THE INTERACTIONS OF E2F WITH PRB AND WITH P107 ARE REGULATED VIA THE PHOSPHORYLATION OF QRB AND P107 BY A CYCLIN-DEPENDENT KINASE

It has been postulated that the product (pRB) of the retinoblastoma ge ne dissociates from the E2F-pRB complex upon phosphorylation by cyclin -dependent kinase(s) (cdk). However, there is no direct evidence for t he regulation of formation of the E2F-pRB complex via phosphorylation by purified cdk, Therefore,we investigated the regulation of formation of this complex by phosphorylation using pRB and purified cyclin A-cd k2, cyclin E-cdk2 or cyclin D1-cdk4. Purified pRB was incubated with n uclear extracts prepared from pRB-defective cells and then subjected t o gel mobility shift assays, We confirmed that unphosphorylated pRB as sociated with various types of E2F but pRB has been phosphorylated by cyclin A-cdk2 did not. We found that E2F-pRB complexes were disrupted as a consequence of phosphorylation by cyclin A-cdk2, and the levels o f the free forms of E2Fs increased. We also found that not only the E2 F-pRB complexes but also the E2F-p107 complexes were disrupted upon ph osphorylation by cyclin A-cdk2. Furthermore, E2F-pRB complexes were di srupted through phosphorylation by cyclin D1-cdk4 and cyclin E-cdk2, a s web as by cyclin A-cdk2. These results clearly demonstrate that the phosphorylation of pRB and p107 by cdks regulates the formation of com plexes between E2F and pRB or p107.