The Cellular Protein Complex Associated with a Transforming Region of E1A Contains c-MYC

Abstract

The cell-transforming activity of human adenovirus 5 (hAd5) E1A is mediated by the N-terminal half of E1A, which interacts with three different major cellular protein complexes, p300/CBP, TRRAP/p400, and pRb family members. Among these protein interactions, the interaction of pRb family proteins with conserved region 2 (CR2) of E1A is known to promote cell proliferation by deregulating the activities of E2F family transcription factors. The functional consequences of interaction with the other two protein complexes in regulating the transforming activity of E1A are not well defined. Here, we report that the E1A N-terminal region also interacted with the cellular proto-oncoprotein c-MYC and the homolog of enhancer of yellow 2 (ENY2). Our results suggested that these proteins interacted with an essential E1A transforming domain spanning amino acid residues 26 to 35 which also interacted with TRRAP and p400. Small interfering RNA (siRNA)-mediated depletion of TRRAP reduced c-MYC interaction with E1A, while p400 depletion did not. In contrast, depletion of TRRAP enhanced ENY2 interaction with E1A, suggesting that ENY2 and TRRAP may interact with E1A in a competitive manner. The same E1A region additionally interacted with the constituents of a deubiquitinase complex consisting of USP22, ATXN7, and ATXN7L3 via TRRAP. Acute short hairpin RNA (shRNA)-mediated depletion of c-MYC reduced the E1A transforming activity, while depletion of ENY2 and MAX did not. These results suggested that the association of c-MYC with E1A may, at least partially, play a role in the E1A transformation activity, independently of MAX.

Importance: The transforming region of adenovirus E1A consists of three short modules which complex with different cellular protein complexes. The mechanism by which one of the transforming modules, CR2, promotes cell proliferation, through inactivating the activities of the pRb family proteins, is better understood than the activities of the other domains. Our analysis of the E1A proteome revealed the presence of the proto-oncoprotein c-MYC and of ENY2. We mapped these interactions to a critical transforming module of E1A that was previously known to interact with the scaffolding molecule TRRAP and the E1A-binding protein p400. We showed that c-MYC interacted with E1A through TRRAP, while ENY2 interacted with it independently. The data reported here indicated that depletion of c-MYC in normal human cells reduced the transforming activity of E1A. Our result raises a novel paradigm in oncogenic transformation by a DNA viral oncogene, the E1A gene, that may exploit the activity of a cellular oncogene, the c-MYC gene, in addition to inactivation of the tumor suppressors, such as pRb.

FIG 1

Domain-specific transforming activities of E1A. (A) Domain map of S-E1A and the interaction of major cellular proteins. The exons of E1A 12S mRNA and the conserved regions (CR) of S-E1A are indicated. The deleted amino acid regions in the E1A mutant constructs are underlined. (B) BRK cells were transfected with plasmids that express E1A mutants with the indicated deletions and activated H-Ras. The transformation assays were carried out as described previously (26). The transfected cells were stained 10 days after transfection and photographed. (C) Quantification of the transformed colonies. (D) Expression of E1A and Ras in BRK cells. BRK cells were transfected with the indicated plasmids, and 48 h after transfection, the cells were analyzed by Western blotting using a mixture of E1A Abs (M58 and Ab 1-80) and Abs specific for Ras and actin.

FIG 2

Interaction of c-MYC with E1A. (A to C) Interaction of exogenously expressed c-MYC and MYCBP. HeLa cells were transfected with the expression vectors for Flag-MYC, EGFP, EGFP-MYC, and Flag-MYCBP. Twenty-four hours after transfection, the cells were infected with hAd5 dl312 (E1A null), 12S wt, or dl1102 (100 PFU/cell). The whole-cell lysates (WCL) were immunoprecipitated (IP) with the Flag Ab (A and B) or the E1A Ab (M73) (C). The blots were probed with the E1A Ab (M73) (A to C) or the EGFP Ab (C). (D and E) Interaction of endogenous c-MYC and MAX. (D) HeLa cells were infected with hAd5 12S wt or mutant dl1102, and the WCLs were immunoprecipitated with the E1A Ab (M73). The Western blots were probed with the Abs for pRb (IF8), c-MYC, MAX, p400, and TRRAP. (E) Interaction of c-MYC with E1A in transformed cells. The WCLs of HeLa or 293 cells were immunoprecipitated with the E1A Ab (M73), and the Western blots were probed for pRb (C15), c-MYC, and MAX.

