In eukaryotes, sister chromatids remain connected from the time of their synthesis until they are separated in anaphase. This cohesion depends on a complex of proteins called cohesins. In budding yeast, the anaphase-promoting complex (APC) pathway initiates anaphase by removing cohesins from chromosomes. In vertebrates, cohesins dissociate from chromosomes already in prophase. To study their mitotic regulation we have purified two 14S cohesin complexes from human cells. Both complexes contain SMC1, SMC3, SCC1, and either one of the yeast Scc3p orthologs SA1 and SA2. SA1 is also a subunit of 14S cohesin in Xenopus. These complexes interact with PDS5, a protein whose fungal orthologs have been implicated in chromosome cohesion, condensation, and recombination. The bulk of SA1- and SA2-containing complexes and PDS5 are chromatin-associated until they become soluble from prophase to telophase. Reconstitution of this process in mitotic Xenopus extracts shows that cohesin dissociation does neither depend on cyclin B proteolysis nor on the presence of the APC. Cohesins can also dissociate from chromatin in the absence of cyclin-dependent kinase 1 activity. These results suggest that vertebrate cohesins are regulated by a novel prophase pathway which is distinct from the APC pathway that controls cohesins in yeast.

The separation of sister chromatids in anaphase depends on the dissociation of cohesin from chromosomes. In vertebrates, some cohesin is removed from chromosomes at the onset of anaphase by proteolytic cleavage. In contrast, the bulk of cohesin is removed from chromosomes already in prophase and prometaphase by an unknown mechanism that does not involve cohesin cleavage. We show that Polo-like kinase is required for the cleavage-independent dissociation of cohesin from chromosomes in Xenopus. Cohesin phosphorylation depends on Polo-like kinase and reduces the ability of cohesin to bind to chromatin. These results suggest that Polo-like kinase regulates the dissociation of cohesin from chromosomes early in mitosis.

Our results imply that Plk1 is not essential for mitotic entry and APC/C activation but is required for proper spindle assembly and function. In Plk1-depleted cells spindles may not be able to create enough tension across sister kinetochores to stabilize microtubule-kinetochore interactions and to silence the spindle checkpoint.

In vitro studies suggest a role for c-Jun N-terminal kinases (JNKs) in proatherogenic cellular processes. We show that atherosclerosis-prone ApoE-/- mice simultaneously lacking JNK2 (ApoE-/- JNK2-/- mice), but not ApoE-/- JNK1-/- mice, developed less atherosclerosis than do ApoE-/- mice. Pharmacological inhibition of JNK activity efficiently reduced plaque formation. Macrophages lacking JNK2 displayed suppressed foam cell formation caused by defective uptake and degradation of modified lipoproteins and showed increased amounts of the modified lipoprotein-binding and -internalizing scavenger receptor A (SR-A), whose phosphorylation was markedly decreased. Macrophage-restricted deletion of JNK2 was sufficient to decrease atherogenesis. Thus, JNK2-dependent phosphorylation of SR-A promotes uptake of lipids in macrophages, thereby regulating foam cell formation, a critical step in atherogenesis.

SummaryDysfunction and loss of insulin-producing pancreatic β cells represent hallmarks of diabetes mellitus. Here, we show that mice lacking the mitogen-activated protein kinase (MAPK) p38δ display improved glucose tolerance due to enhanced insulin secretion from pancreatic β cells. Deletion of p38δ results in pronounced activation of protein kinase D (PKD), the latter of which we have identified as a pivotal regulator of stimulated insulin exocytosis. p38δ catalyzes an inhibitory phosphorylation of PKD1, thereby attenuating stimulated insulin secretion. In addition, p38δ null mice are protected against high-fat-feeding-induced insulin resistance and oxidative stress-mediated β cell failure. Inhibition of PKD1 reverses enhanced insulin secretion from p38δ-deficient islets and glucose tolerance in p38δ null mice as well as their susceptibility to oxidative stress. In conclusion, the p38δ-PKD pathway integrates regulation of the insulin secretory capacity and survival of pancreatic β cells, pointing to a pivotal role for this pathway in the development of overt diabetes mellitus.

Faithful cell-cycle progression is tightly controlled by the ubiquitin-proteasome system. Here we identify a human Cullin 3-based E3 ligase (Cul3) which is essential for mitotic division. In a complex with the substrate-specific adaptors KLHL9 and KLHL13, Cul3 is required for correct chromosome alignment in metaphase, proper midzone and midbody formation, and completion of cytokinesis. This Cul3-based E3 ligase removes components of the chromosomal passenger complex from mitotic chromosomes and allows their accumulation on the central spindle during anaphase. Aurora B directly binds to the substrate-recognition domain of KLHL9 and KLHL13 in vitro, and coimmunoprecipitates with the Cul3 complex during mitosis. Moreover, Aurora B is ubiquitylated in a Cul3-dependent manner in vivo, and by reconstituted Cul3/KLHL9/KLHL13 ligase in vitro. We thus propose that the Cul3/KLHL9/KLHL13 E3 ligase controls the dynamic behavior of Aurora B on mitotic chromosomes, and thereby coordinates faithful mitotic progression and completion of cytokinesis.

Sister chromatid separation in anaphase depends on the removal of cohesin complexes from chromosomes. In vertebrates, the bulk of cohesin is already removed from chromosome arms during prophase and prometaphase, whereas cohesin remains at centromeres until metaphase, when cohesin is cleaved by the protease separase. In unperturbed mitoses, arm cohesion nevertheless persists throughout metaphase and is principally sufficient to maintain sister chromatid cohesion. How arm cohesion is maintained until metaphase is unknown. Here we show that small amounts of cohesin can be detected in the interchromatid region of metaphase chromosome arms. If prometaphase is prolonged by treatment of cells with microtubule poisons, these cohesin complexes dissociate from chromosome arms, and arm cohesion is dissolved. If cohesin dissociation in prometaphase-arrested cells is prevented by depletion of Plk1 or inhibition of Aurora B, arm cohesion is maintained. These observations imply that, in unperturbed mitoses, small amounts of cohesin maintain arm cohesion until metaphase. When cells lacking Plk1 and Aurora B activity enter anaphase, chromatids lose cohesin. This loss is prevented by proteasome inhibitors, implying that it depends on separase activation. Separase may therefore be able to cleave cohesin at centromeres and on chromosome arms.

Protein ubiquitylation is a post-translational modification that controls all aspects of eukaryotic cell functionality, and its defective regulation is manifested in various human diseases. The ubiquitylation process requires a set of enzymes, of which the ubiquitin ligases (E3s) are the substrate recognition components. Modular CULLIN-RING ubiquitin ligases (CRLs) are the most prevalent class of E3s, comprising hundreds of distinct CRL complexes with the potential to recruit as many and even more protein substrates. Best understood at both structural and functional levels are CRL1 or SCF (SKP1/CUL1/F-box protein) complexes, representing the founding member of this class of multimeric E3s. Another CRL subfamily, called CRL3, is composed of the molecular scaffold CULLIN3 and the RING protein RBX1, in combination with one of numerous BTB domain proteins acting as substrate adaptors. Recent work has firmly established CRL3s as major regulators of different cellular and developmental processes as well as stress responses in both metazoans and higher plants. In humans, functional alterations of CRL3s have been associated with various pathologies, including metabolic disorders, muscle, and nerve degeneration, as well as cancer. In this review, we summarize recent discoveries on the function of CRL3s in both metazoans and plants, and discuss their mode of regulation and specificities.