Phosphorylation and Stabilization of TAp63γ by IκB Kinase-β

Mary Macpartlin,Shelya X Zeng,Hua Lu

doi:10.1074/jbc.m801394200

Mary Macpartlin, Shelya X Zeng + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m801394200

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Abstract

Post-translational modification of the p53 family members is key to their regulation. Here we report the phosphorylation of TAp63gamma, but not DeltaNp63gamma, by IkappaB kinase beta (IKKbeta). Activation of IKKbeta by gamma radiation or tumor necrosis factor-alpha led to increased TAp63gamma protein levels in cells. IKKbeta, but not its kinase-defective mutant IKKbeta-K44A, led to this observed stabilization of TAp63gamma. This stabilization of TAp63gamma in response to gamma radiation was significantly decreased in the absence of IKKbeta. Phosphorylation of TAp63gamma blocks ubiquitylation and possible degradation of this protein. We postulate that phosphorylation of TAp63gamma by IKKbeta stabilizes the TAp63gamma protein by blocking ubiquitylation-dependent degradation of this protein.

Highlights

P63, unlike p53, is rarely mutated in cancer [9]
IKK␣ along with IKK␤ form the catalytic component of the I␬B kinase (IKK) complex along with the regulatory IKK␥ subunit
Human epidermal keratinocytes (HEK) cells were cultured in keratinocyte/SFM medium supplemented with L-glutamine, epidermal growth factor, and bovine pituitary extract (Invitrogen). ␥-Irradiated cells were treated with ␥ radiation (20 Gy) just prior to harvest (48 h posttransfection) at the time points shown, and cell lysates were analyzed on an SDS-8% gel

Summary

EXPERIMENTAL PROCEDURES

Plasmids and Antibodies—The pEGFP-C1-⌬Np63␥ plasmid was constructed using ⌬Np63␥ cDNA [4, 31]. ␥-Irradiated cells were treated with ␥ radiation (20 Gy) just prior to harvest (48 h posttransfection) at the time points shown, and cell lysates were analyzed on an SDS-8% gel. TNF␣-treated cells were treated with TNF␣ (20 ng/ml) prior to harvest (48 h post-transfection) at the time points shown, and cell lysates were analyzed on an SDS-8% gel. Equal amounts of proteins were analyzed by SDS-PAGE followed by immunoblot using the monoclonal anti-p63 antibody. 48 h after transfection, cells from each plate were harvested and split into 2 aliquots, one for straight immunoblot analysis and the other for detection of ubiquitylated proteins using Ni-NTA-agarose beads (Qiagen). The eluted proteins were analyzed by immunoblot for the polyubiquitylation of p63␥ with monoclonal p63 antibodies

RESULTS

HEK cells were also treated with

Findings

DISCUSSION