Ran-binding Protein M Research Articles

Identifying Cytoplasmic and Nuclear C‐Terminal to LisH (CTLH) Complex Interactors Using High‐Throughput Proteomics

The C-terminal to LisH (CTLH) complex is a tumour-suppressive E3 Really Interesting New Gene (RING) ubiquitin ligase that regulates intracellular signaling pathways and maintains cellular homeostasis. It is comprised of 8 individual proteins that have been shown to mediate a variety of intracellular protein-protein interactions; of these proteins, Required for Meiotic Division 5 Homolog A (RMND5A) and Macrophage Erythroblast Attacher (MAEA) contain the RING domains required for the CTLH complex to mediate ubiquitin transfer to specific protein substrates. Although its E3 ligase activity has been recently characterized, the endogenous subcellular localization of the complex and compartment-specific protein interaction network – which encompasses many unidentified E3 ligase substrates – has not been identified. In this study, we hypothesized that the CTLH complex is present in the cytoplasm and nucleus, and that the CTLH complex mediates compartment-specific protein-protein interactions with cytoplasmic and nuclear proteins. To test this hypothesis, we used a chemical-based subcellular fractionation method combined with low- and high-throughput proteomics. We first optimized the chemical fractionation method and used Western blot to detect each endogenous CTLH complex member in HeLa cell cytoplasmic and nuclear protein extracts. We then identified compartment-specific CTLH complex interactors using Affinity Purification Coupled to Mass Spectrometry (AP-MS) using the main CTLH complex scaffold protein – Ran Binding Protein M (RanBPM) – as bait in cytoplasmic and nuclear HeLa protein extracts. Following Liquid Chromatography Electrospray Ionizing Tandem Mass Spectrometry (LC-ESI-MS/MS) and subsequent in silico filtering using the MiST algorithm, we identified 27 cytoplasmic, 155 nuclear, and 31 nucleocytoplasmic high-confidence (MiST Score ≥ 0.75) RanBPM interactors. STRING enrichment analysis of the high-confidence interactors revealed that RanBPM and its associated CTLH complex may be involved in a variety of conserved Gene Ontology (GO) processes including Epigenetic Regulation of Gene Expression and Chromatin Assembly. As such, we validated a number of newly-identified compartment-specific RanBPM interactors associated with the enriched GO terms by co-immunoprecipitation – using RanBPM as bait – and resulting Western blot. Furthermore, we focussed on an interesting novel putative interactor that is present in the two aforementioned GO processes – histone variant MacroH2A1 – and further validated its interaction with the CTLH complex in vivo using Proximity Ligation Assay (PLA), suggesting that this interaction may be physiologically relevant. Overall, by using this workflow, we identified and validated a number of novel compartment-specific potential CTLH complex interactors in vitro, further validated a promising novel interaction between the CTLH complex and MacroH2A1 in vivo, and successfully implicated the CTLH complex in a number of previously unexplored cellular mechanisms.

Regulation of c-Raf Stability through the CTLH Complex.

c-Raf is a central component of the extracellular signal-regulated kinase (ERK) pathway which is implicated in the development of many cancer types. RanBPM (Ran-Binding Protein M) was previously shown to inhibit c-Raf expression, but how this is achieved remains unclear. RanBPM is part of a recently identified E3 ubiquitin ligase complex, the CTLH (C-terminal to LisH) complex. Here, we show that the CTLH complex regulates c-Raf expression through a control of its degradation. Several domains of RanBPM were found necessary to regulate c-Raf levels, but only the C-terminal CRA (CT11-RanBPM) domain showed direct interaction with c-Raf. c-Raf ubiquitination and degradation is promoted by the CTLH complex. Furthermore, A-Raf and B-Raf protein levels are also regulated by the CTLH complex, indicating a common regulation of Raf family members. Finally, depletion of CTLH subunits RMND5A (required for meiotic nuclear division 5A) and RanBPM resulted in enhanced proliferation and loss of RanBPM promoted tumour growth in a mouse model. This study uncovers a new mode of control of c-Raf expression through regulation of its degradation by the CTLH complex. These findings also uncover a novel target of the CTLH complex, and suggest that the CTLH complex has activities that suppress cell transformation and tumour formation.

