| Cyclin E is a key regulator of the cell cycle that, when partnered with cdk2, facilitates the phosphorylation of retinoblastoma proteins and activates E2F transcription factors in order to promote cells into S phase. While functions of cyclin E1 in cultured cell lines have been determined, little is known about its roles in vivo. Our long term goal is to identify and characterize protein interactors that contribute to cyclin E1 in vivo functions. Given the tissue-specific expression of cyclin E1, we hypothesize that cyclin E1's in vivo functions are mediated by core as well as tissue- and development-specific protein interactors. Our hypothesis provides for the following testable predictions: (1) Cyclin E1 forms complexes with different proteins in different tissues; (2) Cyclin E1 forms complexes with different proteins during brain development; (3) The cyclin E1 interactome changes in the absence of cyclin E2 and cdk2.;We have designed a sensitive and reproducible IP-LC-MS/MS method to analyze cyclin E1-mediated multiprotein complexes from primary tissues (brain, spleen, testis, thymus). Our approach resulted in the identification of a core set (cdk2, cdk5, p27 and p57) as well as-tissue- and development-specific proteins associating with cyclin E1. We show that during brain development, cdk5 replaces cdk2 as the primary cyclin E1 kinase partner, although the function of the cyclin E1/cdk5 complex remains a mystery. Furthermore, our analysis of cyclin E2- and cdk2-single knockouts revealed that while cdk 1, cdk4 and cdk5 replace cdk2 in cdk2 knockouts, the absence of cyclin E2 does not significantly affect cyclin E1's normal protein interactions. Our detailed characterization of cyclin E1 multi-protein complexes affords a unique insight into molecular interactions underlying its functions in different tissues. |
