PURIFICATION, CHARACTERIZATION, AND KINETIC MECHANISM OF CYCLIN D1-CENTER-DOT-CDK4, A MAJOR TARGET FOR CELL-CYCLE REGULATION

The cyclin D1 . CDK4-pRb (retinoblastoma protein) pathway plays a cent ral role iu the cell cycle, and its deregulation is correlated with ma ny types of cancers. As a major drug target, we purified dimeric cycli n D1 . CDK4 complex to near-homogeneity by a four-step procedure from a recombinant baculovirus-infected insect culture, We optimized the ki nase activity and stability and developed a reproducible assay. We exa mined 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 u sed as the phosphoacceptor, By using staurosporine and ADP as inhibito rs, the kinetic mechanism of the complex appeared to be a ''single dis placement or Bi-Bi'' with Mg2+. ATP as the leading substrate and phosp horylated Rb-ING as the last product released. In addition, we purifie d a cyclin D1-CDK4 fusion protein to homogeneity by a three-step proto col from another recombinant baculovirus culture and observed similar kinetic properties and mechanisms as those from the complex. We attemp ted to model staurosporine in the ATP-binding site of CDK4 according t o our kinetic data. Our biochemical and modeling data provide validati on of both the complex and fusion protein as highly active kinases and their usefulness in antiproliferative inhibitor discovery.