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Cloning and characterization of the major promoter of the human protein kinase C beta gene. Regulation by phorbol esters.

Publication ,  Journal Article
Obeid, LM; Blobe, GC; Karolak, LA; Hannun, YA
Published in: J Biol Chem
October 15, 1992

The expression of the beta isoenzyme for protein kinase C is regulated developmentally and in response to inducers of cell differentiation (such as phorbol esters and 1 alpha,25-dihydroxyvitamin D3). The 5' segment of the gene for protein kinase C beta was cloned from a human leukocyte genomic library in EMBL3 bacteriophage. This segment of the gene (greater than 54 kilobases in length) encompassed the coding sequence for the amino-terminal regulatory domain of the enzyme, the 5'-untranslated region, and the 5'-flanking region. Initiation of transcription was identified by S1 nuclease analysis and confirmed by RNase protection analysis at 197 base pairs 5' of the initiator ATG. Sequence analysis of the 5'-flanking region revealed it to be extremely G+C-rich (> 80%) with many features of a CpG island. Comparison of sequence with known cis-regulatory motifs disclosed a number of potential regulatory elements including an octamer binding motif at -76, Sp1-binding sites at -94 and -63, E boxes at -110, -26, and +18, an AP-1 site at -442, and an AP-2 site at -330. To demonstrate promoter activity, a 630-base pair fragment extending from -587 to +43 was subcloned in front of a promoterless luciferase gene. This fragment was able to drive the expression of luciferase in transient transfections of human hematopoietic cells. Deletion analysis demonstrated that a fragment -111 to +43 was necessary and sufficient for promoter activity; this fragment did not contain TATA or CAAT motifs. The promoter was stimulated 8-20-fold by phorbol esters accounting for the previously observed transcriptional activation of protein kinase C beta. This phorbol ester responsiveness was conferred by the basal promoter (-111 to +43) and was independent of the AP-1 site. These results define a novel mechanism of protein kinase C autoregulation at a transcriptional level.

Duke Scholars

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

October 15, 1992

Volume

267

Issue

29

Start / End Page

20804 / 20810

Location

United States

Related Subject Headings

  • beta-Galactosidase
  • Tumor Cells, Cultured
  • Transfection
  • Tetradecanoylphorbol Acetate
  • Restriction Mapping
  • Recombinant Fusion Proteins
  • Rats
  • RNA Probes
  • Protein Kinase C
  • Promoter Regions, Genetic
 

Citation

APA
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ICMJE
MLA
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Obeid, L. M., Blobe, G. C., Karolak, L. A., & Hannun, Y. A. (1992). Cloning and characterization of the major promoter of the human protein kinase C beta gene. Regulation by phorbol esters. J Biol Chem, 267(29), 20804–20810.
Obeid, L. M., G. C. Blobe, L. A. Karolak, and Y. A. Hannun. “Cloning and characterization of the major promoter of the human protein kinase C beta gene. Regulation by phorbol esters.J Biol Chem 267, no. 29 (October 15, 1992): 20804–10.
Obeid LM, Blobe GC, Karolak LA, Hannun YA. Cloning and characterization of the major promoter of the human protein kinase C beta gene. Regulation by phorbol esters. J Biol Chem. 1992 Oct 15;267(29):20804–10.
Obeid, L. M., et al. “Cloning and characterization of the major promoter of the human protein kinase C beta gene. Regulation by phorbol esters.J Biol Chem, vol. 267, no. 29, Oct. 1992, pp. 20804–10.
Obeid LM, Blobe GC, Karolak LA, Hannun YA. Cloning and characterization of the major promoter of the human protein kinase C beta gene. Regulation by phorbol esters. J Biol Chem. 1992 Oct 15;267(29):20804–20810.

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

October 15, 1992

Volume

267

Issue

29

Start / End Page

20804 / 20810

Location

United States

Related Subject Headings

  • beta-Galactosidase
  • Tumor Cells, Cultured
  • Transfection
  • Tetradecanoylphorbol Acetate
  • Restriction Mapping
  • Recombinant Fusion Proteins
  • Rats
  • RNA Probes
  • Protein Kinase C
  • Promoter Regions, Genetic