JBC PeproTech; Our Business is Cytokines!

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Konstantinidis, A. K.
Right arrow Articles by Yeh, W.-K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Konstantinidis, A. K.
Right arrow Articles by Yeh, W.-K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

J Biol Chem, Vol. 273, Issue 41, 26506-26515, October 9, 1998

Purification, Characterization, and Kinetic Mechanism of Cyclin D1·CDK4, a Major Target for Cell Cycle Regulation

Alexandros K. Konstantinidis, R. Radhakrishnan, Fei Gu, R. Nagaraja Rao, and Wu-Kuang Yeh

From Research Technologies and Proteins and  Cancer Research, Lilly Research Laboratories, Division of Eli Lilly and Company, Indianapolis, Indiana 46285

The cyclin D1·CDK4-pRb (retinoblastoma protein) pathway plays a central role in the cell cycle, and its deregulation is correlated with many types of cancers. As a major drug target, we purified dimeric cyclin D1·CDK4 complex to near-homogeneity by a four-step procedure from a recombinant baculovirus-infected insect culture. We optimized the kinase activity and stability and developed a reproducible assay. We examined several catalytic and kinetic properties of the complex and, via steady-state kinetics, derived a kinetic mechanism with a peptide (RbING) and subsequently investigated the mechanistic implications with a physiologically relevant protein (Rb21) as the phosphoacceptor. The complex bound ATP 130-fold tighter when Rb21 instead of RbING was used as the phosphoacceptor. By using staurosporine and ADP as inhibitors, the kinetic mechanism of the complex appeared to be a "single displacement or Bi-Bi" with Mg2+·ATP as the leading substrate and phosphorylated RbING as the last product released. In addition, we purified a cyclin D1-CDK4 fusion protein to homogeneity by a three-step protocol from another recombinant baculovirus culture and observed similar kinetic properties and mechanisms as those from the complex. We attempted to model staurosporine in the ATP-binding site of CDK4 according to our kinetic data. Our biochemical and modeling data provide validation of both the complex and fusion protein as highly active kinases and their usefulness in antiproliferative inhibitor discovery.

Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Cancer Res.Home page
M. McGahren-Murray, N. H.A. Terry, and K. Keyomarsi
The Differential Staurosporine-Mediated G1 Arrest in Normal versus Tumor Cells Is Dependent on the Retinoblastoma Protein
Cancer Res., October 1, 2006; 66(19): 9744 - 9753.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Q. Zhao, F. Boschelli, A. J. Caplan, and K. T. Arndt
Identification of a Conserved Sequence Motif That Promotes Cdc37 and Cyclin D1 Binding to Cdk4
J. Biol. Chem., March 26, 2004; 279(13): 12560 - 12564.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
W. Wei, W. A. Jobling, W. Chen, W. C. Hahn, and J. M. Sedivy
Abolition of Cyclin-Dependent Kinase Inhibitor p16Ink4a and p21Cip1/Waf1 Functions Permits Ras-Induced Anchorage-Independent Growth in Telomerase-Immortalized Human Fibroblasts
Mol. Cell. Biol., April 15, 2003; 23(8): 2859 - 2870.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. M. Clare, R. A. Poorman, L. C. Kelley, K. D. Watenpaugh, C. A. Bannow, and K. L. Leach
The Cyclin-dependent Kinases cdk2 and cdk5 Act by a Random, Anticooperative Kinetic Mechanism
J. Biol. Chem., December 14, 2001; 276(51): 48292 - 48299.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
W. Wei, R. M. Hemmer, and J. M. Sedivy
Role of p14ARF in Replicative and Induced Senescence of Human Fibroblasts
Mol. Cell. Biol., October 15, 2001; 21(20): 6748 - 6757.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
D. A. Enke, P. Kaldis, and M. J. Solomon
Kinetic Analysis of the Cyclin-dependent Kinase-activating Kinase (Cak1p) from Budding Yeast
J. Biol. Chem., October 20, 2000; 275(43): 33267 - 33271.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 1998 by the American Society for Biochemistry and Molecular Biology.