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

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 p21ras. 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 p21ras 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. 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 p21ras. 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 p21ras 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. The NF-κB/Rel family of dimeric transcription factors is involved in the immediate early transcription, i.e.independent of protein synthesis, of a large array of genes induced by mitogenic or pathogen-associated stimuli. In its active form, NF-κB is a nuclear homo- or heterodimeric complex of a number of different Rel family members. The canonical and most abundant form of NF-κB is composed of a 50-kDa (p50, or NFκB1) and a 65-kDa (p65, or RelA) subunit. Both subunits can form homodimers as well as heterodimers with other members of the Rel family i.e. c-Rel (Rel), p52 (NFκB2), and RelB (1Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2011) Google Scholar). All members of the Rel family exhibit extensive sequence similarity in their N-terminal region referred to as the Rel homology domain (RHD) 1The abbreviation used is: 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.1The abbreviation used is: 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. responsible for DNA binding and formation of Rel dimers. Only RelA, Rel, and RelB carry a transcription activating domain, and thus only dimers containing one of these proteins activate the transcription of NF-κB-dependent genes efficiently. With respect to transcription activation, the RelA subunit appears to have the highest activity. In most unstimulated cells, NF-κB is constitutively retained in the cytoplasm by inhibitory proteins of the IκB family, namely IκBα, IκBβ, IκBγ, p100, p105, and IκBε (2Whiteside S.T. Israel A. Semin. Cancer Biol. 1997; 8: 75-82Crossref PubMed Scopus (298) Google Scholar). Formation of NF-κB·IκB complexes masks the nuclear localization signal sequence present in NF-κB molecules and thus prevents their nuclear translocation. One of the key events in the activation of NF-κB is the liberation of functional NF-κB dimers from IκB, which results in the translocation of NF-κB to the nucleus. Cytoplasmic release of NF-κB dimers involves site-specific phosphorylation of IκB by kinases of the IκB signalosome (3Didonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1900) Google Scholar, 4Mercurio F. Zhu H.Y. Murray B.W. Shevchenko A. Bennett B.L. Li J.W. Young D.B. Barbosa M. Mann M. Science. 1997; 278: 860-866Crossref PubMed Scopus (1841) Google Scholar, 5Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1065) Google Scholar, 6Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1575) Google Scholar), ubiquitination (7Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1915) Google Scholar), and subsequent proteolytic degradation by the 26 S proteasome pathway (8Traenckner E.B. Wilk S. Baeuerle P.A. 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I. 1997; PubMed Scopus Google Scholar), on RelA transcriptional activity. of dominant negative mutants of or S. L. I. 1997; PubMed Scopus Google Scholar) not inhibit activation of the in results for TNF-α-induced NF-κB activity not and have been to be involved in p21ras L. M. S. A. M. U. S. A. PubMed Scopus Google J. S. A. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and the also in PKCζ L. P. G. J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) and in NF-κB activity in A.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), the of in NF-κB activity in EC, we used a dominant negative mutant that been to as a dominant repressor of G. PubMed Scopus Google Scholar). mutant a and in not of the dominant negative mutant with RelA only to of activity results a dominant negative mutant of the K. I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of cells with a specific of 3-kinase, on or TNF-α-induced activity. or of 3-kinase, not TNF-α-induced IκBα NF-κB DNA or of NF-κB-dependent and not which domain of RelA is by PKCζ and we different proteins in The composed of the DNA binding domain from the bacterial tetracycline repressor to a transactivation domain from the protein The second composed of the DNA binding domain to the C-terminal region of RelA that the transactivation domain In we a composed of the RelA to the transactivation domain activity of the DNA binding domain by with a containing tetracycline to a activity of the RelA DNA binding domain by with the transcription by and transcription not by these transcription not by or transcriptional activity by mutants in a to RelA These that PKCζ and p21ras RelA transcriptional activity by the RelA that the of PKCζ and p21ras is dependent on RelA RHD, we DNA binding through for inhibition of RelA transcriptional activity. this we a protein that the DNA binding domain and the RelA and DNA binding and consensus binding one to the of and on its transcriptional activity on the binding to different DNA consensus in transcriptional activity of this protein by and with the not the the the kinase the the we the of these for the regulation. we a containing the that the with RelA and or this the as the on the kinase not We that regulation of RelA transcriptional activity by PKCζ and p21ras involves the RelA and is only RelA DNA through a consensus RelA is with M. C. EMBO J. 1994; 13: PubMed Scopus Google Scholar, H. H. P. Ghosh S. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, V. G. de J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). In endothelial cells, RelA phosphorylation as by of RelA from cells other with The of the by and by and as p105, and IκBα not RelA phosphorylation The in of the IκBα to IκBα degradation as by we the of from RelA from by in these composed of and only to a phosphorylation of RelA been to phosphorylation of H. H. P. Ghosh S. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) and A.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Both show for the in RelA We RelA phosphorylation by of from or In EC, RelA is on in Upon or the of RelA phosphorylation as reflected by in signal of several and The of phosphorylation to be on and to in RelA and are to a in or cells, to be in These the of several and phosphorylation in is to RelA phosphorylation or i.e. RelA is to we RelA in and the level of in RelA is in unstimulated not the phosphorylation of RelA not that by not be for RelA We phosphorylation of RelA by IκBα RelA be to IκBα expression in in RelA phosphorylation The in RelA phosphorylation not to a in protein as by of RelA on the used for phosphorylation 1 with These that RelA is not retained by RelA is in the cytoplasm liberation from IκB, in the binding to or binding to In to DNA binding is for RelA we a RelA DNA mutant G. L. Brown K. P. U. 