Selective translocation of beta II-protein kinase C to the nucleus of human promyelocytic (HL60) leukemia cells

• Published in: The Journal of biological chemistry Vol. 266; no. 1; pp. 28 - 33
• Main Authors: Hocevar, B A, Fields, A P
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 05.01.1991; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Antibodies - isolation & purification; Biological and medical sciences; Cell Line; Cell Nucleus - enzymology; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Humans; Isoenzymes - genetics; Isoenzymes - isolation & purification; Isoenzymes - metabolism; Kinetics; Lamin Type B; Lamins; Leukemia, Promyelocytic, Acute; Molecular Sequence Data; Nuclear Envelope - enzymology; Nuclear Proteins - metabolism; Peptides - chemical synthesis; Phosphorylation; Protamine Kinase - metabolism; Protein Kinase C - genetics; Protein Kinase C - isolation & purification; Protein Kinase C - metabolism; Transferases; Translocation; Protein kinase C; Enzyme; Substrate specificity; Immunoblotting assay; Activation; Nuclear envelope; Biological activity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)52396-6

The promyelocytic leukemia (HL60) cell line differentiates into monocyte-like cells after treatment with phorbol dibutyrate (PBt2). In contrast, bryostatin 1 (bryo), a structurally distinct protein kinase C (PKC) activator, does not induce differentiation and blocks the cytostatic effect of PBt2. The divergent responses to these agents correlate with activation of a PKC-like activity at the nucleus in response to bryo but not PBt2 (Fields, A. P., Pettit, G. R., and May, W.S. (1988) J. Biol. Chem. 263, 8253-8260). In the present study, this nuclear PKC-like activity (termed PKCn) was isolated from HL60 cells and shown to phosphorylate its known nuclear substrate, lamin B. PKCn-mediated phosphorylation of nuclear envelope-associated lamin B in vitro is calcium-dependent and is stimulated by bryo and 1,2-dioctanoylglycerol (DiC8), but not PBt2. In contrast, PKCn-mediated phosphorylation of histone IIIS is stimulated equally by all three activators. PKCn mediates calcium- and phosphatidylserine-dependent phosphorylation of both histone IIIS and partially purified lamin B. PKCn activity can be inhibited by an anti-PKC monoclonal antibody which specifically inhibits PKC. Isotype-specific PKC antibodies identify PKCn as beta II-PKC. Immunoblot analysis indicates that HL60 cells express both alpha- and beta II-PKC but no beta I- or gamma-PKC. Treatment of intact cells with bryo for 30 min leads to complete translocation of both alpha- and beta II-PKC from the cytosol to the membrane fractions. Approximately 8-10% of the total beta II-PKC (and less than 0.3% of the alpha-PKC) is found associated with the nuclear membrane of bryo-treated cells. In contrast, PBt2 treatment leads to complete translocation of alpha-PKC, but only partial translocation of beta II-PKC to the plasma membrane fraction. Neither PKC isotype is found associated with the nuclear membrane of PBt2-treated cells. These data demonstrate that alpha- and beta II-PKC differ with respect to activator responsiveness, intracellular distribution, and substrate specificity and indicate that their selective activation at distinct intracellular sites, including the nucleus, can have a dramatic effect on resulting cellular responses.