Selective translocation of beta II-protein kinase C to the nucleus of human promyelocytic (HL60) leukemia cells.
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.
Hocevar
B A
BA
Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Fields
A P
AP
D000595:Amino Acid Sequence; D000906:Antibodies / Q000302:isolation & purification; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D034921:Lamin Type B; D034882:Lamins; D015473:Leukemia, Promyelocytic, Acute; D008969:Molecular Sequence Data; D009685:Nuclear Envelope / Q000201:enzymology*; D009687:Nuclear Proteins / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis; D010766:Phosphorylation; D006656:Protamine Kinase / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D000906:Antibodies; D007527:Isoenzymes; D034921:Lamin Type B; D034882:Lamins; D009687:Nuclear Proteins; D010455:Peptides; D006656:Protamine Kinase; D011493:Protein Kinase C