RESUMO
Retinoblastoma protein (pRB) regulates cell cycle by utilizing different regions of its pocket domain for interacting with E2F family of transcription factors and with cellular and viral proteins containing an LxCxE motif. An LxCxE-like motif, LxCxD, is present in FZR1, an adaptor protein of the multi-subunit E3 ligase complex anaphase-promoting complex/cyclosome (APC/C). The APC/CFZR1 complex regulates the timely degradation of multiple cell cycle proteins for mitotic exit and maintains G1 state. We report that FZR1 interacts with pRB via its LxCxD motif. By using point mutations, we found that the cysteine residue in the FZR1 LxCxD motif is critical for direct interaction with pRb. The direct binding of the LxCxD motif of FZR1 to the pRB LxCxE binding pocket is confirmed by using human papillomavirus protein E7 as a competitor, both in vitro and in vivo. While mutation of the cysteine residue significantly disrupts FZR1 interaction with pRB, this motif does not affect FZR1 and core APC/C association. Expression of the FZR1 point mutant results in accumulation of S-phase kinase-associated protein 2 (SKP2) and Polo-like kinase 1 (PLK1), while p27Kip1 and p21Cip1 proteins are downregulated, indicating a G1 cell cycle defect. Consistently, cells containing point mutant FZR1 enter the S phase prematurely. Together our results suggest that the LxCxD motif of FZR1 is a critical determinant for the interaction between FZR1 and pRB and is important for G1 restriction.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas Cdh1/metabolismo , Ciclo Celular/fisiologia , Proteína do Retinoblastoma/metabolismo , Sequência de Aminoácidos/fisiologia , Ciclossomo-Complexo Promotor de Anáfase/genética , Proteínas de Ciclo Celular/genética , Divisão Celular/fisiologia , Humanos , Proteína do Retinoblastoma/genética , Fatores de Transcrição/metabolismoRESUMO
The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit, multifunctional ubiquitin ligase that controls the temporal degradation of numerous cell cycle regulatory proteins to direct the unidirectional cell cycle phases. Several different mechanisms contribute to ensure the correct order of substrate modification by the APC/C complex. Recent advances in biochemical, biophysical and structural studies of APC/C have provided a deep mechanistic insight into the working of this complex ubiquitin ligase. This complex displays remarkable conformational flexibility in response to various binding partners and post-translational modifications, which together regulate substrate selection and catalysis of APC/C. Apart from this, various features and modifications of the substrates also influence their recognition and affinity to APC/C complex. Ultimately, temporal degradation of substrates depends on the kind of ubiquitin modification received, the processivity of APC/C, and other extrinsic mechanisms. This review discusses our current understanding of various intrinsic and extrinsic mechanisms responsible for 'substrate ordering' by the APC/C complex.