MONOCLONAL-ANTIBODIES SPECIFIC FOR UNDERPHOSPHORYLATED RETINOBLASTOMAPROTEIN IDENTIFY A CELL-CYCLE-REGULATED PHOSPHORYLATION SITE TARGETEDBY CDKS

The growth suppressive activity of the retinoblastoma tumour suppresso r protein is controlled by cell cycle dependent phosphorylation, Howev er, while many in vivo phosphorylation sites have been mapped, the ide ntities of those residues whose phosphorylation is regulated remain el usive, We have mapped the epitopes of three independent monoclonal ant ibodies that recognise a distinction between differentially phosphoryl ated pRB sub-populations. All three antibodies recognise an identical epitope which encompasses an essential serine positioned within a cons ensus site for proline directed kinase phosphorylation, We provide evi dence that this residue, serine 608 of pRB, is an authentic phosphoryl ation site that can be phosphorylated in vitro by cyclin A-CDK2 and cy clin D1-CDK4 kinases but not by cyclin E-CDK2 kinase or the mitogen ac tivated kinase ERK2. Phosphorylation at this residue seems to be cell cycle regulated, occurring prior to entry into the S phase.