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.
Highlights
In the 2nd column (“Location of deletion”), the location of the deletion is shown in parentheses
We examined whether the small segment of Cdc[7] containing DAM-1 and DAM-2 can interact with ASK
Identification of Two ASK-binding Motifs, DAM-1 and DAM-2, on Cdc7—In this study, we examined the segments of human Cdc[7] kinase required for interaction with ASK and kinase activation (Fig. 1)
Summary
Cells—HeLa and 293T cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum (Hana-Nesco Bio). muCdc7(Ϫ/Ϫ) ES cells were maintained in DMEM high glucose (Invitrogen, 11995-065) containing 20% FBS (Invitrogen, 16141-079), 1% nonessential amino acids (Invitrogen 11140-050), 2 mM L-glutamine (Invitrogen, 20530-081), 1% nucleotide mixture (Dainippon Sumitomo Pharma, R-ES-008D), 0.07% -mercaptoethanol (Nacalai 214-38), and 103 units/ml ESGRO (Chemicon, ESG1107). Cdc7T1 and T1-ASK were expressed in BL21-CodonPlus(DE3)-RP strain and were purified by using Qiagen nickel-nitrilotriacetic acid-agarose in buffer A containing 40 mM Hepes-KOH (pH 7.6), 100 mM potassium glutamate, 1 mM EDTA, 10% glycerol, 1 mM DTT, and complete protease inhibitor mixture (Roche Applied Science). In Vivo Photocross-linking Assays—A series of Cdc[7] mutants carrying a termination codon TAG at various amino acid residues were generated by using Phusion site-directed mutagenesis kit (New England Biolabs), as recommended by the supplier. BL21 (DE3), transformed with pSupBpaRS-6TRN (47) and pETDuet-Cdc7Bpa mutant-ASK, was grown, and the two proteins were co-induced by addition of 1 mM isopropyl 1-thio--D-galactopyranoside for 20 h at 20 °C in the presence of 1 mM Bpa. The whole cell lysates were analyzed by SDS-PAGE/Western blotting. The reaction mixtures were run on SDS-PAGE, stained with Coomassie Brilliant Blue, dried, and autoradiographed
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