Ras enhances TGF-β signaling by decreasing cellular protein levels of its type II receptor negative regulator SPSB1
© The Author(s). 2018
Received: 8 February 2018
Accepted: 5 March 2018
Published: 13 March 2018
Transformation by oncogene Ras overcomes TGF-β mediated growth inhibition in epithelial cells. However, it cooperates with each other to mediate epithelial to mesenchymal transition (EMT). The mechanism of how these two pathways interact with each other is controversial.
Molecular techniques were used to engineer expression plasmids for Ras, SPRY, TGF-β receptors, type I and II and ubiquitin. Immunoprecipitation and western blots were employed to determine protein-protein interactions, preotein levels, protein phosphorylation while immunofluorecesent staining for molecular co-localization. TGF-β signalling activities is also determined by its luciferase reporter assay. Trans-well assays were used to measure cell migration and invasion.
Ras interacts with the SPSB1’s SPRY domain to enhance TGF-β signaling. Ras interacts and colocalizes with the TGF-β type II receptor’s (TβRII) negative regulator SPSB1 on the cell membrane, consequently promoting SPSB1 protein degradation via enhanced mono- and di-ubiquitination. Reduced SPSB1 levels result in the stablization of TβRII, in turn the increase of receptor levels significantly enhance Smad2/3 phosphorylation and signaling. Importantly, forced expression of SPSB1 in Ras transformed cells suppresses TGF-β signaling and its mediated migration and invasion.
Ras positively cooperates with TGF-β signaling by reducing the cellular protein levels of TβRII negative regualtor SPSB1.
TGF-β regulates a plethora of cellular processes including cell proliferation, differentiation, migration, organization and death . As one of the most potent inhibitors of normal cell growth, the loss of growth inhibitory responses to TGF-β is often observed in cancer cells [2, 3]. It is widely accepted that TGF-β is a tumor suppressor, given the frequent occurrence of many types of tumors in mice with disruptions of TGF-β signaling components by gene targeting and many types of human cancers containing loss-of-function mutation of TGF-β signaling components . In spite of the tumor suppressor activity of TGF-β, the majority of human tumors have not suffered loss-of function of TGF-β signaling components . Tumor cells, particularly advanced tumor cells, often show increased production of TGF-β while they are insensitive to TGF-β induced growth inhibition . TGF-β acting as an important tumor promoter, particularly at late stages of tumor development, is evidenced by using murine animal models and human cellular systems [7–10], in which TGF-β signaling components are required for tumor invasion in vitro and metastasis in vivo. Clinically, there is a substantial body of evidence that excess TGF-β production is associated with poor prognosis in many types of human tumors . Thus, TGF-β acts as a tumor suppressor in early tumor development, but promotes tumor invasion and metastasis during late stages of tumor progression .
Ras proteins are small GTPases that act as molecular switches by cycling between inactive GDP-bound and active GTP-bound states. It functions as a transducer of the cell signals from the membrane receptor to the intracellular pathway that controls cell proliferation, differentiation, and survival . Constitutive active mutations of Ras are frequently expressed in human cancers— ~ 20 to 30% of all human tumors contain one of the mutated Ras genes, especially in pancreas, thyroid and colon carcinomas (90, 60 and 45% respectively) [13, 14]. Ras plays an important role in tumor initiation as well as in tumor maintenance . Many carcinomas carrying the activated Ras proteins have undergone EMT [16, 17]. It is known that Ras downstream effecter pathway Ras-Raf-MAPK is essential mediating EMT [10, 18]. On the other hand, activation of another Ras downstream effecter pathway PI3K/Akt enhances tumor cell growth and mediates protection from TGF-β induced apoptosis [19, 20].
