Functional analysis of Rb2/p130 interaction with cyclins

Department of Microbiology-Immunology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Cancer Research (Impact Factor: 9.33). 06/1996; 56(9):2003-8.
Source: PubMed


The retinoblastoma (Rb) family consists of the tumor suppressor pRb and related proteins p107 and pRb2/p130. Ectopic expression of pRb and p107 results in a growth arrest of sensitive cells in the G1 phase of the cell cycle. We demonstrated here that the growth-suppressive properties of pRb2/p130 were also specific for the G1 phase. The A-, E-, and D-type cyclins as well as transcription factor E2F1 and the E1A viral oncoprotein were able to rescue the pRb2/p130-mediated G1 growth arrest in SAOS-2 cells. The rescue with cyclins A and E correlated with their physical interaction with pRb2/p130, which surprisingly has been found to occur over all phases of the cell cycle. The phosphorylation status as well as the kinase activity associated with pRb2/p130 dramatically increased near the G1-S-phase transition. This suggests that, like the other Rb family members, pRb and p107, the phosphorylation of pRb2/p130 is controlled by the cell cycle machinery and that pRb2/p130 may indeed be another key G1-S-phase regulator.

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    • "Indeed, it has been described that p130 undergoes phosphorylation at distinctive sites during the G0 phase in a way that characterizes p130 from the other members of the Rb family proteins (Kiess et al., 1995; Canhoto et al., 2000). p130 is phosphorylated by the Cdk4/Cyclin D or Cdk6/Cyclin D and Cdk2/Cyclin E or Cdk2/Cyclin A complexes and its expression levels fall when the cells enter into the S phase (Baldi et al., 1995; Mayol et al., 1995; Claudio et al., 1996; Dong et al., 1998; Tedesco et al., 2002). In vivo phosphorylation mapping of human p130 identified 22 serine and threonine residues, targeted by the kinases Cdk2, Cdk4 and Cdk6 (Hansen et al., 2001). "

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    • "They share the ability to recruit chromatin-remodeling enzymes and their best characterized targets are the members of the E2F/DP family of transcription factors, generally referred to as E2F (Cinti et al., 2005; Tosi et al., 2005; Genovese et al., 2006; Macaluso et al., 2006; Purev et al., 2006). Both pRb2/p130 and p107 are able to bind cdk2/ cyclins A and E (Claudio et al., 1996). We have previously described the cdk2-dependent kinase inhibitory activity shown by the pRb2/p130 spacer domain, which suggests that part of its growth regulatory function could be mediated by the inhibition of this important cell cycle kinase (De Luca et al., 1997). "
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    • "Previous reports showed the presence of multimeric E2F DNA binding activities in other cell types, such as CDK2–cyclin A and CDK2–cyclin E in association with p107–E2F or with p130–E2F (reference 10 [for a review], 18, 19, 45, 46), as well as in the melanoma cell line HO-1 47. Similar analysis revealed that indeed CDK2 and cyclin A were in the E2F4–p130 (or p107) complex c, as visualized by supershifts with the respective antibodies (Fig. 3 B, compare lane 1 to 3 and 4, supershifts shown by bars). "
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    ABSTRACT: Inactivation of the retinoblastoma tumor suppressor protein (pRb) has been implicated in melanoma cells, but the molecular basis for this phenotype has not yet been elucidated, and the status of additional family members (p107 and p130, together termed pocket proteins) or the consequences on downstream targets such as E2F transcription factors are not known. Because cell cycle progression is dependent on the transcriptional activity of E2F family members (E2F1-E2F6), most of them regulated by suppressive association with pocket proteins, we characterized E2F-pocket protein DNA binding activity in normal versus malignant human melanocytes. By gel shift analysis, we show that in mitogen-dependent normal melanocytes, external growth factors tightly controlled the levels of growth-promoting free E2F DNA binding activity, composed largely of E2F2 and E2F4, and the growth-suppressive E2F4-p130 complexes. In contrast, in melanoma cells, free E2F DNA binding activity (E2F2 and E2F4, to a lesser extent E2F1, E2F3, and occasionally E2F5), was constitutively maintained at high levels independently of external melanocyte mitogens. E2F1 was the only family member more abundant in the melanoma cells compared with normal melanocytes, and the approximately fivefold increase in DNA binding activity could be accounted for mostly by a similar increase in the levels of the dimerization partner DP1. The continuous high expression of cyclin D1, A2, and E, the persistent cyclin-dependent kinase 4 (CDK4) and CDK2 activities, and the presence of hyperphosphorylated forms of pRb, p107, and p130, suggest that melanoma cells acquired the capacity for autonomous growth through inactivation of all three pocket proteins and release of E2F activity, otherwise tightly regulated in normal melanocytes by external growth factors.
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