CYCLIN D3-ASSOCIATED KINASE-ACTIVITY IS REGULATED BY P27(KIP1) IN BALB C 3T3 CELLS/

We report that cyclin D3/cdk4 kinase activity is regulated by p27(kip1 ) in BALB/c 3T3 cells. The association of p27(kip1) was found to resul t in inhibition of cyclin D3 activity as measured by immune complex ki nase assays utilizing cyclin D3-specific antibodies. The ternary p27(k ip1)/cyclin D3/cdk4 complexes do exhibit kinase activity when measured in immune complex kinase assays utilizing p27(kip1)-specific antibodi es. The association of p27(kip1) With cyclin D3 was highest in quiesce nt cells and declined upon mitogenic stimulation, concomitantly with d eclines in the total level of p27(kip1) protein. The decline in this a ssociation could be elicited by PDGF treatment alone; this was not suf ficient, however, for activation of cyclin D3 activity, which also req uired the presence of factors in platelet-poor plasma in the culturing medium. Unlike cyclin D3 activity, which was detected only in growing cells, p27(kip1) kinase activity was present throughout the cell cycl e. Since we found that the p27(kip1) activity was dependent on cyclin D3 and cdk4, we compared the substrate specificity of the active terna ry complex containing p27(kp1) and the active cyclin D3 lacking p27(ki p1) by tryptic phosphopeptide mapping of GST-Rb phosphorylated in vitr o and also by comparing the relative phosphorylation activity toward a panel of peptide substrates. We found that ternary p27(kip1)/cyclin D 3/cdk4 complexes exhibited a different specificity than the active bin ary cyclin D3/cdk4 complexes, suggesting that p27(kip1) has the capaci ty to both inhibit cyclin D/cdk4, activity as well as to modulate cycl in D3/cdk4 activity by altering its substrate preference.