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J. 1997; PubMed Scopus Google Scholar). and its phosphorylation by IκBα for RelA, IκBα not protein as by and the of to the RelA RelA from cells with RelA or with a form, by The phosphorylation of RelA as with RelA However, the in phosphorylation not only by a in specific phosphorylation but also by protein of the RelA with a of RelA as with RelA, the level of expression of RelA RelA not We are the of this of from RelA and RelA the specific loss of a in RelA to the a in the RelA which is constitutively in RelA, and its phosphorylation is not by or These that is constitutively in We inhibition of PKCζ or p21ras pathways with RelA of RelA with or RelA phosphorylation as with of RelA or not RelA protein as by of RelA which domain of RelA by or we used different proteins and in The phosphorylation of the RelA by or or not protein of the which the C-terminal RelA transactivation domain, not by or that PKCζ and p21ras are involved in the phosphorylation of RelA RHD, we the phosphorylation of the by from or with or by on with the from or RelA the most is the of the most and and the of a The of phosphorylation with or as with of These to and not in and phosphorylation of in cells with and phosphorylation of and only by and not by These in the phosphorylation that PKCζ and p21ras pathways controlling RelA is that regulation of NF-κB transcriptional activity is mainly by retention of NF-κB in the cytoplasm by members of the IκB In this we that in endothelial cells there is that the transcriptional activity of nuclear NF-κB by the RelA subunit. involves through PKCζ and p21ras. kinases have been implicated in the regulation of nuclear RelA transcriptional activity. kinase A is involved in the regulation of RelA transcriptional activity through phosphorylation of in the consensus in the M. C. EMBO J. 1994; 13: PubMed Scopus Google H. H. P. Ghosh S. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). In also been implicated in RelA transcriptional activity. However, to protein kinase not directly on RelA S. M. M. K. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), as by the that inhibition of not in in RelA phosphorylation A. B.W. S. G. P. W. EMBO J. 1996; PubMed Scopus Google Scholar). kinase also been to with NF-κB in and to the C-terminal transcriptional activation domain of RelA in K. H. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). We that PKCζ and p21ras are in the regulation of RelA transcriptional activity. We show that inhibition of these results in of RelA transcriptional activity that with inhibition of RelA for the of PKCζ or p21ras One by PKCζ and p21ras is the to activate the signal-regulated kinase which been to NF-κB activity (15Berra E. Diaz Meco M.T. Lozano J. Frutos S. Municio M.M. Sanchez P. Sanz L. J. EMBO J. 1995; PubMed Scopus Google Scholar, J. 1995; 9: PubMed Scopus Google Scholar). However, we that in endothelial cells a dominant negative mutant of not with of p21ras and PKCζ is the N-terminal kinase is that this pathway is involved in NF-κB regulation in endothelial cells, a dominant negative N-terminal kinase 1 to inhibit RelA transcriptional activity not a dominant negative mutant of blocked p21ras induced N-terminal kinase activation to inhibit NF-κB activity not and W. J. Immunol. 1997; Google Scholar). Inhibition of by a dominant negative mutant or by to have on inhibition of not TNF-α-induced activity. RelA been to be in several and phosphorylation of the transactivation domain been to be a mechanism by which the activity of several transcription factors is T. Rev. 1995; Google Scholar). phosphorylation of the RelA transactivation domain been M.A. Baeuerle P.A. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). In phosphorylation of the and phosphorylation of the activation have been to RelA transcriptional activity M.A. Baeuerle P.A. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). phosphorylation of the RelA transactivation domain by kinase been K. H. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), and the of phosphorylation of been A.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In this we present evidence that the domain to the phosphorylation of We show that RelA phosphorylation can be by IκBα, which suggests that RelA is liberation from IκB molecules. The that phosphorylation of RelA is only by IκBα phosphorylation of RelA is suggests that the C of RelA is constitutively phosphorylation mainly on the We show that RelA is constitutively and that phosphorylation of but not of this is by or the phosphorylation of of these is regulated by PKCζ and phosphorylation of other is not by these that RelA is constitutively and this phosphorylation is not by or In a RelA mutant retained several which is different from cells, the RelA phosphorylation H. H. P. Ghosh S. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). show a as the RelA mutant can be A.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). as a of RelA transcriptional activity and show that the phosphorylation of can the transcriptional activity of the transactivation domain. to be of the transactivation domain in that acts on the RelA as well as on a transactivation domain is to that RelA and to the of M.A. H. M. Baeuerle P.A. J. Biol. 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A. 1997; PubMed Scopus Google Scholar). not in this is that be a involved in the regulation of RelA transcriptional activity by PKCζ or p21ras. is by the that the transactivation domain, which is thought not to with is by or to RelA the that the RelA and be by protein kinase A and with only to a and not to the a phosphorylation of RelA by PKCζ and p21ras pathways RelA transcriptional activity through of mechanism by which PKCζ and p21ras RelA transcriptional activity is through in the DNA binding activity of been that DNA binding of RelA can be by in phosphorylation through protein kinase A and protein kinase C M. C. 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