The adenovirus (Adv) oncoprotein E1A stimulates cell proliferation and inhibits differentiation. These activities are primarily linked to the N-terminal region (exon 1) of E1A, which interacts with multiple cellular protein complexes. The C terminus (exon 2) of E1A antagonizes these processes, mediated in part through interaction with C-terminal binding proteins 1 and 2 (CtBP1/2). To identify additional cellular E1A targets that are involved in the modulation of E1A C-terminus-mediated activities, we undertook tandem affinity purification of E1A-associated proteins. Through mass spectrometric analysis, we identified several known E1A-interacting proteins as well as novel E1A targets, such as the forkhead transcription factors, FOXK1/K2. We identified a Ser/Thr-containing sequence motif in E1A that mediated interaction with FOXK1/K2. We demonstrated that the E6 proteins of two beta-human papillomaviruses (HPV14 and HPV21) associated with epidermodysplasia verruciformis also interacted with FOXK1/K2 through a motif similar to that of E1A. The E1A mutants deficient in interaction with FOXK1/K2 induced enhanced cell proliferation and oncogenic transformation. The hypertransforming activity of the mutant E1A was suppressed by HPV21 E6. An E1A-E6 chimeric protein containing the Ser/Thr domain of the E6 protein in E1A interacted efficiently with FOXK1/K2 and inhibited cell transformation. Our results suggest that targeting FOXK1/K2 may be a common mechanism for certain beta-HPVs and Adv5. E1A exon 2 mutants deficient in interaction with the dual-specificity kinases DYRK1A/1B and their cofactor HAN11 also induced increased cell proliferation and transformation. Our results suggest that the E1A C-terminal region may suppress cell proliferation and oncogenic transformation through interaction with three different cellular protein complexes: FOXK1/K2, DYRK(1A/1B)/HAN11, and CtBP1/2. Adenovirus (Adv) is a model DNA tumor virus that has been widely used to decipher critical pathways of oncogenesis. The adenovirus early gene E1A is an intensely investigated viral oncogene and has been instrumental in uncovering common cell cycle-regulatory pathways shared by other DNA tumor virus oncogenes such as human papillomavirus (HPV) E7 (reviewed in reference 43) and simian virus 40 (SV40) T antigen (Ag) (reviewed in reference 9). E1A promotes cellular entry into S phase by deregulating the cell cycle and activates other early viral genes to facilitate viral replication. As a consequence of nonproductive infection, E1A immortalizes rodent cells and also oncogenically transforms these cells in cooperation with other viral or cellular oncogenes (27,32,52,62).The E1A gene encodes two major protein isoforms that are expressed from two mRNAs (13S and 12S) generated by alternative RNA splicing. The 13S mRNA encodes a 289-aminoacid ([aa] 289R) protein while the 12S mRNA encodes a 243-amino-acid (243R) protein. The 289R and 243R proteins differ by a 46-amino-acid region that is unique to 289R. The 243R protein is not required for viral replica...