Monoclonal antibodies specific for underphosphorylated retinoblastoma protein identify a cell cycle regulated phosphorylation site targeted by CDKs

The growth suppressive activity of the retinoblastoma tumour suppressor protein is controlled by cell cycle dependent phosphorylation, However, while many in vivo phosphorylation sites have been mapped, the identities of those residues whose phosphorylation is regulated remain elusive, We have mapped the epitopes of three independent monoclonal antibodies that recognise a distinction between differentially phosphorylated pRB sub-populations. All three antibodies recognise an identical epitope which encompasses an essential serine positioned within a consensus site for proline directed kinase phosphorylation, We provide evidence that this residue, serine 608 of pRB, is an authentic phosphorylation site that can be phosphorylated in vitro by cyclin A-CDK2 and cyclin D1-CDK4 kinases but not by cyclin E-CDK2 kinase or the mitogen activated kinase ERK2. Phosphorylation at this residue seems to be cell cycle regulated, occurring prior to entry into the S phase.