Bipartite Binding of a Kinase Activator Activates Cdc7-related Kinase Essential for S Phase
Dfp1/Him1 protein of fission yeast, Schizosaccharomyces pombe , encodes the regulatory subunit for Hsk1 kinase, a homologue of budding yeast Cdc7 kinase essential for initiation and progression of the S phase of the cell cycle. This protein binds and activates Hsk1 kinase, which phosphorylates the M...
Saved in:
Published in | The Journal of biological chemistry Vol. 276; no. 33; pp. 31376 - 31387 |
---|---|
Main Authors | , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Society for Biochemistry and Molecular Biology
17.08.2001
|
Subjects | |
Online Access | Get full text |
Cover
Loading…
Summary: | Dfp1/Him1 protein of fission yeast, Schizosaccharomyces pombe , encodes the regulatory subunit for Hsk1 kinase, a homologue of budding yeast Cdc7 kinase essential for initiation and progression
of the S phase of the cell cycle. This protein binds and activates Hsk1 kinase, which phosphorylates the MCM2 protein. Comparison
of the amino acid sequences of the Cdc7 regulatory subunits from various eukaryotes revealed the presence of three small stretches
of conserved amino acid sequences, namely Dbf4 motifs N, M, and C. We report here that the Dbf4 motif M, a unique proline-rich
motif, and the Dbf4 motif C, a C 2 H 2 -type zinc finger motif, are essential for mitotic functions of Dfp1/Him1 protein as well as for full-level activation of
Hsk1 kinase. In vitro , a small segment containing the Dbf4 motif M or C alone binds to and partially activates Hsk1. Co-expression of these two
segments augments the extent of activation. Furthermore, a fused polypeptide containing only Dbf4 motifs M and C without any
spacer can activate Hsk1 and is capable of rescuing the growth defect of him1 null cells. Insertion of a long stretch of amino acids between the motif M and motif C can be tolerated for mitotic functions.
On the other hand, internal deletion of Dbf4 motif N, which has some similarity with the BRCA C-terminal domain motif, results
in a defect in hydroxyurea-induced checkpoint responses and sensitivity to methyl methane sulfonate, yet mitotic functions
and kinase activation are intact. In one-hybrid assays with budding yeast Dbf4, motif N mutants exhibit reduced interaction
with a replication origin. Our observations suggest the molecular architecture of Cdc7·Dbf4-related kinase complexes at the
origins, in which they are tethered to replication machinery through Dbf4 motif N and the catalytic subunits are activated
through bipartite binding of Dbf4 motifs M and C of the regulatory subunits. |
---|---|
Bibliography: | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2 |
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M102197200 |