One of the hallmarks of cancer cells is their unconstrained proliferation. To get insights into the mechanisms of tumorigenesis, it is necessary to understand how cellular proliferation is regulated. Cyclin-dependent kinases (CDKs) are a group of serine/threonine kinases that control the eukaryotic cell cycle progression. Our laboratory is interested in understanding the mechanisms by which cyclin E-Cdk2 regulates the G1-to-S phase transition in mammalian cells. To this end, we have started to identify and characterize the physiologically relevant substrates of this kinase complex.

We have developed a screening method to identify the potential substrates of cyclin E-Cdk2 kinase. One of the substrates we identified from the screen is NPAT. We showed that NPAT interacts with cyclin E-Cdk2 and can be phosphorylated by this kinase in vivo. The protein level of NPAT peaks at the G1/S phase boundary and overexpression of NPAT promotes entry into S phase, suggesting that NPAT is a rate-limiting factor for S phase entry in mammalian cells. NPAT localizes at histone gene clusters in vivo in a cell cycle-dependent manner and activates histone gene transcription, one of the major S phase-specific events. Thus, NPAT is a cell-cycle regulated transcription factor, and it links cyclin E-Cdk2 to the regulation of histone gene transcriptional activation during the G1-to-S phase transition. We are studying how the localization of NPAT is regulated, how NPAT activates histone gene transcription and how the activities of NPAT is regulated by cyclin E-Cdk2 kinase. We are also pursuing the possibility that there are other NPAT targets, besides histone genes, whose expression is also required for the S phase entry.

In addition to NPAT, we have isolated several other potential substrates for cyclin E-Cdk2 complex. We will examine whether they are also physiologically relevant substrate of this kinase in vivo and how they are involved in the control of S phase entry. Particularly, we are interested in looking for substrates that are involved in DNA replication and centrosome duplication/separation, since the activity of cyclin E-Cdk2 is required for these processes and the kinase substrates involved have not been identified.

1. DeRan, M., Pulvino, M., Greene, E., Su, C., and Zhao, J. (2008) Transcriptional activation of histone genes requires NPAT-dependent recruitment of TRRAP-Tip60 complex to histone promoters during the G1/S phase transition. Molecular and Cellular Biology, 28: 435-447.
   
   
2. Le,XF, Bedrosian, I., Mao, W., Murray, M., Lu, Z., Keyomarsi, K., Lee, MH., Zhao, J. and Bast, RC. (2006) Anti-Her2 antibody transtuzumab inhibits CDK2-mediated NPAT and histone H4 expression via the PI3K pathway. Cell Cycle, 5: 1654-1661.
   
   
3. Kim, S-W., Oleksyn, D.W., Rossi, R., Jordan, C.T., Sanz, I., Chen, L., and Zhao, J. (2008) Protein kinase C-associated kinase PKK is required fro NF-kB signaling and survival in diffuse large B-cell lymphoma cells. Blood (In Press; online Nov. 19, 2007)
   
   
4. Zhao, J. (2005) Identification of protein-protein interactions by overlay screening of phage cDNA expression libraries. in E. Golemis and P. Adams (eds) Protein-Protein Interactions, A Molecular Cloning Manual, 2ed edition. pp 129-140. Cold Spring Harbor Laboratory Press, New York, New York.
   
   
5. Zhao, J. (2004) Coordination of DNA synthesis and histone gene expression during normal cell cycle progression and after DNA damage. Cell Cycle, 3:695-697.
   
   
6. Su, C., Gao, G., Schneider, S., Helt, C, Weiss, C., O'Reilly, M., Bohmann, D. and Zhao, J. (2004) DNA damage induces down-regulation of histone gene expression throught the G1 checkpoint pathway. EMBO J. 23:1133 - 43.
   
   
7. Barbie DA, Kudlow BA, Frock R, Zhao J, Johnson BR, Dyson N, Harlow E, Kennedy BK.(2004) Nuclear reorganization of mammalian DNA synthesis prior to cell cycle exit. Molecular and Cellular Biology, Jan 24 (2): 595 - 607.
   
   
8. Gao, G., Bracken, A., Burkard, K., Pasini, D., Classon, M., Attwooll, C., Sagarav, M., Imai, T., Helin K, and Zhao, J. (2003) NPAT Expression Is Regulated by E2F and is Essential for Cell Cycle Progression. Molecular and Cellular Biology, 23:2821 - 2833.
   
   
9. Jiyong Zhao, Brian Kennedy, Brandon Lawrence, David Barbie, A. Gregory Matera, Jonathan Fletcher and Ed Harlow. (2000) NPAT Links cyclin E-Cdk2 to the regulation of replication-dependent histone gene transcription. Genes & Development. 14: 2283-2297
   
   
10. Jiyong Zhao, Brian Dynlacht, Takashi Imai, Tada-aki Hori and Ed harlow. (1998) Expression of NPAT, a novel substrate of cyclin E-Cdk2, promotes S-phase entry. Genes & Development. 12:456-461.

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