Mps1 is one of the several essential kinases whose activation is required for robust mitotic spindle checkpoint signalling. The activity of Mps1 is tightly regulated and increases dramatically during mitosis or in response to spindle damage. To understand the molecular mechanism underlying Mps1 regulation, we determined the crystal structure of the kinase domain of Mps1. The 2.7‐Å‐resolution crystal structure shows that the Mps1 kinase domain adopts a unique inactive conformation. Intramolecular interactions between the key Glu residue in the αC helix of the N‐terminal lobe and the backbone amides in the catalytic loop lock the kinase in the inactive conformation. Autophosphorylation appears to be a priming event for kinase activation. We identified Mps1 autophosphorylation sites in the activation and the P+1 loops. Whereas activation loop autophosphorylation enhances kinase activity, autophosphorylation at the P+1 loop (T686) is associated with the active kinase. Mutation of T686 autophosphorylation site impairs both autophosphorylation and transphosphorylation. Furthermore, we demonstrated that phosphorylation of T676 may be a priming event for phosphorylation at T686. Finally, we identified two critical lysine residues in the loop between helices αEF and αF that are essential for substrate recruitment and maintaining high levels of kinase activity. Our studies reveal critical biochemical mechanisms for Mps1 kinase regulation.
The Cdc7 kinase is essential for the initiation of DNA replication in eukaryotes. Two regulatory subunits of the Xenopus Cdc7 kinase have been identified: XDbf4 and XDrf1. In this study we determined the expression pattern of XDbf4 and XDrf1 and examined their involvement in DNA replication. We show that XDrf1 expression is restricted to oogenesis and early embryos, whereas XDbf4 is expressed throughout development. Immunodepletion from Xenopus egg extracts indicated that both proteins are only found in complexes with XCdc7 and there is a 5-fold molar excess of the XCdc7/Drf1 over SCdc7/Dbf4 complexes. Both complexes exhibit kinase activity and are differentially phosphorylated during the cell cycle. Depletion of the XCdc7/Drf1 from egg extracts inhibited DNA replication, whereas depletion of XCdc7/Dbf4 had little effect. Chromatin binding studies indicated that XCdc7/Drf1 is required for pre-replication complex activation but not their assembly. XCdc7/Dbf4 complexes bound to the chromatin in two steps: the first step was independent of pre-replication complex assembly and the second step was dependent on pre-replication complex activation. By contrast, binding of XCdc7/Drf1 complexes was entirely dependent on pre-replication complex assembly. Finally, we present evidence that the association of the two complexes on the chromatin is not regulated by ATR checkpoint pathways that result from DNA replication blocks. These data suggest that Cdc7/Drf1 but not Cdc7/Dbf4 complexes support the initiation of DNA replication in Xenopus egg extracts and during early embryonic development.In eukaryotes, initiation of DNA replication requires the assembly and activation of pre-replication complexes (pre-RCs) 5 on chromatin (1). Sequential binding to DNA of the origin recognition complex, Cdc6, Cdt1, and mini-chromosome maintenance proteins (Mcm2-7) lead to formation of pre-RCs. Pre-RC activation is under the control of two kinases, Cdk2 and Cdc7, and ultimately results in the loading of replication factors such as Cdc45 and the unwinding of replication origins by the MCM helicase complex (2-5).Cdc7 is a serine/threonine kinase that is conserved from yeast to human and is essential for cell proliferation and embryonic development (6). Like CDKs (cyclin-dependent kinases), Cdc7 activity is regulated by its association with a regulatory subunit, the Dbf4 protein. This complex is often referred to as DDK (Dbf4-dependent kinase). The existence in fission yeast of a Cdc7/Dbf4 complex paralog, Spo4/Spo6, and the recent discovery of multiple Dbf4-related molecules in animal cells suggest that they belong to a novel DDK protein kinase family (7-10
p27Kip1 is an essential cell cycle inhibitor of Cyclindependent kinases. Ubiquitin-mediated proteolysis of p27Kip1 is an important mechanism for activation of Cyclin E-Cdk2 and facilitates G 1 /S transition. Ubiquitination of p27 is primarily catalyzed by a multisubunit E3 ubiquitin ligase, SCF Skp2 , and requires an adapter protein Cks1. In addition, phosphorylation of p27 at Thr 187 by Cyclin E and Cdk2 is also essential for triggering substrate ubiquitination. Here we investigate the molecular mechanism of p27 ubiquitination. We show that Cyclin E-Cdk2 is essential for targeting the p27 substrate to SCF Skp2 . Direct physical contact between Cyclin E but not Cdk2 and p27 is required for p27 recruitment to SCF Skp2 . In a search for positively charged amino acid residues that may be involved in recognition of the Thr 187 phosphate group, we found that Arg 306 of Skp2 is required for association and ubiquitination of phosphorylated p27 but dispensable for ubiquitination of unphosphorylated p21. Thus, our data unravel the molecular organization of the ubiquitination complex that catalyzes p27 ubiquitination and provide unique insights into the specificity of substrate recognition by SCF Skp2 .
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