TGF-β and Ras signaling are two of the most important molecular pathways mediating the fundamental cellular process, namely EMT, involved in tumor metastasis [21, 22]. Depending on the cellular contexts, Ras signaling antagonizes TGF-β-induced growth arrest and apoptosis  by suppressing the TGF-β-Smad signaling . It was reported that Ras, acting through Mek1 and Erk kinases, induced the phosphorylation of Smad2/3 at a cluster of Ser/Thr-Pro sites in the linker region . The Ras-induced phosphorylation in the linker region prevents the accumulation of Smad2/3 in the nucleus. Prolonged activation of Raf/MAPK pathway in MDCK cells significantly reduces Smad3 levels independently of TGF-β stimulation . Recently, Ras has been shown to induce the down-regulation of TβRII . Induced expression of mutant Ras activates MAPK pathway which leads to the recruitment of histone deacetylase (HDAC). HDAC suppresses the TβRII promoter region (− 127/− 75) and consequently results in the down-regulation of TβRII in lung cancer cells . In contrast, Ras signaling was shown to up-regulate TGF-β production , enhancing endogenous TGF-β signaling . During advanced stage of tumour development, Ras signaling often positively cooperates with TGF-β signaling. Activation of the Ras-MAPK signaling often results in autocrine TGF-β signaling which is critical in EMT maintenance [9, 19, 27–29]. It has been shown that metastasis is driven by sequential elevation of H-Ras and Smad2 levels . During tumour progression from keratinocyte towards squamous carcinoma then to invasive spindle cell carcinoma, TGF-β signaling activity was dramatically increased and the constitutively activated Smad2 was observed in invasive spindle tumour cells only. Activated H-Ras over-expression in squamous carcinoma cells demonstrated that oncogenic Ras stimulated TGF-β-induced transcription and enhanced TGF-β-induced phosphorylated Smad2 levels . However, how Ras positively regulates the TGF-β signaling is unclear. The mechanisms of cross-talk between the Ras and TGF-β signaling are being investigated in a number of cell lines, with controversial results .
Recently, we have identified SPSB1 (SPRY domain-containing a SOCS box protein 1) as a novel negative regulator of the TGF-β signaling pathway . The SPSB1 gene expression is induced by TGF-β and it feeds back to negatively regulate the TGF-β signaling pathway. Interestingly, SPSB1 has also been reported to positively regulate the c-MET-Ras-MAPK signaling . We investigate whether SPSB1 bridges the Ras and TGF-β signaling. This study describes a new mechanism of how Ras up-regulates the TGF-β signaling: Ras interacts with the newly identified TβRII negative regulator SPSB1 and causes its degradation via ubiquitination. This leads to the enhanced TβRII levels and consequently increased TGF-β signaling activity. This is the first to report that Ras is directly targeting TGF-β signaling regulatory components to enhance its signaling activity.
Antibodies and reagents
The mouse anti-FLAG (M2) and anti-Actin monoclonal antibodies were obtained from Sigma-Aldrich (St Louis, MO). Mouse monoclonal anti-MYC and anti-Ras (detects endogenous Ras) antibodies were generated in house. Rabbit polyclonal anti-TβRI, anti-TβRII and mouse monoclonal anti-H-Ras antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-FLAG antibody was obtained from ABR (Affinity BioReagents, Golden, CO). Rabbit polyclonal anti-phospho-Smad2 antibody was kindly provided by Prof Peter ten Dijke (Leiden University Medical Center, Netherlands). Mouse monoclonal anti-Smad2 antibody was obtained from BD Transduction Laboratories (Rockville, MD). Goat anti-mouse IgG HRP conjugated secondary antibody, Goat anti-rabbit IgG HRP conjugated secondary antibody were obtained from Bio-Rad (Bio-Rad Laboratories, Gladesville, N.S.W., Australia). The anti-mouse Alexa488 and Alexa546-conjugated secondary antibodies were from Invitrogen (Invitrogen Corp., Mulgrave, Australia). Human recombinant TGF-β1 was obtained from R&D Systems (Minneapolis, MN). Doxycycline and Cycloheximide were purchased from Sigma-Aldrich, while MG132 was obtained from Merck (Merck, Darmstadt, Germany).
DNA constructs and primers
FLAG-TβRI, HA-TβRII and v-Ha-Ras were cloned into pcDNA3 mammalian cell expression vector as described previously [33, 34]. v-Ha-Ras(N85A), v-Ha-Ras(N86A) and v-Ha-Ras(D120A, R124A) were generated based on v-Ha-Ras using Quick Change® II XL Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) according to the manufacturer’s recommendations. The following pimers were used in the PCR reaction: v-Ha-Ras N85A forward: TGTGTATTTGCCATCGCCAACACCAAGTCCTT. v-Ha-Ras N85A reverse: AAGGACTTGGTGTTGGCGATGGCAAATACACA. v-Ha-Ras N86A forward: GTATTTGCCATCAACGCCACCAAGTCCTTTGA. v-Ha-Ras N86A reverse: TCAAAGGACTTGGTGGCGTTGATGGCAAATAC. v-Ha-Ras D120A, R124A forwards: TGGGCAACAAGTGTGCACTGGCCGCTGCCACTGTTGAGTCTC. v-Ha-Ras D120A, R124A reverse: GAGACTCAACAGTGGCAGCGGCCAGTGCACACTTGTTGCCCA. The sequence of all newly generated v-Ha-Ras mutants were confirmed by direct DNA sequencing. FLAG/MYC-SPSB1, FLAG/MYC-SPSB1∆, MYC-SPSB1(Y129A) and MYC-SPSB1(T160A, Y161A) were all cloned into the pEF-BOS mammalian cell expression vector .
