Stabilizers and Destabilizers Controlling Cell Cycle Oscillators

Various destabilizing factors of the ubiquitin system contribute to the synchrony and unidirectionality of the cell cycle clock by finely tuning the activity of var- ious CDKs. The recent findings of hierarchical and connected waves of cyclin stabilizers highlight the complexity of this network

Oncogenic aberrations of cullin-dependent ubiquitin ligases

Accumulating evidence points to a key role of the ubiquitin\u2013proteasome pathway in oncogenesis. Aberrant proteolysis of substrates involved in cellular processes such as the cell division cycle, gene transcription, the DNA damage response and apoptosis has been reported to contribute significantly to neoplastic transformation. Cullin-dependent ubiquitin ligases (CDLs) form a class of structurally related multisubunit enzymes central to the ubiquitin-mediated proteolysis of many important biolo- gical substrates. In this review, we describe the role of CDLs in the ubiquitinylation of cancer-related substrates and discuss how altered ubiquitinylation by CDLs may contribute to tumor development

Two paths to let the replisome go

Accurate DNA replication is essential for genome mainte- nance. Two recent reports have uncovered new molecular mechanisms controlling the termination phase of DNA replication in higher eukaryotes and established crucial roles for the CRL2LRR1 ubiquitin ligase and the p97 segregase in replisome unloading from chromatin

Inhibition of differentiation in myoblasts deprived of the interferon-related protein PC4

PC4 (pheochromocytoma cell-4) is an immediate early gene related to IFN-gamma, the mRNA of which is induced during the course of neuronal differentiation by nerve growth factor in the PC12 cell line. Here we report that PC4 mRNA is also expressed in the myoblast C2C12 cell line and is regulated during differentiation; its expression decreases within 6 h from the onset of differentiation, attains a minimum after 12 h, and returns to basal level within 36 h. This transient down-regulation of PC4 expression in C2C12 myoblasts is prevented by transforming growth factor beta, a molecule which inhibits the differentiation of muscle. Sense and antisense PC4 cDNA transfection strategies in C2C12 cells were then used to clarify the role of PC4 in muscle differentiation. While no effect was seen by over-expression of PC4, stable transfectants underexpressing PC4 exhibited a delay in attaining the differentiated phenotype, with an impairment of myogenin and myosin expression. Myogenin was also inhibited in C2C12 cells microinjected with the anti-PC4 polyclonal antibody A451. We thus postulate a role for PC4 as a positive regulator during muscle differentiation

PC3 potentiates NGF-induced differentiation and protects neurons from apoptosis

PC3TIS21/BTG2 is member of a novel family of antiproliferative genes (BTG1, ANA/BTG3, PC3B, TOB, and TOB2) that play a role in cellu- lar di\ua1erentiation. We have previously shown that PC3TIS21/BTG2 is induced by nerve growth factor (NGF) at the onset of neuronal di\ua1erentiation in the neural crest-derived PC12 cell line, and is a marker for neuronal birth. We now observe that PC3TIS21/BTG2 ectopically expressed in PC12 cells synergises with NGF, similarly to the cyclin-dependent kinase inhibitor p21, potentiating the induction of the neuronal markers tyrosine hydroxylase and neuro\ua2lament 160 kDa. Furthermore, PC3TIS21/BTG2 protects from apoptosis elicited by NGF deprivation in terminally di\ua1erentiated PC12 cultures. Such e\ua1ects might be a consequence of the arrest of cell cycle exerted by PC3TIS21/BTG2, or expression of a sensitising (neurogenic)propertyofthemolecule

APC/CCdc20 Controls the Ubiquitin-Mediated Degradation of p21 in Prometaphase

During the G1/S transition, p21 proteolysis is mediated by Skp2; however, p21 reaccumu- lates in G2 and is degraded again in prometa- phase. How p21 degradation is controlled in mitosis remains unexplored. We found that Cdc20 (an activator of the ubiquitin ligase APC/C) binds p21 in cultured cells and identi- fied a D box motif in p21 necessary for APC/ CCdc20-mediated ubiquitylation of p21. Overex- pression of Cdc20 or Skp2 destabilized wild- type p21; however, only Skp2, but not Cdc20, was able to destabilize a p21(D box) mutant. Silencing of Cdc20 induced an accumulation of p21, increased the fraction of p21 bound to Cdk1, and inhibited Cdk1 activity in p21+/+ prometaphase cells, but not in p21/ cells. Thus, in prometaphase Cdc20 positively regu- lates Cdk1 by mediating the degradation of p21. We propose that the APC/CCdc20-medi- ated degradation of p21 contributes to the full activation of Cdk1 necessary for mitotic events and prevents mitotic slippage during spindle checkpoint activation

Phosphatidic acid-dependent localization and basal de-phosphorylation of RA-GEFs regulate lymphocyte trafficking

