Cdk1 inactivation terminates mitotic checkpoint surveillance and stabilizes kinetochore attachments in anaphase

Two mechanisms safeguard the bipolar attachment of chromosomes in mitosis. A correction mechanism destabilizes erroneous attachments that do not generate tension across sister kinetochores [1]. In response to unattached kinetochores, the mitotic checkpoint delays anaphase onset by inhibiting the anaphase-promoting complex/cyclosome (APC/CCdc20) [2]. Upon satisfaction of both pathways, the APC/CCdc20 elicits the degradation of securin and cyclin B [3]. This liberates separase triggering sister chromatid disjunction and inactivates cyclin-dependent kinase 1 (Cdk1) causing mitotic exit. How eukaryotic cells avoid the engagement of attachment monitoring mechanisms when sister chromatids split and tension is lost at anaphase is poorly understood [4]. Here we show that Cdk1 inactivation disables mitotic checkpoint surveillance at anaphase onset in human cells. Preventing cyclin B1 proteolysis at the time of sister chromatid disjunction destabilizes kinetochore-microtubule attachments and triggers the engagement of the mitotic checkpoint. As a consequence, mitotic checkpoint proteins accumulate at anaphase kinetochores, the APC/CCdc20 is inhibited, and securin reaccumulates. Conversely, acute pharmacological inhibition of Cdk1 abrogates the engagement and maintenance of the mitotic checkpoint upon microtubule depolymerization. We propose that the simultaneous destruction of securin and cyclin B elicited by the APC/CCdc20 couples chromosome segregation to the dissolution of attachment monitoring mechanisms during mitotic exit

Caracterización funcional de las fosfatasas Cdc14 humanas en la regulación del ciclo de división celular

[ES]Las fosfatasas Cdc14 desempeñan funciones relacionadas con la regulación del ciclo celular en diversos organismos eucariotas, actuando como antagonistas de la actividad CDK (quinasa dependiente de ciclina). En las levaduras Saccharomyces cerevisiae y Schizosaccharomyces pombe, respectivamente, Cdc14 y Flp1/Clp1 participan en la regulación de la salida de mitosis, promoviendo la inhibición de la actividad CDK mitótica mediante mecanismos de acción diferentes.Con el fin de estudiar las funciones de las proteínas Cdc14 humanas, hCdc14A y hCdc14B, comenzamos analizando la homología funcional entre éstas y Flp1. De este modo comprobamos que ambas proteínas humanas complementan el fenotipo correspondiente a la falta de Flp1 en S. pombe, mediante mecanismos diferentes. La fosfatasa hCdc14A, como Flp1, desfosforila a Cdc25 (activador de los complejos CDK mitóticos) e induce así la reducción de sus niveles proteicos; mientras que el mecanismo de la complementación por hCdc14B no se conoce.A continuación, determinamos que hCdc14A también es capaz de desfosforilar in vitro al menos a dos de las proteínas Cdc25 humanas en residuos fosforilados por Cdk1, a hCdc25A (en Serina 115 y Serina 320) y a hCdc25C (en residuos no determinados). Además, probamos que la desfosforilación de hCdc25A por hCdc14A en estos dos residuos identificados promueve su degradación.Por último, estudiamos los efectos que tienen la sobre-expresión y del silenciamiento de la expresión de hCdc14A sobre la progresión por mitosis en varias líneas celulares humanas. Determinamos así que hCdc14A, de forma dependiente de su actividad fosfatasa, participa en la regulación de los complejos CDK mitóticos a la entrada en mitosis, a través de la inhibición de la actividad catalítica de hCdc25A. Obtuvimos también datos que sugieren que hCdc14A podría inducir la inactivación de los complejos CDK mitóticos a la salida de mitosis en parte, posiblemente, a través de la desfosforilación y consiguiente degradación de hCdc25A.[EN]Cdc14 phosphatases are involved in regulation of the eukaryotic cell cycle, acting as antagonists of CDK (Cyclin-Dependent Kinases) activity. In yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively, Cdc14 and Flp1/Clp1 control mitotic exit, promoting mitotic CDK activity inhibition by different mechanisms. In order to study the functions developed by human Cdc14 proteins, hCdc14A and hCdc14B, we analyzed functional homology among hCdc14s and Flp1. Both human proteins complement the lack of Flp1 in S. pombe cells, by different molecular mechanisms. hCdc14A phosphatase, as Flp1 does, dephosphorylates Cdc25 (mitotic CDK activator) and thereby induces its degradation, while how hCdc14B complements the lack of Flp1 remains unknown.hCdc14A also dephosphorylates in vitro at least two of human Cdc25 proteins, previously phosphorylated by Cdk1, hCdc25A (on Serines 115 and 320) and hCdc25C (on unknown residues). hCdc25A dephosphorylation on this two residues by hCdc14A promotes its degradation.Finally, we also studied how the deregulation of hCdc14A levels (by overexpression or gene silencing) affects mitotic progression in human cell lines. We concluded that hCdc14A, in a phosphatase activity-dependent manner, is involved in the regulation of mitotic CDK complexes at the entry into mitosis, by inhibiting hCdc25A phosphatase activity. Our results also suggested that hCdc14A could induce mitotic CDK complexes inactivation at mitotic exit in part, possibly, by dephosphorylating hCdc25A and promoting its degradation

Functional Homology among Human and Fission Yeast Cdc14 Phosphatases

[EN] 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