Purification, characterization, and kinetic mechanism of cyclin D1•CDK4, a major target for cell cycle regulation

被引:38
作者
Konstantinidis, AK
Radhakrishnan, R
Gu, F
Rao, RN
Yeh, WK [1 ]
机构
[1] Lilly Res Labs, Res Technol & Prot, Indianapolis, IN 46285 USA
[2] Lilly Res Labs, Canc Res, Indianapolis, IN 46285 USA
关键词
D O I
10.1074/jbc.273.41.26506
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The cyclin D1 . CDK4-pRb (retinoblastoma protein) pathway plays a central role iu 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 (Rb-ING) and subsequently investigated the mechanistic implications with a physiologically relevant protein (Rb-21) as the phosphoacceptor. The complex bound ATP 130-fold tighter when Rb-21 instead of Rb-ING 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 Rb-ING 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.
引用
收藏
页码:26506 / 26515
页数:10
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