Ran-binding protein M is associated with human spermatogenesis and oogenesis.

The aim of the present study was to explore the underlying mechanism and diagnostic potential of Ran-binding protein M (RanBPM) in human spermatogenesis and oogenesis. RanBPM expression in human testis and ovaries was analysed using polymerase chain reaction (PCR) and western blotting, and immunofluorescence was performed on testis and ovary tissue sections during different developmental stages of spermatogenesis and oogenesis using RanBPM antibodies. Interactions with a variety of functional proteins were also investigated. RanBPM mRNA and protein expression levels were determined by PCR and western blotting in the tissue sections. Results revealed that the mRNA expression levels were highest in the testis followed by the ovary. The RanBPM protein was predominantly localized in the nucleus of germ cells, and the expression levels were highest in pachytene spermatocytes and cells surrounding spermatids in testis tissue. In ovary cells, RanBPM was localized in the nucleus and cytoplasm. In conclusion, the results suggested that RanBPM may have multiple roles in the regulation of germ cell proliferation during human spermatogenesis and oogenesis. This research may provide a novel insight into the underlying molecular mechanism of RanBPM and may have implications for the clinical diagnosis and treatment of human infertility.

Inhibition of HDAC6 activity through interaction with RanBPM and its associated CTLH complex

BackgroundHistone deacetylase 6 (HDAC6) is a microtubule-associated deacetylase that promotes many cellular processes that lead to cell transformation and tumour development. We previously documented an interaction between Ran-Binding Protein M (RanBPM) and HDAC6 and found that RanBPM expression inhibits HDAC6 activity. RanBPM is part of a putative E3 ubiquitin ligase complex, termed the C-terminal to LisH (CTLH) complex. Here, we investigated the involvement of the CTLH complex on HDAC6 inhibition and assessed the outcome of this regulation on the cellular motility induced by HDAC6.MethodsCell lines (Hela, HEK293 and immortalized mouse embryonic fibroblasts) stably or transiently downregulated for several components of the CTLH complex were employed for the assays used in this study. Interactions of HDAC6, RanBPM and muskelin were assessed by co-immunoprecipitations. Quantifications of western blot analyses were employed to evaluate acetylated α-tubulin levels. Confocal microscopy analyses were used to determine microtubule association of HDAC6 and CTLH complex members. Cell migration was evaluated using wound healing assays.ResultsWe demonstrate that RanBPM-mediated inhibition of HDAC6 is dependent on its association with HDAC6. We show that, while HDAC6 does not require RanBPM to associate with microtubules, RanBPM association with microtubules requires HDAC6. Additionally, we show that Twa1 (Two-hybrid-associated protein 1 with RanBPM) and MAEA (Macrophage Erythroblast Attacher), two CTLH complex members, also associate with α-tubulin and that muskelin, another component of the CTLH complex, is able to associate with HDAC6. Downregulation of CTLH complex members muskelin and Rmnd5A (Required for meiotic nuclear division homolog A) resulted in decreased acetylation of HDAC6 substrate α-tubulin. Finally, we demonstrate that the increased cell migration resulting from downregulation of RanBPM is due to the relief in inhibition of HDAC6 α-tubulin deacetylase activity.ConclusionsOur work shows that RanBPM, together with the CTLH complex, associates with HDAC6 and restricts cell migration through inhibition of HDAC6 activity. This study uncovers a novel function for the CTLH complex and suggests that it could have a tumour suppressive role in restricting HDAC6 oncogenic properties.

Ran binding protein 9 (RanBPM) binds IFN-λR1 in the IFN-λ signaling pathway.

