Abstract
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.
Highlights
Retinoblastoma protein (Rb) is a tumor suppressor that binds and represses E2F transcription factors
We found that full-length Rb was stabilized in cells, probably because the negatively charged amino acid sequence at its C-terminus prevents the proteasome from initiating degradation
Calpain and a cullin E3 ligase contribute to Rb degradation
Summary
Retinoblastoma protein (Rb) is a tumor suppressor that binds and represses E2F transcription factors. Human papilloma virus (HPV) protein E7 binds to Rb, releasing it from E2F to promote cell cycle progression, and inducing ubiquitination of Rb. E7mediated proteasomal degradation of Rb requires action by another protease, calpain, which cleaves Rb after Lys 810. E7 activates and recruits calpain-1 to Rb to induce cleavage after residue Lys 810, and the cleavage product Rb1–810 can be detected in vitro and in some HPV-positive cells including HeLa and Caski cells after treatment with proteasome inhibitor[18]. The Rb1–810 fragment does not bind E2F transcription factors and is unstable in cells It is not known how this cleavage destabilizes Rb. Here, we report that calpain cleavage exposes a region in Rb that is recognized by the proteasome and allows it to initiate degradation efficiently, thereby significantly reducing its stability. The molecular mechanism of E7-mediated destabilization of cleaved Rb we present in this study introduces a regulatory mechanism for selective degradation of cellular proteins by the proteasome
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