Selective post-transcriptional down-regulation of protein kinase C isoenzymes in leukemic T cells chronically treated with phorbol ester.

N Isakov,P Mcmahon,A Altman

doi:10.1016/s0021-9258(19)39944-2

N Isakov, P Mcmahon + Show 1 more

Open Access

https://doi.org/10.1016/s0021-9258(19)39944-2

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Abstract

Binding to, and activation of, protein kinase C (PKC) by phorbol ester (PE) tumor promoters may underlie their tumor-promoting activity. To study the effects of long-term PE treatment on regulation of cellular PKC, we adapted the human leukemic T cell line, Jurkat (JK), to continuous growth in the presence of PE. Such cells (JKPE) displayed loss of CD2 and CD3 cell surface molecules, known to play an important role in agonist-stimulated T cell activation, unresponsiveness to stimuli that induce interleukin 2 (IL2) receptor expression or IL2 production, change in the expression of several cell cycle-regulated genes, and a 6-fold reduction in cellular PKC enzymatic activity. This reduction was accompanied by the disappearance of a major approximately 82-kDa immunoreactive protein in JKPE cytosol when cell extracts were immunoblotted with a polyclonal anti-PKC peptide antibody cross-reactive with the PKC isoenzymes, alpha, beta, and gamma. Analysis with additional anti-peptide antibodies specific for alpha, beta, or gamma PKC indicated that all three types of PKC are expressed in JK cells; however, JKPE cells lost a major approximately 82 kDa immunoreactive cytosolic protein detectable with anti-PKC alpha antibody. In contrast, levels of expression and subcellular distribution of immunoreactive PKC beta and PKC gamma, as well as levels of mRNA specific for the three PKC isoenzymes, were not significantly affected by chronic PE treatment. These results indicate that PE-mediated reduction of PKC in JKPE cells is selective and occurs at the protein, not mRNA, level, and support the notion that distinct isoenzymes encoded by the PKC multigene family may be independently regulated. Moreover, the correlation between phenotypic and functional changes on one hand, and the selective reduction of PKC alpha on the other, raises the possibility that expression of CD2 and/or CD3 and functional activation in JKPE cells are preferentially regulated by PKC alpha.

Highlights

Selective Post-transcriptional Down-regulation of Protein Kinase C Isoenzymes in Leukemic T Cells Chronically Treated with Phorbol Ester*
These results indicate that phorbol ester (PE)-mediated reduction of protein kinase C (PKC) in JK cells chronically treated with PE (JKPE) cells is selective and occurs at the protein, not mRNA, level, and support the notion that distinct isoenzymes encoded by the PKC multigene family may be independently regulated
To determine the effects of chronic PE treatment, we measured and compared PKC activity in cytosol and particulate fractions derived from JK and JKPE cells

Summary

AND METHODS

12,13-dibutyrate, 12-O-tetradecanoylphorbol-13-acetate (TPA), phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, dimethyl sulfoxide, bovine serum albumin (fraction V), trichloroacetic acid, ATP, phosphatidylserine, 1,2-diolein, and Hl histone (type III-s) were purkhased from Sigma..Phytohemagglutinin-. PKC Isolation and Enzymatic Assay-Cytosol and particulate fractions of JK and JKPE cells were prepared as described [17] and PKC was partially purified on DE52 columns by one-step elution with 100. Sp&ficsyaid titer of antisera or affinity-purified antibodies were determined by an enzyme-linked immunosorbent assay (ELISA) as described [20], using plates coated with pure bovine spleen PKC (50 rig/well) or synthetic peptides (25 rig/well). Immunoblotting-Mouse brain PKC or protein extracts of JK and JKPE cells were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10 or 12% gels and electrophoretically blotted onto nitrocellulose paper as described [21]. Treatment with a 1:lOOO dilution of alkaline phosphatase-conjugated goat anti-rabbit IgG (Cappel Laboratories, Cochranville, PA) and development of the color reaction with nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indoIy1 phosphate (both from Sigma); 2). An oligonucleotide (PKC-4) corresponding to nucleotides 177-191 of human PKCrv [13], a sequence that is fully conserved in PKCp or PKCy, was synthesized and used as a common probe for all three PKC mRNA species

RESULTS

Anti-PKC- 1 iCommonJ “19

DISCUSSION