María P. Sacristán

Cdc6 cooperates with Sic1 and Hct1 to inactivate mitotic cyclin-dependent kinases.

Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinases (CDKs). In the budding yeast, Saccharomyces cerevisiae, inactivation of CDKs during late mitosis involves degradation of B-type cyclins as well as direct inhibition of cyclin–CDK complexes by the CDK-inhibitor protein Sic1 (refs 1,2,3). Several striking similarities exist between Sic1 and Cdc6, a DNA replication factor essential for the formation of pre-replicative complexes at origins of DNA replication. Transcription of both genes is activated during late mitosis by a process dependent on Swi5 (ref. 10). Like Sic1, Cdc6 binds CDK complexes in vivo and downregulates them in vitro. Here we show that Cdc6, like Sic1, also contributes to inactivation of CDKs during late mitosis in S. cerevisiae. Deletion of the CDK-interacting domain of Cdc6 does not inhibit the function of origins of DNA replication during S phase, but instead causes a delay in mitotic exit; this delay is accentuated in the absence of Sic1 or of cyclin degradation. By contributing to mitotic exit and inactivation of CDKs, Cdc6 helps to create the conditions that are required for its subsequent role in the formation of pre-replicative complexes at origins of DNA replication.

Functional Homology among Human and Fission Yeast Cdc14 Phosphatases

Budding and fission yeast Cdc14 homologues, a conserved family of serine-threonine phosphatases, play a role in the inactivation of mitotic cyclin-dependent kinases (CDKs) by molecularly distinct mechanisms. Saccharomyces cerevisiae Cdc14 protein phosphatase inactivates CDKs by promoting mitotic cyclin degradation and the accumulation of a CDK inhibitor to allow budding yeast cells to exit from mitosis. Schizosaccharomyces pombe Flp1 phosphatase down-regulates CDK/cyclin activity, controlling the degradation of the Cdc25 tyrosine phosphatase for fission yeast cells to undergo cytokinesis. In the present work, we show that human Cdc14 homologues (hCdc14A and hCdc14B) rescued flp1-deficient fission yeast strains, indicating functional homology. We also show that hCdc14A and B interacted in vivo with S. pombe Cdc25 and that hCdc14A dephosphorylated this mitotic inducer both in vitro and in vivo. Our results support a Cdc14 conserved inhibitory mechanism acting on S. pombe Cdc25 protein and suggest that human cells may regulate Cdc25 in a similar manner to inactivate Cdk1-mitotic cyclin complexes.

Synthesis and biological activity of O-glycosylated morphiceptin analogues

The synthesis and biological activity of [Hyp4]morphiceptin and two glycosyl derivatives are reported. Glycopeptide amides were obtained using Fmoc solid-phase chemistry and mild conditions for cleavage from a tris(alkoxy)benzylamide (PAL) resin. Analogues were evaluated in the guinea pig ileum in vitro assay and in in vivo tail-flick and paw-pressure antinociceptive tests after intrathecal administration in rats. Substitution of Pro4 by Hypo4 and further derivatization of Hyp with glucose or galactose, resulted in an unexpected decrease in biological activity with respect to morphiceptin.

Cdc14b regulates mammalian RNA polymerase II and represses cell cycle transcription.

Cdc14 is an essential phosphatase in yeast but its role in the mammalian cell cycle remains obscure. We report here that Cdc14b-knockout cells display unscheduled induction of multiple cell cycle regulators resulting in early entry into DNA replication and mitosis from quiescence. Cdc14b dephosphorylates Ser5 at the C-terminal domain (CTD) of RNA polymerase II, a major substrate of cyclin-dependent kinases. Lack of Cdc14b results in increased CTD-Ser5 phosphorylation, epigenetic modifications that mark active chromatin, and transcriptional induction of cell cycle regulators. These data suggest a function for mammalian Cdc14 phosphatases in the control of transcription during the cell cycle.

Human Cdc14A Phosphatase Modulates the G2/M Transition through Cdc25A and Cdc25B

The Cdc14 family of serine-threonine phosphatases antagonizes CDK activity by reversing CDK-dependent phosphorylation events. It is well established that the yeast members of this family bring about the M/G1 transition. Budding yeast Cdc14 is essential for CDK inactivation at the end of mitosis and fission yeast Cdc14 homologue Flp1/Clp1 down-regulates Cdc25 to ensure the inactivation of mitotic CDK complexes to trigger cell division. However, the functions of human Cdc14 homologues remain poorly understood. Here we have tested the hypothesis that Cdc14A might regulate Cdc25 mitotic inducers in human cells. We found that increasing levels of Cdc14A delay entry into mitosis by inhibiting Cdk1-cyclin B1 activity. By contrast, lowering the levels of Cdc14A accelerates mitotic entry. Biochemical analyses revealed that Cdc14A acts through key Cdk1-cyclin B1 regulators. We observed that Cdc14A directly bound to and dephosphorylated Cdc25B, inhibiting its catalytic activity. Cdc14A also regulated the activity of Cdc25A at the G2/M transition. Our results indicate that Cdc14A phosphatase prevents premature activation of Cdk1 regulating Cdc25A and Cdc25B at the entry into mitosis.

