The p21 family of CDK inhibitors, including p21, p27, and p57, promote the assembly of cdk4/cyclin D complexes. These proteins specifically enhance the association of cdk4 with D-type cyclins, which is inefficient under normal conditions. This effect is not due to a simple cell cycle block but requires the function of both cdk and cyclin-binding domains. Kinetic studies show that p21 and p27 significantly increase the association rate (Ka) of cdk4 and cyclin D1 by reducing the dissociation rate (Koff). At low concentrations, p21 promotes active kinase complexes, while at higher concentrations, it inhibits activity. Immunodepletion experiments reveal that most active cdk4-associated kinase activity is associated with p21. In mammary epithelial cells, cyclin D1 and p21 bind to cdk4 during complex assembly, and most of the cyclin D1-associated kinase activity is also p21-associated. All three CIP/KIP inhibitors target cdk4 and cyclin D1 to the nucleus. These findings suggest that p21 family proteins may act as adaptors to assemble and program kinase complexes for specific functions, in addition to their roles as inhibitors. The results indicate that p21 promotes complex assembly and nuclear localization of cdk4/cyclin D1, while p27 and p57 do not. The data support the idea that p21 can both promote and inhibit kinase activity depending on its concentration, and that it is involved in the assembly of active kinase complexes in vivo. The study also shows that p21 is not essential for normal cell cycle function, as p21-null mice develop normally and are fertile. However, subtle defects may exist in specific cell types or under certain conditions. The findings highlight the dual role of p21 as both an inhibitor and an adaptor protein in the assembly and regulation of cdk4/cyclin D1 complexes.The p21 family of CDK inhibitors, including p21, p27, and p57, promote the assembly of cdk4/cyclin D complexes. These proteins specifically enhance the association of cdk4 with D-type cyclins, which is inefficient under normal conditions. This effect is not due to a simple cell cycle block but requires the function of both cdk and cyclin-binding domains. Kinetic studies show that p21 and p27 significantly increase the association rate (Ka) of cdk4 and cyclin D1 by reducing the dissociation rate (Koff). At low concentrations, p21 promotes active kinase complexes, while at higher concentrations, it inhibits activity. Immunodepletion experiments reveal that most active cdk4-associated kinase activity is associated with p21. In mammary epithelial cells, cyclin D1 and p21 bind to cdk4 during complex assembly, and most of the cyclin D1-associated kinase activity is also p21-associated. All three CIP/KIP inhibitors target cdk4 and cyclin D1 to the nucleus. These findings suggest that p21 family proteins may act as adaptors to assemble and program kinase complexes for specific functions, in addition to their roles as inhibitors. The results indicate that p21 promotes complex assembly and nuclear localization of cdk4/cyclin D1, while p27 and p57 do not. The data support the idea that p21 can both promote and inhibit kinase activity depending on its concentration, and that it is involved in the assembly of active kinase complexes in vivo. The study also shows that p21 is not essential for normal cell cycle function, as p21-null mice develop normally and are fertile. However, subtle defects may exist in specific cell types or under certain conditions. The findings highlight the dual role of p21 as both an inhibitor and an adaptor protein in the assembly and regulation of cdk4/cyclin D1 complexes.