A Novel Growth- and Cell Cycle-Regulated Protein, ASK, Activates Human Cdc7-Related Kinase and Is Essential for G₁/S Transition in Mammalian Cells

Abstract

A novel human protein, ASK (activator of S phase kinase), was identified on the basis of its ability to bind to human Cdc7-related kinase (huCdc7). ASK forms an active kinase complex with huCdc7 that is capable of phosphorylating MCM2 protein. ASK appears to be the major activator of huCdc7, since immunodepletion of ASK protein from the extract is accompanied by the loss of huCdc7-dependent kinase activity. Expression of ASK is regulated by growth factor stimulation, and levels oscillate through the cell cycle, reaching a peak during S phase. Concomitantly, the huCdc7-dependent kinase activity significantly increases when cells are in S phase. Furthermore, we have demonstrated that ASK serves an essential function for entry into S phase by showing that microinjection of ASK-specific antibodies into mammalian cells inhibited DNA replication. Our data show that ASK is a novel cyclin-like regulatory subunit of the huCdc7 kinase complex and that it plays a pivotal role in G₁/S transition in mammalian cells.

ACKNOWLEDGMENTS

We express our indebtedness to Shigeki Jinno and Hiroto Okayama (Tokyo University School of Medicine) for their enormous help in microinjection experiments, without which these experiments would have been impossible. We also thank Takahisa Hichiya and Katsuyuki Tamai (MBL) for generation of antibodies against huCdc7 and ASK proteins. We thank Jung Min Kim, Min Kwan Cho, Tadayuki Takeda, Satoshi Asaro, Masachi Uchiyama, and other members of our laboratory for helpful discussion.

This work was partly supported by grants-in-aid for Cooperative Research and for Special Project Research from the Ministry of Education, Science and Culture, Japan, to H.M. DNAX is funded by Schering-Plough Corporation.