Background: Lymphocytes circulate between peripheral lymphoid tissues via blood and lymphatic systems, and chemokine-induced migration is important in trafficking lymphocytes to distant sites. The small GTPase Rap1 is important in mediating lymphocyte motility, and Rap1-GEFs are involved in chemokine-mediated Rap1 activation. Here, we describe the roles and mechanisms of Rap1-GEFs in lymphocyte trafficking. Results: In this study, we show that RA-GEF-1 and 2 (also known as Rapgef2 and 6) are key guanine nucleotide exchange factors (GEF) for Rap1 in lymphocyte trafficking. Mice harboring T cell-specific knockouts of Rapgef2/6 demonstrate defective homing and egress of T cells. Sphingosine-1-phosphate (S1P) as well as chemokines activates Rap1 in a RA-GEF-1/2-dependent manner, and their deficiency in T cells impairs Mst1 phosphorylation, cell polarization, and chemotaxis toward S1P gradient. On the other hand, B cell-specific knockouts of Rapgef2/6 impair chemokine-dependent retention of B cells in the bone marrow and passively facilitate egress. Phospholipase D2-dependent production of phosphatidic acid by these chemotactic factors determines spatial distribution of Rap1-GTP subsequent to membrane localization of RA-GEFs and induces the development of front membrane. On the other hand, basal de-phosphorylation of RA-GEFs is necessary for chemotactic factor-dependent increase in GEF activity for Rap1. Conclusions: We demonstrate here that subcellular distribution and activation of RA-GEFs are key factors for a directional movement of lymphocytes and that phosphatidic acid is critical for membrane translocation of RA-GEFs with chemokine stimulation

Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division

Rac1 regulates a wide variety of cellular processes. The polybasic region of the Rac1 C terminus functions both as a plasma membrane–targeting motif and a nuclear localization sequence (NLS). We show that a triproline N-terminal to the polybasic region contributes to the NLS, which is cryptic in the sense that it is strongly inhibited by geranylgeranylation of the adjacent cysteine. Subcellular fractionation demonstrated endogenous Rac1 in the nucleus and Triton X-114 partition revealed that this pool is prenylated. Cell cycle–blocking agents, synchronization of cells stably expressing low levels of GFP-Rac1, and time-lapse microscopy of asynchronous cells revealed Rac1 accumulation in the nucleus in late G2 and exclusion in early G1. Although constitutively active Rac1 restricted to the cytoplasm inhibited cell division, activated Rac1 expressed constitutively in the nucleus increased the mitotic rate. These results show that Rac1 cycles in and out of the nucleus during the cell cycle and thereby plays a role in promoting cell division

APC/C (Cdh1) controls the proteasome-mediated degradation of E2F3 during cell cycle exit

E2F transcription factors regulate gene expression in concert with the retinoblastoma tumor suppressor family. These transcriptional complexes are master regulators of cell cycle progression and, in addition, control the expression of genes involved in DNA repair, G 2/M checkpoint and differentiation. E2F3 has recently attracted particular attention, because it is amplified in various human tumors. Here we show that E2F3 becomes unstable as cells exit the cell cycle. E2F3 degradation is mediated by the anaphase-promoting complex/cyclosome and its activator Cdh1 (APC/C (Cdh1) ). E2F3 interacts with Cdh1 but not Cdc20, the other APC/C activator. Enforced expression of Cdh1 results in proteasome-dependent degradation of E2F3, whereas the overexpression of Cdc20 has no effect on E2F3 turnover. Finally, silencing of Cdh1 by RNA interference stabilizes E2F3 in differentiating neuroblastoma cells. These findings indicate that the APC/C (Cdh1) ubiquitin ligase targets E2F3 for proteasome-dependent degradation during cell cycle exit and neuronal differentiation

Control of the SCFSkp2\u2013Cks1 ubiquitin ligase by the APC/CCdh1 ubiquitin ligase

Skp2 and its cofactor Cks1 are the substrate-targeting subunits of the SCFSkp2\u2013Cks1 (Skp1/Cul1/F-box protein) ubiquitin ligase complex that regulates entry into S phase by inducing the degradation of the cyclin-dependent kinase inhibitors p21 and p27 (ref. 1). Skp2 is an oncoprotein that often shows increased expression in human cancers2; however, the mechanism that regulates its cellular abundance is not well understood. Here we show that both Skp2 and Cks1 proteins are unstable in G1 and that their degradation is mediated by the ubiquitin ligase APC/ CCdh1 (anaphase-promoting complex/cyclosome and its activator Cdh1). Silencing of Cdh1 by RNA interference in G1 cells stabilizes Skp2 and Cks1, with a consequent increase in p21 and p27 proteolysis. Depletion of Cdh1 also increases the per- centage of cells in S phase, whereas concomitant downregulation of Skp2 reverses this effect, showing that Skp2 is an essential target of APC/CCdh1. Expression of a stable Skp2 mutant that cannot bind APC/CCdh1 induces premature entry into S phase. Thus, the induction of Skp2 and Cks1 degradation in G1 rep- resents a principal mechanism by which APC/CCdh1 prevents the unscheduled degradation of SCFSkp2\u2013Cks1 substrates and main- tains the G1 state