Interaction of the fork head domain transcription factor MPP2 with the human papilloma virus 16 E7 protein: enhancement of transformation and transactivation

Abstract

The high risk human papillomavirus (HPV) type 16 E7 protein a ects cell growth control and promotes transformation by interfering with functions of cellular proteins. A key target of E7 is the tumor suppressor protein p105RB. Although this interaction is required for E7-dependent transformation, other cellular molecules must also be involved, because some E7 mutants that have reduced transforming abilities still bind to p105RB. In order to identify additional proteins that interact with E7 and that may be responsible to mediate its transforming function, we have used the C-terminal half of E7 in a yeast two-hybrid screen. We identi®ed the fork head domain transcription factor M phase phosphoprotein 2 (MPP2) as an interaction partner of E7. Speci®c interaction of the two proteins both in vitro and in vivo in mammalian cells was detected. The interaction of MPP2 with E7 is functionally relevant since MPP2 enhances the E7/Ha-Ras co-transformation of rat embryo ®broblasts. In addition HPV16 E7, but neither non-transforming mutants of HPV16 E7 nor low risk HPV6 E7, was able to stimulate MPP2-speci®c transcriptional activity. Thus, MPP2 is a potentially important target for E7-mediated transformation.

A functional interaction of E7 with B-Myb-MuvB complex promotes acute cooperative transcriptional activation of both S- and M-phase genes. (129 c)

“…Interestingly, when the same experiments were done with the E7 LXCXE mutant, less cooperativity was seen indicating that the LXCXE motif may have a role in E7 cooperation with B-Myb and FoxM1 and the B-Myb-MuvB complex (Supplementary Figure S1A and S1B). HPV16 E7 interacts with B-MyB and FoxM1 and is recruited to mitotic promoters The synergistic increase of mitotic genes transcript levels by HPV16 E7 and B-Myb or FoxM1 strongly suggested that E7 directly interacts with B-Myb or FoxM1, as demonstrated previously for FoxM1 only, 9 and could perhaps form transcriptional complexes with these proteins. We, therefore, looked for E7:B-Myb interaction by performing immunoprecipitation in extracts of 293T cells that overexpressed E7 and B-Myb and were indeed able to detect an interaction between these two proteins (Figure 3a).…”

“…7 The HPV16 E7 protein had previously been shown to specifically activate B-Myb transcription 8 and to interact with and activate FoxM1. 9 Mammalian B-Myb is a product of the MYBL2 gene that is expressed in all dividing cells, such that loss of function mutations in this gene causes early embryonic lethality. 10 Orthologous MYBL2 in both drosophila [11][12][13] and zebra fish 14 is similarly essential for the regulation of mitotic progression.…”

PARP-10, a novel Myc-interacting protein with poly(ADP-ribose) polymerase activity, inhibits transformation

“…Coimmunoprecipitated proteins were detected by Western blot analysis. PARP-10 expression was analysed by Western blotting using whole-cell extracts prepared in RIPA buffer (Luscher-Firzlaff et al, 1999). For immunoprecipitations and Western blot analysis, anti-HA-tag mAb 3F10 (Roche) and anti-Flag-tag mAb M2 (Sigma) were used.…”

“…The supplements were 100 U of penicillin, 100 mg/ml streptomycin, and 10% fetal calf serum (FCS). Transient transfection assays were performed as described before (Luscher-Firzlaff et al, 1999). Transformation assays using rat embryo cells were carried out essentially as described previously (Cerni et al, 1995).…”

“…Polyclonal antisera and mAbs against PARP-10 were generated by immunizing New Zealand white rabbits and LOU/C rats, respectively, with a bacterially expressed GST-PARP-10(1-907) fusion protein. GST-pulldown assays were performed as described previously (Luscher-Firzlaff et al, 1999). PARP-10 and c-Myc were in vitro transcribed and translated using the TNT-coupled reticulocyte lysate system (Promega).…”