We are testing the hypothesis that cAMP regulation of aging and development in Dictyostelium is mediated by a cyclic AMP dependent protein kinase (cAMPdPK). We purpose to: I. Obtain information on the mechanism by which campdPK regulates cell differentiation: (A) Determine the requirements for the dissociation and reassociation of the catalytic and regulatory subunits. (B) Attempt to inhibit the nuclear kinase activity (that we have observed) with the purified regulatory subunit of cAMPdPK and, therefore, identify the nuclear kinase activity as the catalytic subunit. (C) By using the ultramicrotechniques determine the distribution in the two cell types of holoenzyme vs. catalytically active dissociated subunits. II. Cyclic AMP regulation of glucose mobilization - a key event in differentiation and aging: Under various conditions of exposure of cells to cAMP we will (A) determine the time of synthesis of the two known phosphorylases by incorporation of radioactive amino acids and immunoprecipitation of either the """"""""a"""""""" or """"""""b"""""""" forms, (B) determine the immunological cross-reactivity of the two forms by """"""""Western blotting"""""""" and enzyme-linked immunoassay, (C) determine the levels of translatable mRNA for each form during the time course of the treatments by utilizing in vitro protein synthesis and immunoprecipitation of the reaction products as well as hybridization with cloned cDNA, (D) utilize the ultramicromethods to determine if the specific cells that are undergoing glycogen degradation contain the """"""""a"""""""" form, while other adjacent cells contain the """"""""b"""""""" form. III. Cyclic AMP regulation of cytoplasmic and nuclear events during differentiation and aging: (A) Extend our earlier work in this area by using two-dimensional gel electrophoresis and incorporation of 35S-methionine and 32P to analyze the appearance of phosphoproteins before and after the accumulation of cyclic AMP and cAMPdPK. We will determine if the known maintainance of gene activity by cAMP is correlated with and perhaps mediated by cAMP induced phosphorylation of nuclear proteins. (B) Continue our initial studies on the phosphorylation patterns of nuclear proteins during the time course of differentiation before and after the appearance of cAMPdPK and cAMP. (C) Dissociate 13 hour aggregates in the presence and absence of cAMP. Determine the effect of cAMP on the phosphorylation of histones and non-histone proteins, and determine if the maintainance of late stage gene activity is related to the phosphorylation of certain nuclear proteins.
