Purification, characterization, and kinetic mechanism of cyclin D1. CDK4, a major target for cell cycle regulation.
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.
Konstantinidis
A K
AK
Research Technologies and Proteins, Lilly Research Laboratories, Division of Eli Lilly and Company, Indianapolis, Indiana 46285, USA.
Radhakrishnan
R
R
Gu
F
F
Rao
R N
RN
Yeh
W K
WK
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002453:Cell Cycle; D002460:Cell Line; D019938:Cyclin D1 / Q000378:metabolism*; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism; D018411:Spodoptera; D019311:Staurosporine / Q000378:metabolism; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D019938:Cyclin D1; D000255:Adenosine Triphosphate; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D019311:Staurosporine