Regulation of Nuclear Factor κB Transactivation: IMPLICATION OF PHOSPHATIDYLINOSITOL 3-KINASE AND PROTEIN KINASE C ζ IN c-Rel ACTIVATION BY TUMOR NECROSIS FACTOR α

Angel G Martin,Belén San-Antonio,Manuel Fresno

doi:10.1074/jbc.m011313200

Abstract

Transactivation by c-Rel (nuclear factor kappaB) was dependent on phosphorylation of several serines in the transactivation domain, indicating that it is a phosphorylation-dependent Ser-rich domain. By Ser --> Ala mutational and deletion analysis, we have identified two regions in this domain: 1) a C-terminal region (amino acids 540-588), which is required for basal activity; and 2) the 422-540 region, which responds to external stimuli as tumor necrosis factor (TNF) alpha or phorbol myristate acetate plus ionomycin. Ser from 454 to 473 were shown to be required for TNFalpha-induced activation, whereas Ser between 492 and 519 were required for phorbol myristate acetate plus ionomycin activation. Phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) zeta were identified as downstream signaling molecules of TNFalpha-activation of c-Rel transactivating activity. Interestingly, dominant negative forms of PI3K inhibited PKCzeta activation and dominant negative PKCzeta inhibited PI3K-mediated activation of c-Rel transactivating activity, indicating a cross-talk between both enzymes. We have identified the critical role of different Ser for PKCzeta- and PI3K-mediated responses. Interestingly, those c-Rel mutants not only did not respond to TNFalpha but also acted as dominant negative forms of nuclear factor kappaB activation.

Highlights

Transactivation by c-Rel was dependent on phosphorylation of several serines in the transactivation domain, indicating that it is a phosphorylation-dependent Ser-rich domain
By Ser 3 Ala mutational and deletion analysis, we have identified two regions in this domain: 1) a C-terminal region, which is required for basal activity; and 2) the 422–540 region, which responds to external stimuli as tumor necrosis factor (TNF) ␣ or phorbol myristate acetate plus ionomycin
PKC␨ Involvement in the Activation of the c-Rel Transactivation Domain—PKC␨ has been recently found to be involved in the process of nuclear factor ␬B (NF-␬B) activation by Tumor necrosis factor (TNF)␣ stimulation [25], so we studied the effect of this kinase on c-Rel activation

Summary

EXPERIMENTAL PROCEDURES

Cells and Reagents—Jurkat cells and COS-7 cells were grown in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 5% heat-inactivated fetal bovine serum (FBS; Life Technologies, Inc.) and containing 100 ␮g/ml streptomycin, 100 units/ml penicillin, 2 mM Lglutamine, plus nonessential amino acids, at 37 °C in a 7% CO2-in-air atmosphere saturated with water vapor incubator. Solid Phase in Vitro Phosphorylation Assay—c-Rel transactivation domain constructs from position 422–588 or 422–540 (using as template pRc-hc-Rel wild type) were cloned into the BamHI-EcoRI site of plasmid pGEX2T (Amersham Pharmacia Biotech) in order to express recombinant GST-c-Rel fusion protein. These recombinant proteins were purified from E. coli induced cultures according to the manufacturer’s instructions. For PKC␨ phosphorylation assay, anti-HA precipitates from WCE from transfected COS-7 cells were incubated in the kinase buffer described before, containing 1 ␮g of soluble recombinant GST c-Rel-(422–588)

RESULTS

PMA ϩ ionophorea

DISCUSSION