The small family of polo-like kinases (Plks) includes Cdc5 from Saccharomyces cerevisiae, Plo1 from Schizosaccharomyces pombe, Polo from Drosophila melanogaster and the four mammalian genes Plk1, Prk/Fnk, Snk and Sak. These kinases control cell cycle progression through the regulation of centrosome maturation and separation, mitotic entry, metaphase to anaphase transition, mitotic exit and cytokinesis. Plks are characterized by an N-terminal Ser/Thr protein kinase domain and the presence of one or two C-terminal regions of similarity, termed the polo box motifs. These motifs have been demonstrated for Cdc5 and Plk1 to be required for mitotic progression and for subcellular localization to mitotic structures. Here we report the 2.0 A crystal structure of a novel domain composed of the polo box motif of murine Sak. The structure consists of a dimeric fold with a deep interfacial cleft and pocket, suggestive of a ligand-binding site. We show that this domain forms homodimers both in vitro and in vivo, and localizes to centrosomes and the cleavage furrow during cytokinesis. The requirement of the polo domain for Plk family function and the unique physical properties of the domain identify it as an attractive target for inhibitor design.

Polo-like kinases in yeast, flies, and mammals regulate key events in mitosis. Such events include spindle formation at G2/M, the anaphase-promoting complex (APC) at the exit from mitosis, the cleavage structure at cytokinesis, and DNA damage checkpoints in G2/M. Polo-like kinases are distinguished by two C-terminal polo box (pb) motifs, which localize the enzymes to mitotic structures. We previously identified Sak, a novel polo-like kinase found in Drosophila and mammals. Here, we demonstrate that the Sak kinase has a functional pb domain that localizes the enzyme to the nucleolus during G2, to the centrosomes in G2/M, and to the cleavage furrow during cytokinesis. To study the role of Sak in embryo development, we generated a Sak null allele, the first polo-like kinase to be mutated in mice. Sak(-/-) embryos arrested after gastrulation at E7.5, with a marked increase in mitotic and apoptotic cells. Sak(-/-) embryos displayed cells in late anaphase or telophase that continued to express cyclin B1 and phosphorylated histone H3. Our results suggest that Sak is required for the APC-dependent destruction of cyclin B1 and for exit from mitosis in the postgastrulation embryo.

The presence of late embryogenesis abundant (LEA) proteins in plants and animals has been linked to their ability to tolerate a variety of environmental stresses. Among animals, encysted embryos of the brine shrimp Artemia franciscana are among the most stress resistant eukaryotes, and for that reason it is considered to be an extremophile. The study presented here demonstrates that these embryos contain multiple group 1 LEA proteins with masses of 21, 19, 15.5 and 13 kDa. The LEA proteins first appear in diapause-destined embryos, beginning at ∼4 days post-fertilization, but not in nauplii-destined embryos. After resumption of embryonic development, the LEA proteins decline slowly in the desiccation resistant encysted stages, then disappear rapidly as the embryo emerges from its shell. LEA proteins are absent in fully emerged embryos, larvae and adults. They are abundant in mitochondria of encysted embryos, but barely detectable in nuclei and absent from yolk platelets. LEA proteins were also detected in dormant embryos of six other species of Artemia from hypersaline environments around the world. This study enhances our knowledge of the group 1 LEA proteins in stress tolerant crustacean embryos.