A masked initiation region in retinoblastoma protein regulates its proteasomal degradation.

Retinoblastoma protein (Rb) is a tumor suppressor that binds and represses E2F transcription factors. In cervical cancer cells, human papilloma virus (HPV) protein E7 binds to Rb, releasing it from E2F to promote cell cycle progression, and inducing ubiquitination of Rb. E7-mediated proteasomal degradation of Rb requires action by another protease, calpain, which cleaves Rb after Lys 810. However, it is not clear why cleavage is required for Rb degradation. Here, we report that the proteasome cannot initiate degradation efficiently on full-length Rb. Calpain cleavage exposes a region that is recognized by the proteasome, leading to rapid proteolysis of Rb. These findings identify a mechanism for regulating protein stability by controlling initiation and provide a better understanding of the molecular mechanism underlying transformation by HPV.

A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

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The authors declare that the data supporting the findings of this study are available within the paper. Sample sizes were chosen based on similar published studies elsewhere. All analyzes were performed at least three independent times and each repeat yielded highly similar results.
Experiments were excluded from analysis when either the positive or negative control failed.
All the results were reproduced at least three times for each experiment.
Randomization was not required because we performed all the experiments using specified cell lines.
Blinding was not performed because there is no way to introduce bias into the experiments in this study.
Anti-Flag; the manufacturer validated that this antibody detects only the band(s) of Flag-tagged protein on a Western blot from an E. coli, plant or mammalian crude cell lysate. Anti-actin; the manufacturer validated that this antibody recognizes the 42 kDa actin band using immunoblotting with human or animal tissue extracts. Anti-16E7; the manufacturer validated that this antibody recognizes HPV16 E7 protein expressed in the HPV16-positive Caski cell line on a Western blot.
All cell lines tested negative for micoplasma contamination.
No commonly misidentified cell lines were used in the study.