Like the type I interferons (IFNs), the recently discovered cytokine IFN-λ displays antiviral, antiproliferative, and proapoptotic activities, mediated by a heterodimeric IFN-λ receptor complex composed of a unique IFN-λR1 chain and the IL-10R2 chain. However, the molecular mechanism of the IFN-λ-regulated pathway remains unclear. In this study, we newly identified RAN-binding protein M (RanBPM) as a binding partner of IFN-λR1. The interaction between RanBPM and IFN-λR1 was identified with a glutathione S-transferase pull-down assay and coimmunoprecipitation experiments. IFN-λ1 stimulates this interaction and affects the cellular distribution of RanBPM. However, the interaction between RanBPM and IFN-λR1 does not correlate with their conserved TRAF6-binding sites. Furthermore, we also found that RanBPM is a scaffolding protein with a modulatory function that regulates the activities of IFN-stimulated response elements. Therefore, RanBPM plays a novel role in the IFN-λ-regulated signaling pathway.

RanBPM Likely Mediates Sodium Chloride Cotransporter (NCC) Regulation through a WNK4‐ERK 1/2 Signaling Pathway

Ran‐Binding protein M (RanBPM) is a nucleoplasmic protein. A recent study has shown that RanBPM is an inhibitor of ERK 1/2 signaling pathway by regulating c‐Raf stability. WNK is a serine/threonine kinase. Mutations in WNK1 and WNK4 cause pseudohypoaldosteronism type II (PHA II), characterized by hypertension, hyperkalemia and metabolic acidosis. Our previous study has shown that WNK4 inhibits NCC by activating ERK 1/2 signaling pathway. To determine whether WNK4 inhibits NCC through RanBPM‐dependent ERK 1/2 signaling pathway, we first investigated interaction between WNK4 and RanBPM using yeast‐two hybrid screening technique. We found that WNK4 interacted with RanBPM. RanBPM was also shown to prevent ERK 1/2 phosphorylation in response to EGF stimulation. When mouse distal convoluted cells (mDCT) were transfected with myc‐RanBPM, ERK 1/2 phosphorylation was decreased while NCC protein expression increased. When mDCT cells were transfected with siRNA RanBPM, ERK 1/2 phosphorylation was increased. These data suggest that 1) WNK4 interacts with RanBPM which is an inhibitor of ERK 1/2 phosphorylation; 2) RanBPM mediates NCC regulation likely through ERK1/2 signaling pathway.

Identification of Ran-binding protein M as a stanniocalcin 2 interacting protein and implications for androgen receptor activity

Stanniocalcin (STC), a glycoprotein hormone originally discovered in fish, has been implicated in calcium and phosphate homeostasis. While fishes and mammals possess two STC homologs (STC1 and STC2), the physiological roles of STC2 are largely unknown compared with those of STC1. In this study, we identified Ran-binding protein M (RanBPM) as a novel binding partner of STC2 using yeast two-hybrid screening. The interaction between STC2 and RanBPM was confirmed in mammalian cells by immunoprecipitation. STC2 enhanced the RanBPM-mediated transactivation of liganded androgen receptor (AR), but not thyroid receptor β, glucocorticoid receptor, or estrogen receptor β. We also found that AR interacted with RanBPM in both the absence and presence of testosterone (T). Furthermore, we discovered that STC2 recruits RanBPM/AR complex in T-dependent manner. Taken together, our findings suggest that STC2 is a novel RanBPM-interacting protein that promotes AR transactivation. [BMB Reports 2014; 47(11): 643-648]

Aggresome formation is regulated by RanBPM through an interaction with HDAC6

ABSTRACTIn conditions of proteasomal impairment, the build-up of damaged or misfolded proteins activates a cellular response leading to the recruitment of damaged proteins into perinuclear aggregates called aggresomes. Aggresome formation involves the retrograde transport of cargo proteins along the microtubule network and is dependent on the histone deacetylase HDAC6. Here we show that ionizing radiation (IR) promotes Ran-Binding Protein M (RanBPM) relocalization into discrete perinuclear foci where it co-localizes with aggresome components ubiquitin, dynein and HDAC6, suggesting that the RanBPM perinuclear clusters correspond to aggresomes. RanBPM was also recruited to aggresomes following treatment with the proteasome inhibitor MG132 and the DNA-damaging agent etoposide. Strikingly, aggresome formation by HDAC6 was markedly impaired in RanBPM shRNA cells, but was restored by re-expression of RanBPM. RanBPM was found to interact with HDAC6 and to inhibit its deacetylase activity. This interaction was abrogated by a RanBPM deletion of its LisH/CTLH domain, which also prevented aggresome formation, suggesting that RanBPM promotes aggresome formation through an association with HDAC6. Our results suggest that RanBPM regulates HDAC6 activity and is a central regulator of aggresome formation.

