Tapasin Paul Spearmanin kirjassa maininnan merlin proteineista ja nyt yritän saada niistä käsityksen. Siis tässä kirjoituksen alussa minulla EI ole niistä käsitystä.
Löysin erään Korean kustantaman tutkimuksen netistä ja katson sitä ensiksi. En vielä suomenna. Kerään kuvia tähän artikkeliin tekstin selventämiseksi.
The neurofibromatosis type 2 gene-encoded protein, merlin, is related to the ERM (ezrin, radixin, and moesin) family of membrane-cytoskeleton-associated proteins.
Recent studies suggest that the loss of neurofibromatosis type 2 function contributes to tumor development and metastasis.
Although the cellular functions of merlin as a tumor suppressor are relatively well characterized, the cellular mechanism whereby merlin controls cell proliferation from membrane locations is still poorly understood.
During our efforts to find potential merlin modulators through protein-protein interactions, we identified transactivation-responsive RNA-binding protein (TRBP) as a merlin-binding protein in a yeast two-hybrid screen.
The interaction between TRBP and merlin was confirmed by glutathione S-transferase pull-down assays, co-immunoprecipitation, and co-localization experiments. The carboxyl-terminal regions of each protein were responsible for their interaction.
Cells overexpressing TRBP showed enhanced cell growth in cell proliferation assays and also exhibited transformed phenotypes, such as anchorage-independent cell growth and tumor development in mouse xenografts.
Merlin efficiently inhibited these oncogenic activities of TRBP in our experiments.
These results provide the first clue to the functional interaction between TRBP and merlin and suggest a novel mechanism for the tumor suppressor function of merlin both in vitro and in vivo.
For the better understanding of merlin function, merlin-associated proteins were screened using a yeast two-hybrid interaction trap strategy from a human brain cDNA library.
We identified a human transactivation-responsive RNA-binding protein (TRBP) as a novel protein interacting with merlin via its carboxyl-terminal domain.
TRBP is encoded by the tarbp2 gene localized at human chromosome 12 and mouse chromosome 15 (12, 13). The two isoforms of TRBP, TRBP1 (original TRBP) and TRBP2, are expressed by alternative transcriptional initiation, resulting in the TRBP2 protein that is longer than TRBP1 by 21 amino acids at its amino terminus (14–16).
TRBP was originally cloned as an HIV-1 transactivation-response RNA-binding protein and belongs to the family of double-stranded RNA (dsRNA)-binding proteins with two clearly defined dsRNA-binding domains (dsRBDs) and a carboxyl-terminal basic region (17–21).
TRBP acts in synergy with functional Tat to stimulate the expression of the HIV-1 long terminal repeats LTR in human and murine cells (14, 22). The murine homologue, Prbp, binds the 3′-untranslated region of Prm1 protamine RNA, represses its translation, and plays a physiological role in spermatogenesis (23, 24).
TRBP also directly binds the double-stranded RNA-dependent protein kinase (PKR) that has anti-viral and anti-proliferative effects.
In relation to the inhibition of PKR activity, TRBP was recently demonstrated to play a growth promoting role and has an oncogenic potential (26).
- Therefore, it is possible that the tumor suppressor merlin interacts with oncogenic TRBP and inhibits its function.
- In this report, we present evidence for the direct interaction between merlin and TRBP both in vitro and in vivo and show that merlin reverses the growth promoting and tumorigenic activities of TRBP.
Previously, we reported that
- merlin inhibited the Ras signaling pathway strongly related to cell transformation and tumor development (27, 34).
- Moreover, we recently observed that merlin increased p53 activity by inducing MDM2 degradation in NIH3T3 cells (35).
- Considering all of these results and related papers, it would appear that merlin has multiple activities for tumor suppression, one of which might be the suppression of TRBP function reported herein.
In this study, we showed that TRBP-mediated anchorage-independent cell growth and tumorigenesis in the nude mouse model were suppressed by merlin (Figs. 7 and 8). It was also demonstrated that the cell proliferation (Fig. 6) and foci formation (data not shown) induced by TRBP was inhibited by the co-expression of merlin in NIH3T3 cells. These findings suggest that merlin plays a role in the regulation of the oncogenic activity of TRBP.
TRBP was initially known as a cellular protein that binds HIV-1 transactivation-response RNA and increases viral expression from the long terminal repeat in synergy with functional Tat (14, 22). TRBP is also known to play a role in the cellular down-regulation of the PKR that has anti-viral and anti-proliferative effects. TRBP directly binds PKR and inhibits its ability to phosphorylate eukaryotic translation initiation factor 2, leading to its inactivation (25, 26). In relation to the inhibition of PKR activity, TRBP was recently demonstrated to have a growth promoting role and oncogenic potential; cells that overexpress TRBP exhibit transformed phenotypes (26). Therefore it is possible that the inhibition of TRBP function by merlin leads to PKR activation, which in turn inhibits cell proliferation and transformation. However, the exact role of each protein and the precise mechanism of their interaction in tumor development need further investigation.
The functional counteraction between TRBP and merlin appears to be mediated via direct interaction of these proteins. The physical interaction of merlin and TRBP was demonstrated in vitro, in vivo, and in the physiological condition (Figs. 3 and 4), and domain analysis identified the carboxyl-terminal regions of each protein as being responsible for their interaction (Table I and Figs. 3 and 4).
Both the endogenous and overexpressed merlins in cells seem to exist predominantly in the phosphorylated form, suggesting thatwas observed mainly in the cytoplasm2font-weight: bold;">hypophosphorylated merlin is tightly controlled.
Previously, it was reported that the active hypophosphorylated merlin associates with β-catenin, a potential oncogene, and functions as a tumor and metastasis suppressor by controlling cadherin-mediated cell-cell contact (11).
Merlin associates with β-catenin at sites of cell-cell contact and the loss of merlin at these locations may directly impair the formation of adherens junctions or the stability of their components, leading to the loss of contact-dependent inhibition of cell growth. In view of the results obtained so far, it is plausible to assume that active merlin also interacts with TRBP and inhibits its ability to control cell growth.
It is not yet clear what the regulation mechanism and downstream events of the interaction between TRBP and merlin are. Notably in this study, merlin seemed to have a role in the membrane subcellular localization of TRBP.
Consistent with previous reports (32, 36, 37), this study showed that the overexpressed merlin-RFP was observed primarily at the cytoplasmic membrane (Fig. 5B) and other previously reported sites, such as the membrane ruffling region and granules/vesicles in the perinuclear region (data not shown).
When co-expressed in the same cells, however, merlin-RFP and TRBP-GFP were co-localized mainly in the perinuclear region and partly in the cell membrane (Fig. 5).
In MEF cells, endogenous TRBP was detected in the cellular membrane region only in the wild type but not in the NF2-deficient MEF cells, which supports further the possible role of merlin in the membrane localization of TRBP.
In conclusion, we demonstrated that TRBP, which was previously described as a double-stranded RNA-binding protein and was involved in tumorigenesis, also interacts specifically with the carboxyl terminus of merlin via its carboxyl-terminal region. The consequences of their interaction include inhibitory effects on TRBP-mediated cell proliferation, anchorage-independent cell growth, oncogenic transformation, and tumor development in nude mice.