Fig. 2.
Cdc6 activates p21-associated Cdk2 in an ATP-dependent manner in vitro. (A) p21-bound Cdk2 in MMS-treated S phase-arrested NRK cell lysates and in vitro synthesized His-tagged Cdc6 preparation were assayed for Cdc6, Cdk2, cyclin A, cyclin E, p21, and p27KIP1 by immunoblot before and after anti-Cdk2 immunoprecipitation (BIP and AIP) or affinity-purification (BAP and AAP). (B) (Left) The immunoprecipitated p21-bound Cdk2 (equivalent to the Cdk2 amount in 30 μl of the S-phase-arrested cell lysate) was incubated in the activation buffer with 5 μl (equivalent to the Cdc6 mount in 25 μl of the S-phase cell lysate) or 10 μl of the purified His-tagged Cdc6 preparation, 10 μl of either heat-inactivated purified His-tagged Cdc6 or similarly affinity-purified EV product in the presence or absence of ATP, followed by assays for the activity and amount of Cdk2 as described in Materials and Methods. (Right) p21-bound Cdk2 before immunoprecipitation (50 μl of the S-phase-arrested cell lysate) was incubated in an 80-μl activation buffer with 5 or 10 μl of the purified Cdc6, immunoprecipitated with the anti-Cdk2 antibody, and assayed for the activity and amount of Cdk2. In parallel, the same amount of p21-associated Cdk2 as in Left was incubated with 10 μl of the purified Cdc6 or the EV product in the absence of ATP, immunoprecipitated, and assayed. (C) The relative Cdk2 activity obtained by in vitro activation as in B was compared with that in a lysate of logarithmically proliferating NRK cells. (D) p21-bound Cdk2 was immunoprecipitated with the anti-cyclin A or the anti-cyclin E antibody from the same NRK cell lysate as in A and subjected to the activation assay as in B. (E) Baculovirus-expressed affinity-purified Cdk2–cyclin A complexes were preincubated at 30°C for 30 min in 50 mM Tris·HCl (pH 7.5) containing 150 mM NaCl and 10 mM MgCl2 with or without E. coli-expressed double affinity-purified p21 (the same amount as in MMS-treated S-phase cells, relative to Cdk2), immunoprecipitated with the anti-Cdk2 antibody, incubated with double affinity-purified FLAG- and His-tagged Cdc6 (≈2 fold excess) or its control vector product, and assayed for Cdk2 activity as in B.