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Characterization of nuclear protein kinase C

Hocevar, Barbara Ann
http://rave.ohiolink.edu/etdc/view?acc_num=case1057177248

Abstract Details

1993, Doctor of Philosophy, Case Western Reserve University, Pharmacology.
The calcium- and phospholipid-dependent protein kinase, protein kinase C (PKC), is a family of isozymes which function in cell surface transduction for a variety of external stimuli. In the present study, the role of nuclear PKC in HL60 promyelocytic and K562 erythroleukemia cell line proliferation and differentiation is investigated. In addition, the phosphorylation sites mediated by nuclear PKC on the previously identified nuclear envelope substrate lamin B are identified and a role for PKC in the modulation of nuclear structural dynamics during cell cycle is suggested. Treatment of HL60 and K562 cells with phorbol esters induces differentiation, while treatment with bryostatin 1 (bryo) fails to induce differentiation and can block the cytostatic effect of the phorbol esters. The divergent biological effects of phorbol esters and bryo correlate with the translocation and activation of a PKC-like activity at the nucleus. While these cells express both the α and β II PKC isotypes, this nuclear PKC activity was purified and identified as the β II isotype. Nuclear PKC-mediated phosphorylation of nuclear-envelope associated lamin B is stimulated by bryo and 1,2-dioctanoylglycerol, but not by phorbol ester. In contrast, nuclear PKC-mediated phosphorylation of histone IIIS is stimulated equally by all th ree activators. Treatment of cells with phorbol ester or bryo leads to the rapid translocation of both α and β II PKC to the non-nuclear particulate fraction. However, bryo also induces selective translocation of β II PKC to the nuclear membrane. These data demonstrate that α and β 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. Using purified human β II PKC and isolated HL60 nuclear envelopes, the major sites of phosphorylation on human lamin B mediated by β II PKC have been determined. Using a combination of cyanogen bromide cleavage, direct microsequencing, tryptic phosphopeptide mapping, and phosphate release analyses, two major sites of PKC-mediated phosphorylation have been identified as Ser395 and Ser405. Functionally, β II PKC-mediated lamin B phosphorylation leads to the time-dependent solubilization of lamin B indicative of mitotic nuclear envelope breakdown in-vitro, which is attributed to direct phosphorylation by β II PKC rather than indirect activation of cdc2 kinase. Therefore, we conclude that β II PKC represents a physiologically relevant lamin kinase that can directly modulate nuclear lamina structure in-vitro.
Alan Fields (Advisor)
166 p.
Biology, Cell
Characterization nuclear protein kinase C

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Citations

  • Hocevar, B. A. (1993). Characterization of nuclear protein kinase C [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1057177248

    APA Style (7th edition)

  • Hocevar, Barbara. Characterization of nuclear protein kinase C. 1993. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1057177248.

    MLA Style (8th edition)

  • Hocevar, Barbara. "Characterization of nuclear protein kinase C." Doctoral dissertation, Case Western Reserve University, 1993. http://rave.ohiolink.edu/etdc/view?acc_num=case1057177248

    Chicago Manual of Style (17th edition)

case1057177248
408
© 1993, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.
Release 3.2.12