Purification and assay of a phosphatidylglycerol-stimulated protein kinase from murine leukemic cells and its perturbation in response to IL-3 and PMA treatment

• Published in: Experimental hematology Vol. 16; no. 10; p. 855
• Main Authors: Klemm, D J, Elias, L
• Format: Journal Article
• Language: English
• Published: Netherlands 01-11-1988
• Subjects: Cell Line; Electrophoresis, Polyacrylamide Gel; Enzyme Activation - drug effects; Interleukin-3 - pharmacology; Leukemia, Experimental - enzymology; Phosphatidylglycerols; Phosphorylation; Protein Kinase C - metabolism; Protein Kinases - isolation & purification; Protein Kinases - metabolism; Protein-Serine-Threonine Kinases; Tetradecanoylphorbol Acetate - pharmacology
• ISSN: 0301-472X

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.