DIFFERENTIAL PHOSPHORYLATION OF THE RETINOBLASTOMA PROTEIN BY G(1) S CYCLIN-DEPENDENT KINASES/

The retinoblastoma tumor suppressor protein, pRB, is inactivated by ph osphorylation. While existing evidence is strong that such phosphoryla tion is mediated by one or more cyclin dependent kinases (CDKs) active during G(1)/S-1 it remains unclear which of the various CDKs is respo nsible. We show here that three candidate pRB inactivating kinases, CD K4-cyclin D1, CDK2-cyclin E, and CDK2-cyclin A, phosphorylate pRB diff erentially, each on a subset of authentic pRB phosphorylation sites. N otably, two neighboring pRB phosphate accepters, threonine 821 and thr eonine 826, which have previously been implicated in the regulation of LXCXE protein binding, are phosphorylated by different CDKs. We demon strate that phosphorylation by either CDK2-cyclin A, which phosphoryla tes T821, or CDK4-cyclin D1, which phosphorylates threonine 826, can d isable pRB for subsequent binding of an LXCXE, protein. However, only one of these two kinases, CDK2-cyclin A, can dissociate a pre-existing LXCXE protein-pRB complex. We provide evidence that prior binding of an LXCXE protein blocks access to certain residues specifically target ed by CDK4-cyclin D1, explaining the inability of this kinase to resol ve such complexes. While these results are not direct proof of the rel evance of differential pRB phosphorylation in cells, our findings supp ort a model whereby full phosphorylation of pRB may require the action of more than one kinase and explains how such differential phosphoryl ation by different CDKs might translate into a differential regulation of downstream effector pathways.