Cell lines, cell culture and treatments
The human embryonic kidney cell line HEK-293 T (293 T), the Madin Darby Canine Kidney (MDCK) cell line, the v-Ha-Ras stable transformed MDCK (21D1) cell line have all been previously described [32, 34]. To generate the doxycycline inducible SPSB1 cell line in 21D1 cells, a tetracycline-inducible vector, pTRE was utilized . Briefly, pTRE-FLAG-SPSB1 and pEFpurop-Tet-on  were co-transfected into 21D1 cells by using FuGENE HD transfection reagent (Roche, Basel, Switzerland) following the manufacturer’s instructions and selected for using puromycin (Roche, Basel, Switzerland). To generate the doxycycline inducible v-Ha-Ras cell line in MDCK cells, pTRE-v-Ha-Ras and pEFpurop-Tet-on were co-transfected into MDCK cells by using FuGENE 6 transfection reagent (Roche, Basel, Switzerland) following the manufacturer’s instructions and selected for using puromycin (Roche, Basel, Switzerland). All positive clones were selected by Western blot analysis using FLAG antibody (Sigma-Aldrich) or Ras antibody (In house made). All cells were maintained in Dulbecco’s Modified Eagle’s Medium contained 10% foetal bovine serum (FBS) (DKSH, Hallam, Victoria, Australia), 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptoMYCin (Invitrogen).
Cells were transiently transfected with firefly luciferase (luc) construct pCAGA 12 -luc , along with other DNA constructs as indicated using FuGENE HD transfection kit for 293 T cells and METAFECTENE PRO (Biontex Laboratories, San Diego, CA) for all other cells. Twenty-four hours after transfection, cells were stimulated with ± TGF-β at indicated concentration in medium containing 10% FCS for a further 24 h. Thereafter, cells were lysed and assessed for luciferase activity using the Luciferase Reporter Assay Kit (Promega Corp, Madison, WI) following the manufacturers’ instructions.
Immunoprecipitation and immunoblotting
After transfection, cells were lysed in lysis buffer (50 mM Tris, 150 mM NaCl, 1% Triton-X-100, 50 mM NaF, 2 mM MgCl2, 1 mM Na3VO4, 25 μg/ml leupeptin and 25 μg/ml aprotinin) and cell lysates were subjected to immunoprecipitation with appropriate antibody conjugated sepharose protein G bead or anti-FLAG beads (Sigma-Aldrich) for 4 h. Immunoprecipitates were washed three times with ice-cold PBS containing 0.5% Tween-20 and immunoprecipitated proteins were separated by SDS-PAGE (Invitrogen) and blotted onto nitrocellulose membrane and probed with the indicated primary antibodies. The signal was visualized using the ECL chemoluminescence detection kit (GE Healthcare, Rydelmere, N.S.W., Australia) following incubation with appropriate secondary antibodies.
Qualitative analysis for protein half life
The intensity of the bands in western blot images was measured using image J. Rectangular selection tool was used to select the area where the bands were located (the intensity of bands that were used to calculate the half-life of the protein was measured together in one selected area). The gaps between each band were used as relative background. The intensity of each band was measured 3 times by selecting three different gap intensities as the relative background (background intensity selected at low, medium and high). Protein stability curves were generated by smoothly joining the intensity values of each set of bands in the Y-axis, with their corresponding treatment times in the X-axis using Microsoft excel. Half-life was determined as the time at which protein band intensities were 50% of the starting level (time 0). The value of half-life shown in the results is the mean of the three estimated half-life values for each four bands. The results are shown as the mean of estimated half-life values +/− SD.
