Journal Article

Insufficient p65 phosphorylation at S536 specifically contributes to the lack of NF-κB activation and transformation in resistant JB6 cells

Jing Hu, Hiroyasu Nakano, Hiroaki Sakurai and Nancy H. Colburn

in Carcinogenesis

Volume 25, issue 10, pages 1991-2003
Published in print October 2004 | ISSN: 0143-3334
Published online October 2004 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/bgh198
Insufficient p65 phosphorylation at S536 specifically contributes to the lack of NF-κB activation and transformation in resistant JB6 cells

More Like This

Show all results sharing this subject:

  • Clinical Cytogenetics and Molecular Genetics

GO

Show Summary Details

Preview

NF-κB activation is required for TNF-α-induced transformation of JB6 mouse epidermal cells. Deficient activation of p65 contributes to the lack of NF-κB activation in transformation-resistant (P−) cells. We hypothesized that the differential NF-κB activation involves differential p65 phosphorylation arising from enzyme activity differences. Here we show that TNF-α induces greater ERK-dependent p65 phosphorylation at S536 in transformation sensitive (P+) cells than in P− cells. Our results establish that limited ERK content contributes to a low IκB kinase (IKKβ) level, in turn resulting in insufficient p65 phosphorylation at S536 upon TNF-α stimulation in P− cells. Phosphorylation of p65 at S536 appears to play a role in TNF-α-induced p65 DNA binding and recruitment of p300 to the p65 complex as well as in release of p65 bound to HDAC1 and 3. Blocking p65 phosphorylation at S536, but not at S276 or S529, abolishes p65 transactivational activity. Over-expression of p65 but not p65 phosphorylation mutant (S536A) in transformation-resistant P− cells renders these cells sensitive to TNF-α-induced transformation. Over-expression of p65 phosphorylation mimics p65-S536D or p65-S536E in P− cells and also rescues the transformation response. These findings provide direct evidence that phosphorylation of p65 at S536 is required for TNF-α-induced NF-κB activation in the JB6 transformation model. The lack of NF-κB activation seen in P− cells can be attributed to an insufficient level of p65 phosphorylation on S536 that arises from insufficient IKKβ that in turn arises from insufficient ERK. Thus, p65 phosphorylation at S536 offers a potential molecular target for cancer prevention.

Keywords: EMEM, Eagles Minimum Essential Media; FBS, fetal bovine serum; IKKβ, IκB kinase; IP, immunoprecipitation; MEKK1, mitogen-activated protein kinase/ERK kinase kinase-1; NIK, NF-κB inducing kinase

Journal Article.  8698 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

Full text: subscription required

How to subscribe Recommend to my Librarian

Users without a subscription are not able to see the full content. Please, subscribe or login to access all content.