MN2+ -PHOSPHOLIPID STIM PK AND LEUKEMIC DIFFERENTIATION

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This project deals with the biochemistry and cellular physiology of a newly identified protein kinase, referred to as "PK-P", for phospholipid-modulated protein kinase, and its major endogenous substrate pp73. This system is detectable in murine and human leukemic cells, wherein it may play a role in multi-colony stimulating factor (IL-3) signal transduction and chemically inducible differentiation. pp73 has been purified, and will be characterized further, with respect to the proteolytic activity which it has been observed to exhibit; its interaction with phospholipids and divalent cations, and with protein kinases as a substrate; and its structure as determined by partial amino acid sequencing and immunologic analysis. PK-P has also been found to readily phosphorylate ribosomal protein S6. This reaction will be further quantitated and compared to phosphorylation by other kinases, and its effects upon in vitro translation determined. Precursors, other subunits or interacting activities, or modifications modulating PK-P will be identified. The pattern of response of the PK-P/pp73 system to chemical and biologic differentiation inducing agents of the HL-60 human myeloid leukemic cell line, and to IL-3 treatment of the DA-1 murine leukemic cell line will be further characterized using direct measurement of protein and phosphorylation levels and subcellular distribution by protein immunoblotting techniques. Interaction of these components with membranes from such cells subjected to various such treatments will also be studied. Molecular biologic studies aimed towards the cloning of the cDNA gene for PK-P will be undertaken, so that the complete primary structure of PK-P can ultimately be determined.