Immunofluorescence staining and confocal microscopy
After transfection with appropriate DNA constructs using FuGENE HD for 48 h, cells were washed once in pre-heated 37°C PBS and fixed with 3.7% formaldehyde (Sigma) in PBS for 7 min. Following two PBS washes, cells were permeabilized with 0.2% Triton-X-100 (Merck) in PBS for another 7 min. Cells were then wash 3 times with PBS and blocked with PBS containing 5% BSA for 1 h at room temperature. Following another 3 washes in PBS, cells were stained with relevant primary antibody (diluted in PBS containing 2% BSA) for 1 h at room temperature and washed in PBS 3 more times. Visualisation was achieved with either Alexa546 or Alexa488-conjugated secondary antibody using the Nikon TE2000-E & C1 Confocal Microscope with a Nikon 60X water immerged lens. Nikon confocal EZ-C1 v.1.4 was used to collate images.
In vitro scratch assay and fluorescence microscopy
21D1 cells were transfected with indicated DNA constructs and seeded onto 12-well culture plate until 100% confluent. Forty-eight hours post-transfection, scratches were created using a P1000 pipette tip to scratch a straight line on the culture plate. The culture medium was replaced with fresh medium to remove detached cells. Phase-contrast and fluorescence images were acquired at 0 and 24 h post-scratch using an inverted microscope (IX50, Olympus) equipped with a CCD camera (Model 11.3, Diagnostics instruments, MI), and SPOT advanced imaging software (v4.0.4) was used to acquire and process images.
In vitro invasion and migration assay
21D1 cells were transfected with indicated DNA constructs in a 6-well plate. Forty-eight hours post-transfection, 21D1 (20,000 cells/chamber) cells were resuspended in serum free DMEM and seeded in the top chamber of a 70 μl solidified matrigel (BD Biosciences, 1:1 mixed with DMEM) coated, 8 μm, polycarbonate membrane transwell insert (Corning Incorporated, Corning NY). Serum free DMEM ±2 ng/ml of TGF-β was added to the bottom chamber. Cells were then incubated for 24 h at 37 °C with 10% CO2. Thereafter, cells that invaded through the coated matrigel and migrated to the other side of the membrane of the transwell insert were fixed with 3.7% formaldehyde (Sigma) for 7 min. Cells were then washed and stained with Hoechst for 5 min. Any remaining cells in the top chamber of the transwell insert were removed by using a cotton swab. Only cells in the bottom side of the transwell insert were counted. Fluorescent images were taken in three random fields (20×) per insert. Assays were performed in triplicate.
All statistical analyses were performed using a two-tail Students’ T-test (P < 0.05 indicating statistical significance).
Ras reduces SPSB1 expression levels
To investigate the effect of Ras on the expression of SPSB1, we co-transfected FLAG-SPSB1 with v-Ha-Ras in 293 T cells. As shown in lane 2 of Fig. 1c, SPSB1 was readily expressed in the absence of Ras. However, the expression level of SPSB1 was reduced in the presence of Ras (lane 1, Fig. 1c), suggesting that Ras suppresses SPSB1 expression directly. Indeed, when Ras expression levels were progressively reduced, the expression levels of SPSB1 were progressively restored with or without TGF-β treatment (Fig. 1d), confirming the notion that Ras reduces SPSB1 expression level. Furthermore, EGF-induced activation of endogenous Ras has no effect on the degradation rate of SPSB1 (Additional file 1: Figure S1), suggesting a Ras activation independent mechanism.
Ras interacts with SPSB1 through the SPRY domain
Ras N85 and N86 are not responsible for its interaction with SPSB1
We and others have previously shown that SPSB1 could recognize D-I-N-N-N-X or similar sequence motifs present in multiple target proteins [31, 39]. In order to further investigate the Ras-SPSB1 interaction, we searched for similar motifs in Ras. Our sequence alignment shows Ras contains a stretch of Ile84-Asn-Asn-Thr-Lys88 (I-N-N-T-K) in the middle region of the protein. Based on the importance of the asparagine residues in other SPSB1 interacting proteins (TβRII, Par4, VASA and iNOS) [31, 35, 40–42], we generated two mutant v-Ha-Ras DNA constructs, one containing a N85A substitution and the other N86A. Neither mutation disrupted the function of the v-Ha-Ras in mediating Erk1/2 phosphorylation (Additional file 1: Figure S3), indicating correct folding of the expressed mutants. Anti-Ras immuoprecipitation showed that the SPSB1 was co-precipitated with Ras(N85A) and Ras(N86A) at similar potency as the wild-type Ras (Fig. 2f). This non-disruption of interaction was confirmed by performing a reciprocal immunoprecipitation of SPSB1 (Additional file 1: Figure S4). This suggests that although Ras protein contains a similar motif as D-I-N-N-N-X, the asparagine residues are unlikely to be involved in the Ras-SPSB1 interaction.
