Function of cyclin D1 in development and cancer.

摘要

Cyclin D1 is a multifunctional protein that is able to interact with pRb via a conserved LxCxE pRb binding motif, activate a kinase partner and direct the phosphorylation of pRb, activate cyclin E-cdk2 by titrating Cip/Kip cdk inhibitors, and modulate the activity of a variety of transcription factors. To date, the specific importance of each of these functions of cyclin D1 in the processes of development and tumorigenesis has not been described. To test the importance of the LxCxE mediated pRb binding and kinase activating functions of cyclin D1, we have engineered two "knock-in" mice with mutant alleles of cyclin D1 that specifically abrogate each of these functions.; To test the importance of the LxCxE domain in vivo, we have generated a knock-in mouse by replacing the wild-type cyclin D1 gene with a mutant allele exactly lacking the nucleotides encoding the LxCxE domain. Analysis of this mouse has demonstrated that the LxCxE protein is biochemically similar to wild-type cyclin D1 in all tested respects. Moreover, we were unable to detect phenotypic abnormalities in growth, retinal development, or mammary gland development, all of which are affected by deleting cyclin D1. Although we cannot exclude the presence of subtle defects, these results suggest that the LxCxE domain of cyclin D1 is not necessary for function, despite the absolute conservation of this motif in the D-type cyclins from plants and vertebrates.; The most widely accepted and well-understood function of cyclin D1 is to direct the phosphorylation of pRb, inactivating it and allowing S-phase entry. To specifically test the importance of cyclin D1-associated kinase activity, we have replaced the wild-type mouse cyclin D1 gene with an allele containing a point mutation of K112 to E (cyclin D1KE/KE). Comparison of these mice to wild-type and cyclin D1-/- mice has demonstrated that the cyclin D1KE/KE mice have some phenotypes that are similar to phenotypes of the cyclin D1 knock-out. In contrast, other tissues profoundly affected by loss of cyclin D1 are normal or nearly normal in the cyclin D1KE/KE mice. Analysis of the retinas of cyclin D1KE/KE mice shows decreased levels of pRb phosphorylation, consistent with our expectation that the cyclin D1 K112E protein is deficient in its ability to activate cdk4/6. In addition, cyclin D1 K112E-cdk4-p27 tripartite complexes are formed in the retina, and cdk-2 associated kinase activity is increased relative to knock-out retinas, consistent with the idea that cyclin D1 K112E retains its titration function. Significantly, although the mammary glands of cyclin D1KE/KE mice develop normally in response to pregnancy, these mice are completely resistant to MMTV-neu mediated tumorigenesis. These data indicate that cyclin D1-associated kinase activity is important for development of some tissues, while kinase-independent functions suffice in others. Further, kinase activation is critical for the function of cyclin D1 in MMTV-neu induced tumorigenesis.