Regulation of Ubiquitination and Degradation of p53 in Unstressed Cells through C-terminal Phosphorylation

Abstract

Previously, we found that the protein kinase C (PKC) inhibitor H7 stimulates p53 to accumulate in a form incapable of inducing transcription from p53-dependent promoters. We concluded that H7 inhibits constitutive C-terminal phosphorylation of p53, which regulates its turnover in unstressed cells. We now show that p53 and its inhibitor MDM2 (HDM2 in human cells) are together in the nuclei of H7-treated cells and can be co-immunoprecipitated. Despite this association of p53 with the ubiquitin ligase MDM2, ubiquitinated p53 was not detected in H7-treated cells. Furthermore, co-treatment with H7 and the proteosome inhibitor LLnL prevented the accumulation of ubiquitinated p53 that was observed in cells treated solely with LLnL. In addition, treatment of cells with the PKC activator phorbol ester stimulated the ubiquitination of p53 and reduced its ability to accumulate after stress. H7 did not induce the phosphorylation of human p53 on Ser-15 (Ser-18 in mouse protein), a modification that occurs in response to DNA damage and leads to the release of MDM2 and to transactivation by p53. We conclude that phosphorylation of the C-terminal domain of p53 by PKC increases its ubiquitination and degradation in unstressed cells.

Retinoic acid mediates degradation of IRS-1 by the ubiquitin–proteasome pathway, via a PKC-dependant mechanism

“…Consistent with the inability of RA to regulate IRS-1 levels in the RA-resistant MDA-MB-231 breast cancer cell line, we also failed to observe any regulation of PKC-d in these cells. Although activating PKC has not been previously reported to be required for the ubiquitination of IRS-1, the phosphorylation of p53 by PKC was shown to regulate p53 degradation by stimulating its ubiquitination (Chernov et al, 2001). Interestingly, three of the serine sites (serines 307, 323, and 574) phosphorylated by PKC-d lie within the C-terminal domain of IRS-1 that contains potential PEST sequences, as identified by a PEST-FIND program (http://emb1.bcc.univie.ac.at/ embnet/tools/bio/PESTfind/) (Figure 7a).…”

A p53-independent G1 Cell Cycle Checkpoint Induced by the Suppression of Protein Kinase C α and θ Isoforms

“…In the case of persistent suppression of PKC ␣ and isoforms, cellular protective mechanisms may be activated, resulting in p21 WAF1/CIP1 induction and subsequently causing prolonged growth arrest, perhaps by blocking the CDK/Rb/E2F axis of cell cycle progression machinery. It has been observed that the inhibition of PKC activity increases p53 stability, whereas the activation of PKC by phorbol esters blocks the DNA damage-induced accumulation of p53 (60). It is conceivable that PKC inhibition activates the counteracting phosphatase activity, which affects protein stability, through an unknown mechanism.…”

Initiation factor modifications in the preapoptotic phase

“…Examples are the destabilization of the inhibitor of NFkB (IkB), as a result of phosphorylation-dependent polyubiquitination and subsequent degradation via the proteasome pathway, 120,121 and regulation of the turnover of bcatenin and p53. 122,123 However, it remains to be established whether 4E-BP1 is ubiquitinated prior to its degradation ( Figure 2) and whether the phosphorylation status of 4E-BP1 influences its turnover in a TRAIL-sensitive manner.…”