Functional Interactions between BM88/Cend1, Ran-Binding Protein M and Dyrk1B Kinase Affect Cyclin D1 Levels and Cell Cycle Progression/Exit in Mouse Neuroblastoma Cells

BM88/Cend1 is a neuronal-lineage specific modulator with a pivotal role in coordination of cell cycle exit and differentiation of neuronal precursors. In the current study we identified the signal transduction scaffolding protein Ran-binding protein M (RanBPM) as a BM88/Cend1 binding partner and showed that BM88/Cend1, RanBPM and the dual specificity tyrosine-phosphorylation regulated kinase 1B (Dyrk1B) are expressed in mouse brain as well as in cultured embryonic cortical neurons while RanBPM can form complexes with either of the two other proteins. To elucidate a potential mechanism involving BM88/Cend1, RanBPM and Dyrk1B in cell cycle progression/exit, we transiently co-expressed these proteins in mouse neuroblastoma Neuro 2a cells. We found that the BM88/Cend1-dependent or Dyrk1B-dependent down-regulation of cyclin D1 is reversed following their functional interaction with RanBPM. More specifically, functional interaction of RanBPM with either BM88/Cend1 or Dyrk1B stabilizes cyclin D1 in the nucleus and promotes 5-bromo-2'-deoxyuridine (BrdU) incorporation as a measure of enhanced cell proliferation. However, the RanBPM-dependent Dyrk1B cytosolic retention and degradation is reverted in the presence of Cend1 resulting in cyclin D1 destabilization. Co-expression of RanBPM with either BM88/Cend1 or Dyrk1B also had a negative effect on Neuro 2a cell differentiation. Our results suggest that functional interactions between BM88/Cend1, RanBPM and Dyrk1B affect the balance between cellular proliferation and differentiation in Neuro 2a cells and indicate that a potentially similar mechanism may influence cell cycle progression/exit and differentiation of neuronal precursors.

RanBPM Is an Inhibitor of ERK Signaling

Ran-binding protein M (RanBPM) is a nucleocytoplasmic protein of yet unknown function. We have previously shown that RanBPM inhibits expression of the anti-apoptotic factor Bcl-2 and promotes apoptosis induced by DNA damage. Here we show that the effects of RanBPM on Bcl-2 expression occur through a regulation of the ERK signaling pathway. Transient and stable down-regulation of RanBPM stimulated ERK phosphorylation, leading to Bcl-2 up-regulation, while re-expression of RanBPM reversed these effects. RanBPM was found to inhibit MEK and ERK activation induced by ectopic expression of active RasV12. Activation of ERK by active c-Raf was also prevented by RanBPM. Expression of RanBPM correlated with a marked decrease in the protein levels of ectopically expressed active c-Raf and also affected the expression of endogenous c-Raf. RanBPM formed a complex with both active c-Raf, consisting of the C-terminal kinase domain, and endogenous c-Raf in mammalian cells. In addition, RanBPM was found to decrease the binding of Hsp90 to c-Raf. Finally, we show that loss of RanBPM expression confers increased cell proliferation and cell migration properties to HEK293 cells. Altogether, these findings establish RanBPM as a novel inhibitor of the ERK pathway through an interaction with the c-Raf complex and a regulation of c-Raf stability, and provide evidence that RanBPM loss of expression results in constitutive activation of the ERK pathway and promotes cellular events leading to cellular transformation and tumorigenesis.

