The specific aims of this research cover four major areas of the newly-identified phospholipid/Ca?2+?-stimulated protein phosphorylation system in human leukemic cells. (1) Phospholipid-sensitive Ca?2+?-dependent protein kinase will be purified to homogeneity from leukemic cells from patients, and its properties and mechanism of activation by phospholipid and Ca?2+? will be studied. Immunocytochemistry of the enzyme in normal neutrophils, leukemic cells from patients, and HL60 and K562 cells in culture will be studied using polyclonal antibodies to the enzyme already developed in this lab. (2) Endogenous substrate proteins for the enzyme will be searched for, identified, and characterized. Attention will be paid to the variation in the substrates so that their pathophysiologic relevance can be suggested. Monoclonal antibodies to the substrates """"""""specific"""""""" to leukemic cells will be developed; and their ability to inhibit certain leukocyte functions, as well as their diagnostic and therapeutic usefulness in leukemias, will be explored. Endogenous protein phosphorylation stimulated by calmodulin/Ca?2+?, cAMP, and cGMP also will be carried out so that the interplays among the cellular mediators at the level of protein phosphorylation in the biology and pathology of human leukocytes can be studied. (3) Alterations in the enzyme (activity level and subcellular distribution) and substrates will be investigated in HL-60 stimulated to terminally differentiate by dimethylsulfoxide and phorbol ester. (4) Structure-activity relationship of alkyl-lysophospholipid analogs to inhibit the enzyme system will be examined, and the results will be compared with their clinical efficacy (if known) and their ability to affect leukemic cell growth in culture. (B)

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA036777-03
Application #
3174347
Study Section
Experimental Therapeutics Subcommittee 2 (ET)
Project Start
1984-03-01
Project End
1988-02-29
Budget Start
1986-03-01
Budget End
1987-02-28
Support Year
3
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Emory University
Department
Type
Schools of Medicine
DUNS #
042250712
City
Atlanta
State
GA
Country
United States
Zip Code
30322
Chambers, T C; Pohl, J; Glass, D B et al. (1994) Phosphorylation by protein kinase C and cyclic AMP-dependent protein kinase of synthetic peptides derived from the linker region of human P-glycoprotein. Biochem J 299 ( Pt 1):309-15
Zheng, B; Chambers, T C; Raynor, R L et al. (1994) Human leukemia K562 cell mutant (K562/OA200) selected for resistance to okadaic acid (protein phosphatase inhibitor) lacks protein kinase C-epsilon, exhibits multidrug resistance phenotype, and expresses drug pump P-glycoprotein. J Biol Chem 269:12332-8
Venema, R C; Raynor, R L; Noland Jr, T A et al. (1993) Role of protein kinase C in the phosphorylation of cardiac myosin light chain 2. Biochem J 294 ( Pt 2):401-6
Chambers, T C; Pohl, J; Raynor, R L et al. (1993) Identification of specific sites in human P-glycoprotein phosphorylated by protein kinase C. J Biol Chem 268:4592-5
Venema, R C; Kuo, J F (1993) Protein kinase C-mediated phosphorylation of troponin I and C-protein in isolated myocardial cells is associated with inhibition of myofibrillar actomyosin MgATPase. J Biol Chem 268:2705-11
Chambers, T C; Raynor, R L; Kuo, J F (1993) Multidrug-resistant human KB carcinoma cells are highly resistant to the protein phosphatase inhibitors okadaic acid and calyculin A. Analysis of potential mechanisms involved in toxin resistance. Int J Cancer 53:323-7
Noland Jr, T A; Kuo, J F (1993) Phosphorylation of cardiac myosin light chain 2 by protein kinase C and myosin light chain kinase increases Ca(2+)-stimulated actomyosin MgATPase activity. Biochem Biophys Res Commun 193:254-60
Chiou, S H; Raynor, R L; Zheng, B et al. (1993) Cobra venom cardiotoxin (cytotoxin) isoforms and neurotoxin: comparative potency of protein kinase C inhibition and cancer cell cytotoxicity and modes of enzyme inhibition. Biochemistry 32:2062-7
Vogler, W R; Olson, A C; Hajdu, J et al. (1993) Structure-function relationships of alkyl-lysophospholipid analogs in selective antitumor activity. Lipids 28:511-6
Noland Jr, T A; Kuo, J F (1993) Protein kinase C phosphorylation of cardiac troponin I and troponin T inhibits Ca(2+)-stimulated MgATPase activity in reconstituted actomyosin and isolated myofibrils, and decreases actin-myosin interactions. J Mol Cell Cardiol 25:53-65

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