title: Molecular mechanism of activation of human Cdc7 kinase: bipartite interaction with Dbf4/activator of S phase kinase (ASK) activation subunit stimulates ATP binding and substrate recognition.
abstract: Cdc7 is a serine/threonine kinase conserved from yeasts to human and is known to play a key role in the regulation of initiation at each replication origin. Its catalytic function is activated via association with the activation subunit Dbf4/activator of S phase kinase (ASK). It is known that two conserved motifs of Dbf4/ASK are involved in binding to Cdc7, and both are required for maximum activation of Cdc7 kinase. Cdc7 kinases possess unique kinase insert sequences (kinase insert I-III) that are inserted at defined locations among the conserved kinase domains. However, precise mechanisms of Cdc7 kinase activation are largely unknown. We have identified two segments on Cdc7, DAM-1 (Dbf4/ASK interacting motif-1; amino acids 448-457 near the N terminus of kinase insert III) and DAM-2 (C-terminal 10-amino acid segment), that interact with motif-M and motif-C of ASK, respectively, and are essential for kinase activation by ASK. The C-terminal 143-amino acid polypeptide (432-574) containing DAM-1 and DAM-2 can interact with Dbf4/ASK. Characterization of the purified ASK-free Cdc7 and Cdc7-ASK complex shows that ATP binding of the Cdc7 catalytic subunit requires Dbf4/ASK. However, the "minimum" Cdc7, lacking the entire kinase insert II and half of kinase insert III, binds to ATP and shows autophosphorylation activity in the absence of ASK. However, ASK is still required for phosphorylation of exogenous substrates by the minimum Cdc7. These results indicate bipartite interaction between Cdc7 and Dbf4/ASK subunits facilitates ATP binding and substrate recognition by the Cdc7 kinase.
authors.affiliation: Genome Dynamics Project, Department of Genome Medicine, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Kitamura
authors.forename: Rino
authors.initials: R
authors.lastname: Fukatsu
authors.forename: Naoko
authors.initials: N
authors.lastname: Kakusho
authors.forename: Yong-Soon
authors.initials: YS
authors.lastname: Cho
authors.forename: Chika
authors.initials: C
authors.lastname: Taniyama
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yamazaki
authors.forename: Gaik-theng
authors.initials: GT
authors.lastname: Toh
authors.forename: Kazuo
authors.initials: K
authors.lastname: Yanagi
authors.forename: Naoko
authors.initials: N
authors.lastname: Arai
authors.forename: Ho-Jin
authors.initials: HJ
authors.lastname: Chang
authors.forename: Hisao
authors.initials: H
authors.lastname: Masai
mesh_terms: D000255:Adenosine Triphosphate / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D010766:Phosphorylation / Q000502:physiology; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; C485678:DBF4 protein, human; D000255:Adenosine Triphosphate; C068619:CDC7 protein, human; D017346:Protein Serine-Threonine Kinases