Degradation of PAR by the ubiquitin-proteasome pathway Free

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PAR is a novel gene encoding a 146 amino acid protein involved in cell cycle and in positive regulation of proliferation in normal and malignant cells. Its expression level is low at the G1/S phase, increases as the cells progress through S phase, reaches the highest level at G2/M transition and returns to low level at the beginning of G1 phase.

PAR destruction at the end of mitosis seems to be important since PAR overexpression is associated with increased cell proliferation and cancers. Our data showed that ectopic increase of PAR in normal cells induced their malignant transformation. Furthermore, PAR is overexpressed in more than 60% of tumor specimens of various histologies, compared with the normal counterparts.

Here we present data showing that the ubiquitin-proteasome pathway mediated by the anaphase-promoting complex (APC) plays an important role in the degradation of PAR.

The first indication about the involvement of this mechanism came from the identification of two D boxes RxxL and RxxLxxV, at amino and C-terminal, respectively, in the aminoacid sequence of PAR protein.

Since most of the proteins that are degraded in a cell cycle dependent manner during mitosis are substrates of APC, an ubiquitin-ligase, we tried to see if PAR is ubiquitinated. MCF7 cells were treated with the proteasome inhibitor LLnL, or its less potent analog ALLM. An accumulation of high molecular weight ubiquitinated forms of PAR was detected only after LLnL, but not after ALLM treatment, as showed by Western blots immunobloted with anti-ubiquitin and anti-PAR antibodies.

Then, we examined the effect of various proteasome and calpain inhibitors on the steady-state level of PAR protein. Treatment of MCF7 cells with MG132, lactacystin, and proteasome inhibitor I resulted in accumulation of PAR protein, compared with the control untreated cells and cells treated with calpain inhibitor II, as showed by Western blots immunoblotted with an anti PAR antibody.

To further investigate the mechanism of PAR degradation we looked at the effect of cyclohexamide on PAR level in MCF7 cells treated and not treated with proteasome inhibitors. MCF7 cells were subjected to MG132, lactacystin, and proteasome inhibitor I inhibition followed by treatment with cycloheximide. Western blot analysis with an anti-PAR antibody revealed that PAR protein was stabilized in the presence of the proteasome inhibitors, while in the cells treated only with cyclohexamide the PAR level decreased quickly. The stabilization of PAR level after blocking the protein synthesis with cyclohexamide strongly suggests the proteasome pathway as the major mechanism of PAR degradation in these cells.

Similar results were obtained with DU145 prostate cancer cell line.

Further studies to determine the role of each of the two D boxes in the degradation of PAR protein are in progress.

In conclusion, these data provide evidence that PAR is multi ubiquitinated and is degraded by the proteasome pathway.