The retinoblastoma-related pocket proteins pRb, p107, and p130 are implicated in the control of cell proliferation, differentiation, and transformation. The function of pocket proteins is in part mediated by their ability to inhibit specific E2F transcription factors. The transcriptional activity of E2Fs is controlled by alteration of their nucleocytoplasmic localization during the cell cycle. p130 was observed to shuttle between the nucleus and cytoplasm in a heterokaryon fusion assay, suggesting the presence of nuclear and cytoplasmic localization signals. Two independent nuclear localization signals (NLS) that could target reporter proteins to the nucleus in transient transfection and microinjection experiments were identified in the C terminus of p130. In addition to the C-terminal NLS, the intact pocket domain of p130 itself was sufficient for nuclear translocation. Moreover, an additional functional NLS was mapped within the unique Loop region of p130. An N-terminal domain that conferred cytoplasmic localization was identified. Removal of the entire N terminus did not affect the ability of p130 to interact with E2F and to induce growth arrest. A model suggesting that the activity of pRb family members can be regulated by intracellular trafficking of the proteins is proposed.The retinoblastoma family of proteins is comprised of pRb and the related proteins p107 and p130 (also known as pRb2 and RBL2) (24,28,32,47,54). Rb-1 has been identified as a tumor suppressor gene deleted or mutated in childhood retinoblastoma and in a wide variety of adult cancers (28,80). Although the role of p107 and p130 in tumor suppression is less clear than that of pRb, there are several reports of p130 inactivating mutations identified in human cancers (10,13,14,37). As is the case for pRb, overexpression of p107 and p130 can induce growth arrest in certain cell types (12, 91) and can bind to and inhibit transcriptional activity of E2F transcription factors. All three members of the pRb family undergo cell cycle-dependent phosphorylation (6,19,51,52,85). It has been proposed that phosphorylation during the transition from G 0 /G 1 to S phase inactivates the growth-suppressive properties of pRb family members (11,40,91) by dissociation from E2F transcription factors.All three pRb family members share a high degree of sequence homology. The characteristic pocket domain is comprised of A and B boxes separated by a spacer domain. The A and B boxes define the minimal region essential for binding to the LXCXE-containing proteins (41, 44) including adenovirus E1A, simian virus 40 (SV40) large T antigen (T-Ag), and human papillomavirus E7 (18, 23, 81), as well as cellular proteins such as histone deacetylase 1 and 2 (5, 50), BRG1, BRM (22,73,76), and others (20). The ability of the LXCXE motif of viral oncoproteins to interact with pRb family members is indispensable for virus-mediated transformation (9,74,83,88,89). However, pRb mutants defective in binding to LXCXEcontaining proteins were capable of inducing growth arrest and remained...
In all eukaryotes, anaphase is triggered by the activation of a protease called separase. Once activated, separase cleaves a subunit of cohesin, a complex that links replicated chromatids before anaphase. Separase and cohesin are conserved from yeasts to humans. Although the machinery for dissolving sister cohesion is conserved, the regulation of this process appears to be more complex in higher eukaryotes than in yeast. Here we report the cloning of full-length human separase cDNA and the characterization of the encoded protein. Human separase was observed at the poles of the mitotic spindle until anaphase, at which time its association with the mitotic spindle was abruptly lost. The dynamic pattern of localization of human separase during cell cycle progression differs from that of fungal separases. Human separase also appears to undergo an autocatalytic processing on anaphase entry. The processed forms of human separase were isolated and the identity of the cleavage sites was determined by N-terminal sequencing and site-directed mutagenesis. The processed catalytic domain was found to be stably associated with the processed N-terminal fragment. Finally, by depletion of endogenous separase with antisense oligonucleotides, we report direct evidence that separase is required for high-fidelity chromosome separation in human cells.
The retinoblastoma family of proteins including pRB, p107 and p130 undergoes cell cycle dependent phosphorylation during the mid-G1 to S phase transition. This phosphorylation is dependent upon the activity of cyclin D/cdk4. In contrast to pRB and p107, p130 is phosphorylated during G0 and the early G1 phase of the cell cycle. We observed that p130 is speci®cally phosphorylated on serine and threonine residues in T98G cells arrested in G0 by serum deprivation or density arrest. Identi®cation of the phospho-serine and phosphothreonine residues revealed that most were clustered within a short co-linear region unique to p130, de®ned as the Loop. Deletion of the Loop region resulted in a change in the phosphorylation status of p130 under growth arrest conditions. Notably, deletion of the Loop did not aect the ability of p130 to bind to E2F-4 or SV40 Large T antigen, to induce growth arrest in Saos-2 cells, and to become hyperphosphorylated during the proliferative phase of the cell cycle. p130 undergoes speci®c G0 phosphorylation in a manner that distinguishes it from pRB and p107. Oncogene (2000) 19, 5116 ± 5122.
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