Stability and Function of Mammalian Lethal Giant Larvae-1 Oncoprotein Are Regulated by the Scaffolding Protein RanBPM

The evolutionarily conserved lethal giant larvae (Lgl) tumor suppressor gene has an essential role in establishing apical-basal cell polarity, cell proliferation, differentiation, and tissue organization. However, the precise molecular mechanism by which the Lgl carries out its function remains obscure. In the current study, we have identified Ran-binding protein M (RanBPM) as a novel binding partner of Mgl-1, a mammalian homolog of Drosophila tumor suppressor protein lethal (2) giant larvae (L(2)gl) by yeast two-hybrid screening. RanBPM seems to act as a scaffolding protein with a modulatory function with respect to Mgl-1. The Mgl-1 and RanBPM association was confirmed by co-immunoprecipitation and GST pull-down experiments. Additionally, expression of RanBPM resulted in inhibition of Mgl-1 degradation, and thereby extended the half-life of Mgl-1. Furthermore, the ability of Mgl-1 activity in cell migration and colony formation assay was enhanced by RanBPM. Taken together, our findings reveal that RanBPM plays a novel role in regulating Mgl-1 stability and contributes to its biological function as a tumor suppressor.

RanBPM regulates cell shape, arrangement, and capacity of the female germline stem cell niche in Drosophila melanogaster

Experiments in cultured cells with Ran-binding protein M (RanBPM) suggest that it links cell surface receptors and cell adhesion proteins. In this study, we undertake a genetic study of RanBPM function in the germline stem cell (GSC) niche of Drosophila melanogaster ovaries. We find that two RanBPM isoforms are produced from alternatively spliced transcripts, the longer of which is specifically enriched in the GSC niche, a cluster of somatic cells that physically anchors GSCs and expresses signals that maintain GSC fate. Loss of the long isoform from the niche causes defects in niche organization and cell size and increases the number of GSCs attached to the niche. In genetic mosaics for a null RanBPM allele, we find a strong bias for GSC attachment to mutant cap cells and observe abnormal accumulation of the adherens junction component Armadillo (β-catenin) and the membrane skeletal protein Hu-li tai shao in mutant terminal filament cells. These results implicate RanBPM in the regulation of niche capacity and adhesion.

Serine/Threonine Kinase Mirk/Dyrk1B Is an Inhibitor of Epithelial Cell Migration and Is Negatively Regulated by the Met Adaptor Ran-binding Protein M

Minibrain-related kinase (Mirk)/Dyrk1B is an arginine-directed serine/threonine kinase that is active in skeletal muscle development but is also expressed in various carcinomas. In the current study, the Met adaptor protein Ran-binding protein M (RanBPM) was identified as a Mirk-binding protein by yeast two-hybrid analysis. The Mirk-RanBPM association was confirmed by glutathione S-transferase pull-down assays, co-immunoprecipitation studies, and in vivo cross-linking. Met plays an important role in tumor cell invasion and cell migration. RanBPM has been reported to bind to the tyrosine kinase domain of the hepatocyte growth factor (HGF) receptor Met, enhance Met downstream signaling, and enhance HGF-induced A704 kidney carcinoma cell invasion (Wang, D., Li, Z., Messing, E. M., and Wu, G. (2002) J. Biol. Chem. 277, 36216-36222). We made a stable Mirk-inducible subline from nontransformed Mv1Lu lung epithelial cells and now demonstrate that induction of Mirk inhibited the migration of these cells in wounding experiments and inhibited their invasion through polycarbonate Transwell filters. Furthermore the ability of Mirk to inhibit Mv1Lu cell migration was attenuated when cells were exposed to HGF or to elevated levels of transiently expressed RanBPM. RanBPM inhibited the kinase activity of Mirk/Dyrk1B and Dyrk1A. In addition, RanBPM and HGF inhibited the function of Mirk as a transcriptional coactivator. Our findings suggest that Mirk plays a role in modulating cell migration through opposing the action of the Met signaling cascade adaptor protein RanBPM.