Murine erythroleukemic (MEL) cells are an established model system for studying hematopoietic cell differentiation in vitro; they can be induced to differentiate into hemoglobin-producing cells by hexamethylene bisacetamide (HMBA). MEL cells deficient in cAMP- dependent protein kinase (A-Kinase) activity are impaired in their ability to differentiate in response to HMBA and the greater their enzyme deficiency, the less they differentiate. This suggested that there are A-Kinase substrates that need to be phosphorylated during HMBA-induced differentiation. By 2-D PAGE a 22 kDa protein (pp22) was identified that was phosphorylated within hours of adding HMBA to parental cells but not to A-Kinase-deficient cells; in vitro experiments confirmed that pp22 was an A-Kinase substrate. By immunoblots of 2-D gels and by immunoprecipitation followed by 2-D gels it was found that pp22 belonged to the ras superfamily but that it was not one of the four ras proteins known to be an A-Kinase substrate. PP22 thus appears to be a ras-related protein important to chemically- induced differentiation of MEL cells. In the proposed work bio- chemical, structural and functional studies of pp22 will be performed.
The specific aims are: (1) To partially sequence pp22 and to isolate a pp22 cDNA. (2) To measure the kinetics of GTP binding to pp22 and the intrinsic GTPase activity of pp22 in the absence and presence of MEL cytosol and purified rasGAP; to determine the effect of HMBA on pp22-bound guanine nucleotides; to identify the amino acid(s) of pp22 phosphorylated by A-Kinase and study the kinetics of phosphorylation; and to examine the posttranslational lipid modifications of pp22. (3) To determine the tissue distribution of pp22, the regulation of its expression in HMBA-treated MEL cells and the effect of overexpressing wild type pp22 and pp22 mutated in the A-Kinase phosphorylation site on MEL cell growth and differentiation. (4) To determine the effect of inhibiting pp22 expression on MEL cell growth and differentiation. These studies should define the role of pp22 in MEL cell different- iation and lead to a better understanding of the function of ras proteins in hematopoietic cell differentiation.
