CYCLIN D1 CDK4 REGULATES RETINOBLASTOMA PROTEIN-MEDIATED CELL-CYCLE ARREST BY SITE-SPECIFIC PHOSPHORYLATION/

The retinoblastoma protein (pRb) inhibits progression through the cell cycle. Although pRb is phosphorylated when G(1) cyclin-dependent kina ses (Cdks) are active, the mechanisms underlying pRb regulation are un known. In vitro phosphorylation by cyclin D1/Cdk4 leads to inactivatio n of pRb in a microinjection-based in vivo cell cycle assay. In contra st, phosphorylation of pRb by Cdk2 or Cdk3 in complexes with A- or E-t ype cyclins is not sufficient to inactivate pRb function in this assay , despite extensive phosphorylation and conversion to a slowly migrati ng ''hyperphosphorylated form.'' The differential effects of phosphory lation on pRb function coincide with modification of distinct sets of sites. Serine 795 is phosphorylated efficiently by Cdk4, even in the a bsence of an intact LXCXE motif in cyclin D, but not by Cdk2 or Cdk3. Mutation of serine 795 to alanine prevents pRb inactivation by Cdk4 ph osphorylation in the microinjection assay. This study identifies a res idue whose phosphorylation is critical for inactivation of pRb-mediate d growth suppression, and it indicates that hyperphosphorylation and i nactivation of pRb are not necessarily synonymous.