Identification of the E2A gene products as regulatory targets of the G1 cyclin-dependent kinases

The E2A gene products, E12 and E47, are multifunctional transcription factors that as homodimers regulate B cell development, growth, and survival. In this report, the E2A gene products are shown to be targets for regulation by the G(1) cyclin-dependent kinases. Two novel G(1) cyclin-dependent kinase sites are identified on the N-terminal domain of E12/E47, One site displays homology to a preferential D-type cyclin-dependent kinase site (serine 780) on the retinoblastoma susceptibility gene product (pRB) and, consistent with this homology, is more efficiently phosphorylated by cyclin D1-CDK4 than by the other cyclin-dependent kinases (CDK) that were tested. The second kinase site is phosphorylated by both cyclin D1-CDK4 and cyclin A/E-CDK2 complexes. Mutation studies indicated that phosphorylation of the cyclin D1-CDK4 site, or more potently, of both the cyclin D1-CDK4 and cyclin A/E-CDK2 sites, negatively regulates the growth suppressor function associated with the N-terminal domain of E12/E47, Transient expression studies showed that ectopic expression of cyclin D1 or E negatively regulates sequence-specific activation of gene transcription by E12/E47, Analysis of site mutants, however, indicated that inhibition of E12/E47 transcriptional activity did not require the N-terminal G(1) cyclin-dependent kinase sites. Together, the results suggest that the growth suppressor and transcriptional activator functions of E12/E47 are targets for regulation by G(1) cyclin-dependent kinases but that the mechanisms of regulation for each function are distinct.