Structure of the Saccharomyces cerevisiae Hrr25:Mam1 monopolin subcomplex reveals a novel kinase regulator.

In budding yeast, the monopolin complex mediates sister kinetochore cross-linking and co-orientation in meiosis I. The CK1δ kinase Hrr25 is critical for sister kinetochore co-orientation, but its roles are not well understood. Here, we present the structures of Hrr25 and its complex with the monopolin subunit Mam1. Hrr25 possesses a "central domain" that packs tightly against the kinase C-lobe, adjacent to the binding site for Mam1. Together, the Hrr25 central domain and Mam1 form a novel, contiguous embellishment to the Hrr25 kinase domain that affects Hrr25 conformational dynamics and enzyme kinetics. Mam1 binds a hydrophobic surface on the Hrr25 N-lobe that is conserved in CK1δ-family kinases, suggesting a role for this surface in recruitment and/or regulation of these enzymes throughout eukaryotes. Finally, using purified proteins, we find that Hrr25 phosphorylates the kinetochore receptor for monopolin, Dsn1. Together with our new structural insights into the fully assembled monopolin complex, this finding suggests that tightly localized Hrr25 activity modulates monopolin complex-kinetochore interactions through phosphorylation of both kinetochore and monopolin complex components.

Facile preparation of highly active casein kinase 1 using Escherichia coli constitutively expressing lambda phosphatase.

Casein kinase 1 (CK1) is a widely expressed Ser/Thr kinase in eukaryotic organisms that is involved in various cellular processes (e.g., circadian rhythm and apoptosis). Therefore, preparing highly active CK1 and investigating its properties in vitro have important implications for understanding the biological roles of the kinase. However, recombinant CK1 undergoes autoinactivation via autophosphorylation in Escherichia coli cells and thus is undesirably prepared as a phosphorylated and inactivated kinase. To circumvent this problem, we established a protein expression system using E. coli strain BL21(DE3)pλPP in which λ protein phosphatase (λPPase) is constitutively expressed. Using this system, recombinant CK1 isoforms (α, δ and ε) were readily prepared as unphosphorylated forms. Furthermore, we found that CK1s prepared using BL21(DE3)pλPP showed markedly higher activity than those prepared by the conventional BL21(DE3). Finally, we demonstrated that the kinase activity of CK1δ from BL21(DE3)pλPP was higher than that prepared by a conventional method consisting of troublesome steps such as in vitro λPPase treatment. Thus, this simple method using BL21(DE3)pλPP is valuable for preparing highly active CK1s. It may also be applicable to other kinases that are difficult to prepare because of phosphorylation in E. coli cells.

Inhibitors of casein kinase 1 block the growth of Leishmania major promastigotes in vitro.

Casein kinase 1 (CK1) is a family of multifunctional Ser/Thr protein kinases that are ubiquitous in eukaryotic cells. Recent studies have demonstrated the existence of, and role for, CK1 in protozoan parasites such as Leishmania, Plasmodium and Trypanosoma. The value of protein kinases as potential drug targets in protozoa is evidenced by the successful exploitation of cyclic guanosine monophosphate-dependent protein kinase (PKG) with selective tri-substituted pyrrole and imidazopyridine inhibitors. These compounds exhibit in vivo efficacy against Eimeria tenella in chickens and Toxoplasma gondii in mice. We now report that both of these protein kinase inhibitor classes inhibit the growth of Leishmania major promastigotes and Trypanosoma brucei bloodstream forms in vitro. Genome informatics predicts that neither of these trypanosomatids codes for a PKG orthologue. Biochemical studies have led to the unexpected discovery that an isoform of CK1 represents the primary target of the pyrrole and imidazopyridine kinase inhibitors in these organisms. CK1 from extracts of L. major promastigotes co-fractionated with [(3)H]imidazopyridine binding activity. Further purification of CK1 activity from L. major and characterization via liquid chromatography coupled tandem mass spectrometry identified CK1 isoform 2 as the specific parasite protein inhibited by imidazopyridines. L. major CK1 isoform 2 expressed as a recombinant protein in Escherichia coli displayed biochemical and inhibition characteristics similar to those of the purified native enzyme. The results described here warrant further evaluation of the activity of these kinase inhibitors against mammalian stage Leishmania parasites in vitro and in animal models of infection, as well as studies to genetically validate CK1 as a therapeutic target in trypanosomatid parasites.

Methods to Purify and Assay Secretory Pathway Kinases.

Members of the four-jointed and VLK families of secretory pathway kinases appear to be responsible for the phosphorylation of secreted proteins and proteoglycans. These enzymes have been implicated in many biological processes and mutations in several of these kinases cause human diseases. Here, we describe methods to purify and assay two members of the four-jointed family of secretory kinases: the Fam20C protein kinase and the Fam20B proteoglycan kinase.

Purification of CK1 by affinity chromatography on immobilised axin.

Members of the Casein Kinase 1 (CK1) family are implicated in the regulation of a variety of physiological processes like development and circadian rhythm, as well as in diseases like cancer and Alzheimer's disease. From that perspective, CK1 family members are interesting targets for potential chemotherapy. We describe here a rapid and efficient method for the purification of CK1 by affinity chromatography on an immobilised fragment of axin. Axin is a scaffolding protein that interacts with a multitude of proteins, amongst them APC, GSK-3, beta-catenin, CK1alpha, delta, and epsilon, and PP2A. A GST-tagged axin peptide (residues 495-684) was produced in Escherichia coli and either immobilised on glutathione agarose beads or purified and immobilised on CNBr-activated sepharose 4B. These "GST-axin" matrices were found to selectively bind native CK1alpha and CK1epsilon from porcine brain. The affinity-purified enzymes displayed high kinase activity. This single step purification method provides a convenient tool to efficiently purify large amounts of active native CK1 for screening purposes. This single step purification method also provides a convenient tool to follow the status of the axin-binding CK1 isoforms alpha, delta, and epsilon (protein levels, composition of isoforms, kinase activity) under different physiological settings.

Nup53p is a target of two mitotic kinases, Cdk1p and Hrr25p.

Nuclear pore complexes (NPCs) form channels across the nuclear envelope and provide the sole sites of molecular exchange between the cytoplasm and nucleoplasm. The NPC is a target of a number of post-translational modifications, including phosphorylation, yet the functions of these modifications are ill defined. Here, we have investigated the mitotic specific phosphorylation of a yeast nucleoporin Nup53p. Two kinases were identified that phosphorylate Nup53p: the mitotic kinase Cdk1p/Cdc2p/Cdc28p and the casein kinase Hrr25p. Hrr25p was identified by screening 119 yeast kinases for their ability to phosphorylate Nup53p in vitro. Conditional alleles of Hrr25p support the conclusion that Hrr25p phosphorylates Nup53p in vivo. We further demonstrated using solution binding and affinity purification assays, that Hrr25p directly binds Nup53p in an interaction that is destabilized by the phosphorylation of Nup53p. Consistent with this observation, we observed that Hrr25p moves between distinct locations in the cell during the cell cycle including the nucleus, the cortex of the emerging bud and the spindle pole bodies. Cdk1p also contributes to Nup53p phosphorylation as specific inhibition of Cdk1p or mutation of Cdk1p consensus sites partially blocked its phosphorylation. The ability of nup53 alleles containing Cdk1p site mutations to complement synthetic defects of nup53 Delta nup170 Delta strains is linked to a function for Nup53p in the spindle assembly checkpoint.