Regulation of NF-κB RelA Phosphorylation and Transcriptional Activity by p21 ras and Protein Kinase Cζ in Primary Endothelial Cells

doi:10.1074/jbc.274.19.13594

Regulation of NF-κB RelA Phosphorylation and Transcriptional Activity by p21^ras and Protein Kinase Cζ in Primary Endothelial Cells*

From the Immunobiology Research Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115

Abstract

The activity of the transcription factor NF-κB is thought to be regulated mainly through cytoplasmic retention by IκB molecules. Here we present evidence of a second mechanism of regulation acting on NF-κB after release from IκB. In endothelial cells this mechanism involves phosphorylation of the RelA subunit of NF-κB through a pathway involving activation of protein kinase Cζ (PKCζ) and p21^ras. We show that transcriptional activity of RelA is dependent on phosphorylation of the N-terminal Rel homology domain but not the C-terminal transactivation domain. Inhibition of phosphorylation by dominant negative mutants of PKCζ or p21^ras results in loss of RelA transcriptional activity without interfering with DNA binding. Raf/MEK, small GTPases, phosphatidylinositol 3-kinase, and stress-activated protein kinase pathways are not involved in this mechanism of regulation.

Footnotes

↵* This work was supported in part by National Institutes of Health Grant 1R01HL59476 (to J. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ To whom correspondence should be addressed: Immunobiology Research Center, Beth Israel Deaconess Medical Center, Harvard Medical School, 99 Brookline Ave., Boston, MA. E-mail:janrathe{at}caregroup.harvard.edu
↵§ Present address: Leukosite, Cambridge, MA 02142.
↵¶ Present address: Zeneca Pharmaceuticals, Macclesfield, Cheshire SK10 4TG3, United Kingdom.
Abbreviations:

RHD

Rel homology domain

PKCζ

protein kinase Cζ

MAPK

mitogen-activated protein kinase

MEK

mitogen-activated protein and extracellular signal-regulated kinase kinase

LPS

bacterial lipopolysaccharide

TNF-α

tumor necrosis factor-α

BAEC

bovine aortic endothelial cell(s)

PAEC

porcine aortic endothelial cell(s)

HUVEC

human umbilical vein endothelial cell(s)

PVDF

polyvinylidene difluoride

EC

endothelial cell(s)

TET

bacterial tetracycline repressor

PI

phosphatidylinositol
- Received August 7, 1998.
- Revision received January 18, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.