Mitotic entry is controlled by the plant-specific phosphatase BSL1 and cyclin-dependent kinase B

Cell cycle regulation is well understood in opisthokonts (fungi and metazoans) but not in plants and Apicomplexa, as some cell cycle regulators are not conserved^1-3. In opisthokonts, cell cycle progression requires dephosphorylation of cyclin-dependent kinase (CDK) by the CDC25 phosphatase⁴. Plants have no CDC25, and thus their mechanisms of cell cycle regulation remain elusive^1,5,6. Here, we show that the BSL1 phosphatase dephosphorylates CDKB1 to promote mitotic entry in Chlamydomonas. Mutations of BSL1 or CDKB1 block mitotic entry after DNA replication. BSL1 shows dynamic localization through the cell cycle at the basal bodies, spindle poles, and cleavage furrow. CDKB1 is hyperphosphorylated at T14 and Y15 residues in the bsl1 mutant and in wild-type cells treated with DNA replication inhibitors. BSL1 binds to CDKB1 and dephosphorylates CDKB1 pT14/pY15 in vitro. Phospho-mimicking mutations of T14/Y15 inactivate CDKB1 function, whereas phospho-blocking mutations cause sensitivity to DNA replication inhibitors, which delay cytokinesis in wild-type cells more than cells expressing unphosphorylatable mutant CDKB1. These results indicate that CDKB1 T14/Y15 is phosphorylated to block mitotic entry before DNA replication is complete, and BSL1 dephosphorylates CDKB1 to promote mitosis. Our study demonstrates that BSL1, a phosphatase conserved in plants and Apicomplexa but absent in fungi and animals, is a CDKB1-activating mitosis-promoting factor that has evolved additional signaling functions in receptor kinase pathways in higher plants.