cAMP-dependent protein kinase in D. discoideum was demonstrated earlier and we suggested that it may mediate the intracellular effects of cAMP on the development of the organism, particularly since an increase in the amount of the enzyme during development was noted. In D. discoideum cAMP plays a dual role insofar as it serves both as chemotactic agent and as second messenger; it was of interest therefore, to determine whether a cAMP-dependent protein kinase occurred in P. pallidum. We found a cAMP-dependent protein kinase in P. pallidum using Kemptide as substrate. The regulatory subunit of the enzyme has an apparent molecular weight of 41,000 and seems to be similar in its properties with that isolated earlier from D. discoideum. The cAMP-dependent protein kinase catalytic subunits from the two species are also similar. Furthermore, there is a developmentally regulated, parallel, two- to threefold increase in the two subunits of the cAMP-dependent protein kinase in P. pallidum. The increase occurs before aggregates are formed. These findings are compatible with a role of the intracellular cAMP and of the cAMP-dependent protein kinase in the development of P. pallidum.

Differential cellular distribution of cAMP-dependent protein kinase during development of Dictyostelium discoideum.

It was shown previously by us that cAMP-dependent protein kinase activity in the cellular slime mold Dictyostelium discoideum increased during the early stages of development. Results from other laboratories showed that during the subsequent stage of cell differentiation and positioning, the accumulation of a number of prespore mRNAs and proteins (but not prestalk mRNAs and proteins) was dependent upon cAMP. The present communication describes the cellular distribution of the cAMP-dependent protein kinase at that stage of development. Pseudoplasmodia were disrupted, and prespore cells were separated from prestalk cells by sedimentation through a Percoll gradient. Prespore cells had approximately 4-5 times as much of both the catalytic and regulatory subunits of the cAMP-dependent protein kinase as did the prestalk cells. That the increase of cAMP-dependent protein kinase during development reflected de novo synthesis of the enzyme in both prespore and prestalk cells was demonstrated on the basis of [(3)H]leucine incorporation into the regulatory subunit. The findings are consistent with a role of the cAMP-dependent protein kinase in mediating the effects of cAMP on the synthesis of prespore-specific mRNAs and proteins at the stage at which cAMP appears to be required for the cell type-specific syntheses.

A cytosolic cyclic AMP-dependent protein kinase in Dictyostelium discoideum. II. Developmental regulation.

The cAMP-dependent protein kinase of the cellular slime mold, Dictyostelium discoideum, is developmentally regulated; there is an approximately 4-fold increase in activity during development. The incorporation of [3H]leucine into the enzyme demonstrates that there is de novo synthesis of the cAMP-dependent protein kinase. The activities of the catalytic and regulatory subunits increase in parallel. The maximal rate of increase of cAMP-dependent protein kinase activity precedes "tip" formation, a stage of development characterized by a sharp increase in mRNA complexity. The high level of cAMP-dependent protein kinase activity, attained at this stage of development, persists when aggregates are dispersed and the amoebae are kept in suspension without added cAMP. The synthesis of the developmentally regulated mRNAs under these conditions is dependent on exogenous cAMP. The increase in cAMP-dependent protein kinase activity during development does not require sustained cell-cell contact insofar as it occurs in single cell suspensions of amoebae. Furthermore, the increase does not require exogenous cAMP, although added cAMP stimulates the synthesis of the enzyme to a level higher than that found, when cAMP is not added. These observations support the hypothesis that in D. discoideum cAMP-dependent protein kinase mediates the effects of cAMP on development.

A cytosolic cyclic AMP-dependent protein kinase in Dictyostelium discoideum. I. Properties.

A cAMP-dependent protein kinase was isolated and partially purified from Dictyostelium discoideum. The cytosolic holoenzyme has an apparent Mr = 160,000-180,000; its activity was stimulated significantly by cAMP when Kemptide served as substrate. The enzyme was dissociated and the regulatory subunit purified by affinity chromatography on 8-aminoethylamino-cAMP. Only one type of regulatory subunit was found; it has an apparent Mr = 41,000 and is a substrate for the in vitro phosphorylation by the homologous catalytic subunit and by purified bovine catalytic subunit. Antibody against the regulatory subunit was prepared. The D. discoideum catalytic subunit was separated from cAMP-independent protein kinase by chromatofocusing. The apparent molecular weight of the catalytic subunit of the D. discoideum cAMP-dependent protein kinase is 33,000 and its pI is 6.4. The enzyme catalyzed the phosphorylation of bovine RII but not of RI regulatory subunit and was inhibited by high concentrations of the inhibitor of mammalian cAMP-dependent protein kinase. The evolution of the functional domains of cAMP-dependent protein kinases is discussed on the basis of a comparison of the analogous D. discoideum and vertebrate enzymes.

Regulation of the synthesis of adenylate cyclase in Escherichia coli by the cAMP -- cAMP receptor protein complex.

The synthesis of the adenylate cyclase [ATP pyrophosphatelyase-(cyclizing), E.C. 4.6.1.1.] of Escherichia coli, appears to be regulated negatively by the cAMP receptor protein, CRP. This conclusion is based on a comparison of adenylate cyclase activities measured in vitro with the rates of cAMP synthesis by intact bacteria. The activity of adenylate cyclase, depending on conditions of growth, is also regulated by CRP; this effect, however, is indirect insofar as it is mediated by a protein or proteins under CRP control.

The effects of coumarin on the frequency of deletions in a duplication strain of Aspergillus nidulans.

Strains of A. nidulans with a chromosome segment in duplicate show instability resulting from deletions in either of the duplicate segments. In Dp (I, II) strains, with the terminal segment of IR attached terminally to IIR, spontaneous deletions occur most frequently, though not exclusively, from the translocated segment. Coumarin, at concentrations which did not affect viability viability or growth rate, enhanced the instability of Dp (I, II) strains by selectively increasing only the deletion class of highest spontaneous frequency. This selective action is interpreted tentatively as due to inhibition of the repair of a particular class of DNA lesion occurring spontaneously in the attachment region of Dp (I, II) strains.