To further investigate the Ras-SPSB1 interaction, an alternative strategy was employed. Infact, Ras is not the first protein identified to interact with SPSB1 without carrying the D-I-N-N-N-X motif. The HGF receptor c-Met has been reported to interact with SPSB1, however, sequence alignment suggests that c-Met does not contain any sequence similar to the D-I-N-N-N-X motif . We hypothesize that Ras may share a similar interaction motif with c-Met to interact with SPSB1. Sequence alignment between Ras and c-Met identified an identical short stretch of Asp120-Leu-Ala-Ala-Arg124 (D-L-A-A-R). The charged residues in the D-L-A-A-R sequence were mutated into alanine (v-Ha-Ras(D120A, R124A)). Surprisingly, the mutant protein expression level was low (Additional file 1: Figure S3), however, the double amino acid substitutions did not impair the ability of oncogenic Ras to mediate Erk1/2 phosphorylation (Additional file 1: Figure S3). Interestingly, while Ras(D120A, R124A) was expressed at much lower levels than its wild-type conterpart, a significantly more Ras(D120A, R124A) was detected in the anti-MYC immunoprecipitates (Additional file 1: Figure S5). Furthermore, this interaction between Ras(D120A, R124A) and SPSB1 was confirmed by performing a reciprocal immunoprecipitation of Ras (Additional file 1: Figure S6). As such, the D-L-A-A-R sequence in Ras may be involved for the Ras-SPSB1 interaction.
Ras down-regulates the expression levels of SPSB1 by enhancing SPSB1 ubiquitination
Since early result indicates that oncogenic Ras does not require the I-N-N-T-K sequence for its interaction with SPSB1, Ras(N85A) and Ras(N86A)’s ability to enhance SPSB1 ubiquitination were examined. As we expected, both Ras(N85A) and Ras(N86A) enhanced the levels of mono-ubiquitinated, di-ubiquitinated and possibly tri- ubiquitinated SPSB1 (Fig. 3a). Again, the result was confirmed by reciprocal immunoprecipitation of MYC for ubiquitin (Fig. 3b). Collectively, these results suggest that oncogenic Ras has the ability to induce the mono-, di- and possibly tri-ubiquitination of SPSB1.
To further confirm the ubiquitination degradation of SPSB1 by oncogenic Ras, we used proteasome inhibitor MG132 to block the protein degradation. As expected, oncogenic Ras induced protein degradation of SPSB1 slowed by treatment of MG132 (Fig. 4d). Collectively, our results suggest that the oncogenic Ras-SPSB1 interaction results in the enhanced ubiquitination and degradation of SPSB1.
Ras inhibits SPSB1 mediated ubiquitination of TβRII and hence stabilizes TβRII
Since oncogenic Ras inhibits SPSB1-mediated TβRII ubiquitination, we next investigated the effect of oncogenic Ras on the SPSB1-mediated TβRII degradation using cyclohexmide treatment. As shown in Fig. 5b, in the presence of SPSB1, the half-life of TβRII was measured at ~ 6 h. In contrast, when oncogenic Ras was co-expressed, the degradation rate of TβRII was slowed with its half-life increased to more than 8 h (Fig. 5b). Consistent with those over-expression data, the endogenous TβRI and TβRII levels in the 21D1 cells were observed to be higher than the ones in the partental MDCK cells (Fig. 1a). Collectively, our results suggest that oncogenic Ras compromises the ability of SPSB1-mediated TβRII ubiquitination, and hence, stabilizes TβRII.
TGF-β receptor levels regulate TGF-β signaling sensitivity and duration
While pCAGA-luciferase reporter measures Smad3 transcriptional activity, we further investigated the effect of the receptor levels on their another effector Smad2. Without over-expression of either TβRI or TβRII, TGF-β treatment of 293 T cells resulted in phosphorylation of the Smad2 (Fig. 6b). A slight increase of Smad2 phosphorylation levels were observed when TβRI was over-expressed (Fig. 6b). However, over-expression of TβRII alone in 293 T cells resulted in substantial increase of Smad2 phosphorylation levels and the effect lasted 12 h (the maximum duration examined in experiment). Similarly, such increase of Smad2 phosphorylation levels were also obvious when both TβRI and TβRII were over-expressed (Fig. 6b). Those results are consistent with above observations using Smad3 reporter assay, further suggesting the important regulatory role played by the levels of TGF-β receptors in its signaling.
