Iron-Inducible Nuclear Translocation of a Myb3 Transcription Factor in the Protozoan Parasite Trichomonas vaginalis

Abstract

cIn Trichomonas vaginalis, a novel nuclear localization signal spanning the folded R2R3 DNA-binding domain of a Myb2 protein was previously identified. To study whether a similar signal is used for nuclear translocation by other Myb proteins, nuclear translocation of Myb3 was examined in this report. When overexpressed, hemagglutinin-tagged Myb3 was localized to nuclei of transfected cells, with a cellular distribution similar to that of endogenous Myb3. Fusion to a bacterial tetracycline repressor, R2R3, of Myb3 that spans amino acids (aa) 48 to 156 was insufficient for nuclear translocation of the fusion protein, unless its C terminus was extended to aa 167. The conserved isoleucine in helix 2 of R2R3, which is important for Myb2's structural integrity in maintaining DNA-binding activity and nuclear translocation, was also vital for the former activity of Myb3, but less crucial for the latter. Sequential nuclear influx and efflux of Myb3, which require further extension of the nuclear localization signal to aa 180, were immediately induced after iron repletion. Sequence elements that regulate nuclear translocation with cytoplasmic retention, nuclear influx, and nuclear efflux were identified within the C-terminal tail. These results suggest that the R2R3 DNAbinding domain also serves as a common module for the nuclear translocation of both Myb2 and Myb3, but there are intrinsic differences between the two nuclear localization signals.

Signal Transduction Triggered by Iron to Induce the Nuclear Importation of a Myb3 Transcription Factor in the Parasitic Protozoan Trichomonas vaginalis

“…Oligonucleotides-Sequences of the oligonucleotides used in the present study are listed in Table 1 unless otherwise reported elsewhere (26,27).…”

“…Plasmid Construction-The HA-Myb3 expression plasmid, pAP65-2.1-ha-myb3, was obtained from a previous study (27). To produce a point mutation in HA-Myb3, a 5Ј-DNA fragment was amplified by polymerase chain reaction (PCR) from pAP65-2.1-ha-myb3 using a primer pair, tub90f and Myb3-(XnXЈ)-3Ј (where X indicates the amino acid to be mutated, n indicates the location of the residue, and XЈ is the mutated residue), and a 3Ј-DNA fragment was amplified by PCR using a primer pair, Myb3-(XnXЈ)-5Ј and SP6.…”

“…Gel-purified PCR products were mixed, denatured, and annealed for a second PCR using a primer pair, tub90f and SP6. The PCR product digested by BglII and NsiI was cloned into a BglII/NsiI restricted pAP65-2.1.ha-Myb3 backbone (27) to generate pAP65-2.1-ha-myb3-T156A, pAP65-2.1-ha-myb3-T156D, and pAP65-2.1-ha-myb3-K143R.…”

“…Studies on transcription regulation of an iron-inducible ap65-1 gene in the parasite showed that upon overnight exposure, iron could exert differential effects on three transcription factors, Myb1, Myb2, and Myb3, which are not iron-binding proteins, at the levels of gene expression, nuclear translocation, and promoter entry (24 -26). In contrast, iron was recently shown to induce a nuclear influx of Myb3 in T. vaginalis within several min (27), implying that the parasite might elicit immediate cellular responses right after a sudden iron overload. With an estimated 46000 -60000 protein genes and a vast array of Ͼ900 protein kinase genes in the genome of T. vaginalis (28,29), the signal transduction network might be crucial for the unicellular parasite to quickly adapt to and thrive in the ever-changing host environments.…”

The cell survival pathways of the primordial RNA-DNA complex remain conserved in the extant genomes and may function as proto-oncogenes

“…Of the three tvMybs, the tvMyb3 protein shows 83% aa sequence identity with human cMyb. Myb proteins are widely expressed in various avian and mammalian hosts and in plants [48][49][50][51]. The ciliates Sterkiella (also known as Oxytricha) and the ciliaphora Euplotes both carry myb genes in their genomes [52].…”