FIG 3

Mode of c-MYC interaction with E1A. (A) HeLa cells were transfected with siRNAs directed against TRRAP, p400, or MYCBP or with control siRNA. Thirty-six hours after transfection, cells were infected with hAd5 12S wt, and WCLs were immunoprecipitated with the E1A Ab (M73). The Western blots were probed with Abs against TRRAP, p400, c-MYC, and MYCBP.

FIG 4

Interaction of ENY2-USP22 complex with E1A. (A) KB cells were infected with hAd5 12S wt or mutant dl1102, and the WCLs were immunoprecipitated with the E1A Ab (M73). The Western blots were probed with Abs for ENY2, ATXN7L3, ATXN7, USP22, GCN5, or E1A (M73). (B) 293 or HeLa cells were either mock infected or infected with hAd5 12S wt, and the E1A-interacting proteins were analyzed as described for panel A.

FIG 5

Mode of interaction of ENY2 with E1A. (A) Effect of TRRAP depletion. HeLa cells were transfected with siRNAs directed against TRRAP or control siRNA, the WCLs were immunoprecipitated with the E1A Ab (M73), and the Western blots were probed with Abs against TRRAP, USP22, ATXN7L3, ENY2, and E1A (M73). (B) GST pulldown assay for E1A-interacting proteins. The WCLs from HeLa cells were incubated with GST or GST-E1A recombinant proteins, and the interacting proteins were purified by using the glutathione-agarose affinity beads. The proteins bound to the beads were analyzed in the flowthrough fractions by Western blotting using Abs against various E1A-interacting proteins. (C) Competitive interaction of ENY2 and TRRAP. HeLa cells were transfected with E1A 12S wt (pLPC-12S) with or without TRRAP and two different concentrations of ENY2. The interactions of TRRAP, ENY2, and pRb with E1A were analyzed by immunoprecipitation with the E1A Ab (M73) and Western blotting.

FIG 6

Effect of c-MYC depletion on E1A-Ras cooperative transformation. (A to C) HNK cells were infected with retroviral vectors that express the Δ178–238 E1A mutant and the activated H-Ras oncogene, along with lentiviral vectors that express specific shRNAs targeted against c-MYC (A), ENY2 (B), MAX (C), or GFP. The cells were then selected with puromycin. The foci were stained with crystal violet and counted (graphs). The transduction of retroviral vectors that expressed only E1A-12S-177-9 (Δ178–238) or H-Ras induced low numbers of lightly stained (with crystal violet) colonies that were readily distinguished and discarded during quantification. The activities of shRNA vectors in the downregulation of c-MYC (A), ENY2 (B), and MAX (C) were determined in HeLa cells with different lentiviral vectors and selection with puromycin (blots). (D) Western blot analysis of E1A and Ras proteins. Seventy-two hours after transduction with different retroviral vectors and shRNA lentiviral vectors, HNK cells were analyzed by Western blotting using the E1A Ab (M58) and Ras antibodies.

FIG 7

Interaction of cellular proteins with the transforming domain of E1A. The interactions of TRRAP, p400, and GCN5 with the E1A domain spanning residues 26 to 35 were previously known. These interactions were not seen with the mutant dl1102. Our present results revealed the interactions of other proteins shown in the figure via TRRAP and p400. The interaction of Tip60 (indicated by the question mark) was not detected in our LC-MS or by E1A co-IP experiments. Our results suggest that ENY2 may interact with E1A independently of TRRAP. Since ENY2 is an integral part of the USP22 DUB complex, we postulate that ENY2 may additionally interact with E1A via the USP22 DUB complex.