Human Cdc14A Reverses CDK1 Phosphorylation of Cdc25A on Serines 115 and 320

Human Cdc14A is an evolutionary conserved dual-specificity protein phosphatase that reverses the modifications effected by cyclin-dependent kinases and plays an important role in centrosome duplication and mitotic regulation. Few substrates of Cdc14A have been identified, some of them with homologues in yeast that, in turn, are substrates of the Saccharomyces cerevisiae Cdc14 homologue, a protein phosphatase essential for yeast cell viability owing its role in mitotic exit regulation. Identification of the physiological substrates of human Cdc14A is an immediate goal in order to elucidate which cellular processes it regulates. Here, we show that human Cdc14A can dephosphorylate Cdc25A in vitro. Specifically, the Cdk1/Cyclin-B1-dependent phosphate groups on Ser115 and Ser320 of Cdc25A were found to be removed by Cdc14A. Cdc25A is an important cell cycle-regulatory protein involved in several cell cycle transitions and checkpoint responses and whose function and own regulation depend on complex phosphorylation/dephosphorylation-mediated processes. Importantly, we also show that the upregulation of Cdc14A phosphatase affects Cdc25A protein levels in human cells. Our results suggest that Cdc14A may be involved in the cell cycle regulation of Cdc25A stability.

The Flp1/Clp1 phosphatase cooperates with HECT-type Pub1/2 protein-ubiquitin ligases in Schizosaccharomyces pombe

The Schizosaccharomyces pombe Flp1p serine-threonine phosphatase is required for the degradation of the mitotic inducer Cdc25p at the end of mitosis. Cdc25p degradation prevents Cdc2p-tyrosine 15 dephosphorylation and, thus, contributes to the timely inactivation of mitotic CDK-associated kinase activity. Both RING- and HECT-type protein-ubiquitin ligases are involved in Cdc25p destabilization. Flp1p function is required for Cdc25p ubiquitination via anaphase-promoting complex/cyclosome or APC/C (RING-type) and the absence of Pub1p (HECT-type) stabilizes the mitotic inducer. In the present report, we study the functional relationship of Flp1p with Pub1p and Pub2p HECT-type-protein ubiquitin ligases. We show that Flp1p is required for the rapid degradation of Cdc25p while Pub1p is responsible for the long-term destabilization of the mitotic inducer. Accordingly, flp1 and pub1 mutants have a strong genetic interaction, correlating defects in the coordination of mitosis and cytokinesis with the stabilization of hyperactive Cdc25p. However, we also show that Flp1 and Pub2p proteins functionally interact in vivo suggesting that both proteins belong to the same regulatory network in S. pombe cells. Thus Flp1p appears to have an important role in integrating HECT- and RING-type ubiquitin ligases in cell cycle control.

Human Cdc14A regulates Wee1 stability by counteracting CDK-mediated phosphorylation

Cdc14A phosphatase regulates Wee1 kinase through dephosphorylation of two Cdk phosphorylation sites in its regulatory domain, Ser-123 and -139, both involved in the degradation of Wee1 at the entry into mitosis. In this way, Cdc14A interferes with the negative feedback loop between Wee1 and Cdk1 to regulate the mitotic switch.

Human Cdc14A becomes a cell cycle gene in controlling Cdk1 activity at the G2/M transition

Cdc14 belongs to a dual-specificity phosphatase family highly conserved through evolution that preferentially reverses CDK (Cyclin dependent kinases) –dependent phosphorylation events. In the yeast Saccharomyces cerevisiae, Cdc14 is an essential regulator of late mitotic events and exit from mitosis by counteracting CDK activity at the end of mitosis. However, many studies have shown that Cdc14 is dispensable for exiting mitosis in all other model systems analyzed. In fission yeast, the Cdc14 homologue Flp1/Clp1 regulates the stability of the mitotic inducer Cdc25 at the end of mitosis to ensure Cdk1 inactivation before cytokinesis. We have recently reported that human Cdc14A, the Cdc14 isoform located at the centrosomes during interphase, down-regulates Cdc25 activity at the G2/M transition to prevent premature activation of Cdk1-Cyclin B1 complexes and untimely entry into mitosis. Here we speculate about new molecular mechanisms for Cdc14A and discuss the current evidence suggesting that Cdc14 phosphatase plays a role in cell cycle control in higher eukaryotes.

Orderly progression through S-phase requires dynamic ubiquitylation and deubiquitylation of PCNA

Proliferating-cell nuclear antigen (PCNA) is a DNA sliding clamp with an essential function in DNA replication and a key role in tolerance to DNA damage by ensuring the bypass of lesions. In eukaryotes, DNA damage tolerance is regulated by ubiquitylation of lysine 164 of PCNA through a well-known control mechanism; however, the regulation of PCNA deubiquitylation remains poorly understood. Our work is a systematic and functional study on PCNA deubiquitylating enzymes (DUBs) in Schizosaccharomyces pombe. Our study reveals that the deubiquitylation of PCNA in fission yeast cells is a complex process that requires several ubiquitin proteases dedicated to the deubiquitylation of a specific subnuclear fraction of mono- and di-ubiquitylated PCNA or a particular type of poly-ubiquitylated PCNA and that there is little redundancy among these enzymes. To understand how DUB activity regulates the oscillatory pattern of ubiquitylated PCNA in fission yeast, we assembled multiple DUB mutants and found that a quadruple mutation of ubp2+, ubp12+, ubp15+, and ubp16+ leads to the stable accumulation of mono-, di-, and poly-ubiquitylated forms of PCNA, increases S-phase duration, and sensitizes cells to DNA damage. Our data suggest that the dynamic ubiquitylation and deubiquitylation of PCNA occurs during S-phase to ensure processive DNA replication.