RESUMEN
The PTEN tumor suppressor functions as a phosphatase of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and negatively regulates the PI 3-kinase signaling pathway. Our previous studies showed that PTEN expression causes accumulation of cyclin-dependent kinase inhibitor p27(Kip1) and G(1) cell cycle arrest. Here, we show that PTEN negatively regulates expression of cyclin D1 and that cyclin D1 plays a unique role in p27 proteolysis. Coexpression of cyclin D1, but not cyclin E, is sufficient to restore p27 levels in PTEN-expressing cells. Conversely, loss of cyclin D1 by siRNA causes p27 accumulation. Silencing of the cyclin D1 gene or inhibition of the PI 3-kinase pathway prevents formation of the SCF(SKP2) complex, with a simultaneous increase in CUL1 binding to CAND1. CAND1-CUL1 binding is known to block the accessibility of CUL1 to SKP1 and (SKP2). We have found that CUL1 is less neddylated in cells that have lost cyclin D1 expression. Using an in vitro extract system, we found that the extracts prepared from cells lacking cyclin D1 have reduced activity to neddylate CUL1, in a manner similar to extracts from cells treated with a PI 3-kinase inhibitor or in G(0) resting cells. Consistently, the steady state levels of CUL1 neddylation were found lower under these conditions. Our studies reveal that PTEN/PI 3-kinase signaling and cyclin D1 control a novel pathway that regulates assembly of the SCF(SKP2) complex by modulating cullin neddylation and CAND1 binding at the G(1)/S cell cycle transition.
Asunto(s)
Ciclina D1/fisiología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Fosfohidrolasa PTEN/fisiología , Procesamiento Proteico-Postraduccional , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Ciclina D1/antagonistas & inhibidores , Ciclina D1/genética , Células HeLa , Humanos , Fosfohidrolasa PTEN/antagonistas & inhibidores , Fosfohidrolasa PTEN/genética , Unión Proteica , ARN Interferente Pequeño/farmacología , Transducción de Señal , Transfección , Células Tumorales CultivadasRESUMEN
The CUL4-ROC1 E3 ligase complex regulates genome stability, replication and cell cycle progression. A novel WD40 domain-containing protein, L2DTL/CDT2 and PCNA were identified as proteins associated with CUL4/DDB1 complexes. Inactivation of CUL4A, L2DTL, PCNA, DDB1 or ROC1 induced p53 stabilization and growth arrest. L2DTL, PCNA and DDB1/CUL4A complexes were found to physically interact with p53 tumor suppressor and its regulator MDM2/HDM2. The isolated CUL4A complexes display potent and robust polyubiquitination activity towards p53 and this activity is dependent on L2DTL, PCNA, DDB1, ROC1 and MDM2/HDM2. We also found that the interaction between p53 and CUL4 complex is regulated by DNA damage. Our data further showed that MDM2/HDM2 is rapidly proteolyzed in response to UV irradiation and this process is regulated by CUL4/DDB1 and PCNA. Our studies demonstrate that PCNA, L2DTL and the DDB1-CUL4A complex play critical and differential roles in regulating the protein stability of p53 and MDM2/HDM2 in unstressed and stressed cells.