RESUMO
Cyclin A and CDC25A are both activators of cyclin-dependent kinases (CDKs): cyclin A acts as an activating subunit of CDKs and CDC25A a phosphatase of the inhibitory phosphorylation sites of the CDKs. In this study, we uncovered an inverse relationship between the two CDK activators. As cyclin A is an essential gene, we generated a conditional silencing cell line using a combination of CRISPR-Cas9 and degron-tagged cyclin A. Destruction of cyclin A promoted an acute accumulation of CDC25A. The increase of CDC25A after cyclin A depletion occurred throughout the cell cycle and was independent on cell cycle delay caused by cyclin A deficiency. Moreover, we determined that the inverse relationship with cyclin A was specific for CDC25A and not for other CDC25 family members or kinases that regulate the same sites in CDKs. Unexpectedly, the upregulation of CDC25A was mainly caused by an increase in transcriptional activity instead of a change in the stability of the protein. Reversing the accumulation of CDC25A severely delayed G2-M in cyclin A-depleted cells. Taken together, these data provide evidence of a compensatory mechanism involving CDC25A that ensures timely mitotic entry at different levels of cyclin A.
Assuntos
Ciclina A , Quinases Ciclina-Dependentes , Fosfatases cdc25 , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismo , Ciclo Celular , Divisão Celular , Ciclina A/metabolismo , Quinases Ciclina-Dependentes/metabolismo , FosforilaçãoRESUMO
Characterizing the functions of essential cell cycle control genes requires tight and rapid inducible gene inactivation. Drawbacks of current conditional depletion approaches include slow responses and incomplete depletion. We demonstrated that by integrating the tetracycline-controlled promoter system and the auxin-inducible degron (AID) system together, AID-tagged proteins can be downregulated more efficiently than the individual technology alone. When used in conjunction with CRISPR-Cas9-mediated disruption of the endogenous locus, this system facilitates the analysis of essential genes by allowing rapid and tight conditional depletion, as we have demonstrated using several cell cycle-regulatory genes including cyclin A, CDK2, and TRIP13. The vectors constructed in this study allow expression of AID-fusion proteins under the control of tetracycline-controlled promoters and should be useful in studies requiring rapid and tight suppression of gene expression in mammalian cells.