title: Purification and assay of a phosphatidylglycerol-stimulated protein kinase from murine leukemic cells and its perturbation in response to IL-3 and PMA treatment.
abstract: Protein kinase P (PK-P), which has previously been detected from both human and murine leukemic cells, is characterized by distinctive patterns of phospholipid stimulation and substrate preferences, and is chromatographically separable from protein kinase C (PK-C). We have developed a three-step purification of PK-P from the interleukin 3 (IL-3)-dependent DA-1 murine leukemic cell line, entailing DEAE-Sephacel chromatography followed by TSK-3000 size exclusion and Mono-Q anion exchange HPLC steps. This yielded a 27-kd protein (from sodium dodecyl sulfate polyacrylamide gel electrophoresis) capable of preferentially phosphorylating the characteristic 75.5- and 77-kd endogenous substrates of PK-P previously noted. These observations were the basis for the development of a quantitative assay for PK-P, utilizing its separation from PK-C upon DEAE-Sephacel minicolumns followed by measurement of phosphatidylglycerol-stimulated histone H2B phosphorylation. This assay, and an analogous assay for PK-C, was then used to study the response of IL-3-starved DA-1 cells to IL-3 restimulation. PK-C exhibited cytosol to particulate redistribution following either IL-3 or phorbol 12-myristate 13-acetate (PMA) treatment, as has been previously described by others using similar systems. PK-P exhibited a rapid decrease in total activity following either IL-3 or PMA treatment, suggesting a response to PK-C activation. This was followed by a recovery phase during which PK-P activity slowly increased, with preferential redistribution into the particulate fraction of IL-3- but not PMA-treated cells. This difference in redistribution was thus likely to be under the control of signal transduction events other than PK-C activation. DA-1 PK-P thus exhibits a complex pattern of modulation by IL-3 and PMA, and may therefore constitute a new component of the cellular signal transduction cascade.
authors.affiliation: Department of Medicine, University of New Mexico School of Medicine, Albuquerque.
authors.forename: D J
authors.initials: DJ
authors.lastname: Klemm
authors.forename: L
authors.initials: L
authors.lastname: Elias
mesh_terms: D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D007377:Interleukin-3 / Q000494:pharmacology; D007942:Leukemia, Experimental / Q000201:enzymology*; D010715:Phosphatidylglycerols; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D007377:Interleukin-3; D010715:Phosphatidylglycerols; D011494:Protein Kinases; C054566:protein kinase P; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate