P107 AND P130 ASSOCIATED CYCLIN-A HAS ALTERED SUBSTRATE-SPECIFICITY

We demonstrate that p107 and p130 immune complexes exhibit kinase acti vity. We have tested such immune complexes with four substrates common ly utilized to assay Cdk activity, including all three known members o f the retinoblastoma family. Immunodepletion revealed this kinase acti vity could be abolished by removal of either cyclin A or Cdk2 but was unaffected by removal of Cdk4 or any D-type cyclin. The appearance of p101 associated activity followed the accumulation of p107 protein. In contrast, the kinase activity associated with p130 immune complexes b ecame apparent after mid-G(1), coincident with p130 hyperphosphorylati on. GST-Rb, GST-p107, and GST-p130 (where GST indicates glutathione S- transferase) were equally suitable substrates in p107 and p130 immune complex kinase assays, yielding activity equal to 25% of the cyclin A activity present. The p107 and p130 associated activity was unable to phosphorylate histone H1, suggesting the p107 and p130 associated cycl in A/Cdk2 may represent a distinct pool with a distinct substrate spec ificity. The p107 and p130 associated activity was released from the i mmune complexes upon incubation with ATP and Mg2+ and exhibited the sa me substrate preference observed with the untreated immune complex. Ou r data suggest that p107 and p130 recognize, or form by association, a distinct pool of cyclin A/Cdk2 that preferentially phosphorylates ret inoblastoma family members.