Protein kinase C in tumoricidal activation of mouse macrophage cell lines.
A potential role of protein kinase C (PKC) in lipopolysaccharide- (LPS-) induced tumoricidal activation of macrophages was investigated by using two mouse macrophage cell lines (P388D1 and J774). J774 cells are stimulated by LPS to kill target P815 mastocytoma cells, whereas P388D1 cells fail to develop such an ability. Pretreatment of J774 cells with H-7 or phorbol myristate acetate resulted in a significant inhibition of LPS-induced cytotoxicity, whereas pretreatment with H-8, ML-7, HA1004, or W-7 did not. Since these results suggested a critical role of PKC in the activation process, the properties of PKC in the two cell lines were compared. Western blotting with rabbit antiserum specific for the PKC beta regulatory domain allowed detection of a protein of 79 kilodaltons (kDa) in the detergent lysates of both cell lines that were not stimulated by LPS. However, LPS treatment resulted in the appearance of a second protein of 40 kDa only in J774 cells and not in P388D1 cells. Furthermore, two forms of protein kinase (one basic and the other acidic) were identified in the cytosol of J774 cells by HPLC on DEAE-5PW, whereas only the basic form was found in P388D1 cells. On the basis of the response of the basic and acidic form protein kinases to phosphatidylserine (PS), diolein, and Ca2+, the basic form was found to contain both regulatory and catalytic domains of PKC, whereas the acidic form was suggested to represent the PKC catalytic domain.(ABSTRACT TRUNCATED AT 250 WORDS)
Novotney
M
M
Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City 66103.
Chang
Z L
ZL
Uchiyama
H
H
Suzuki
T
T
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D003602:Cytotoxicity, Immunologic; D004075:Diglycerides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D007546:Isoquinolines / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects*; D012515:Mast-Cell Sarcoma / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D010718:Phosphatidylserines / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000470:Alkaloids; D004075:Diglycerides; D007546:Isoquinolines; D008070:Lipopolysaccharides; D010718:Phosphatidylserines; D010879:Piperazines; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; C013965:diolein