Ras enhances TGF-β signaling through increase of TβRII levels
To further investigate the interplay among Ras, SPSB1 and TβRII in TGF-β signaling, we used over-expression and TGF-β-Smad3 reporter assay in 293 T cells. While exprssion of SPSB1 suppressed the reporter activity as expected, however, further over-expression of Ras negated SPSB1’s suppressive effect (Fig. 7c). On the other hand, co-expression of Ras with TβRII markedly increased the reporter activity and that increase was largely suppressed by over-expression of SPSB1 (Fig. 7d).
Reducing TGF-β signaling in Ras transformed 21D1 cells by SPSB1 suppresses cell migration and invasion
We then examined the effect of SPSB1 on the invasion property of 21D1 cells using matrigel coated transwell chamber. Again, cells were co-transfected with eGFP construct to mark SPSB1 expressing cells. In the absence of TGF-β, there were hardly any eGFP labelled 21D1 cells on the bottom side of the membrane regardless SPSB1 expression (Fig. 8c). In contrast, with TGF-β treatment but without SPSB1 expression, many eGFP expressing cells moved through the matrigel to the bottom side of the membrane (Fig. 8c). With SPSB1 expression, the number of eGFP labelled cells were reduced by ~ 50% (Fig. 8c). Collectively, those results demonstrate that SPSB1-mediated reduction of TGF-β signaling in Ras transformed 21D1 cells suppresses cell migration and invasion.
TGF-β signaling both suppresses and promotes tumor progression . Functionally, Ras activation overides the cytostatic growth regulation of TGF-β in early tumor development yet synergises with TGF-β signaling to mediate epithelial to mesenchymal transition (EMT) which is the basic cellular process for tumor invasion and metastasis [10, 11, 19]. At molecular level, it has been demonstrated that persistent activation of the MAPK pathway by oncogenic Ras suppresses TGF-β/Smad signaling by inhibiting the nuclear accumulation of R-Smads [24, 43, 44]. Indeed, human colon carcinoma cell lines of known Ras activating mutations show a correlation between the oncogenic Ras and a deficient nuclear accumulation of activated Smad2 and Smad3 [24, 45]. In contrast, Ras signaling has also been shown to up-regulate TGF-β production , to enhance endogenous TGF-β signaling . A number of reports have shown that activation of Ras-MAPK pathway can enhance TGF-β-mediated responses in a cell-specific manner. In human mesangial cells, activation of the Erk signaling mediates the TGF-β-induced phosphorylation of Smad3 and leads to the induction of α2(I) collagen promoter activity . Furthermore, in a series of well-characterized tumour cell line derived from sequential stages of mouse skin carcinogensis, activated H-Ras over-expression in squamous carcinoma cells demonstrate that Ras stimulates TGF-β-induced transcription and enhances TGF-β-induced phosphorylated Smad2 levels . However, how Ras positively regulates the TGF-β signaling is not clear. The mechanisms of cross-talk between the Ras and TGF-β signaling are being investigated in a number of cell lines, with controversial results .
Recently, SPSB2 has been shown to interact with iNOS and targets iNOS for proteasomal degradation, hence, regulating nitric oxide (NO) production in parasite killing . Soon after, the same group has also demonstrated that SPSB1 is the only SPSB family member to be regulated by the same toll-like receptor (TLR) pathways that induce iNOS expression. And SPSB1 acts through a negative-feedback loop that, together with SPSB2, controls the extent of iNOS induction and NO production . On the other hand, Ras proteins have been shown to positively regulate the nitric oxide synthase family proteins [48, 49]. Our identification of the negative regulatory effect of Ras on SPSB1 may provide a molecular link between the Ras pathway and the nitric oxide synthase pathway.
We identify Ras as the first negative regulator of SPSB1. In addition, we also uncover a new mechanism of how Ras up-regulates the TGF-β signaling. Ras down-regulates SPSB1 by inducing its protein degradation. This leads to the up-regulation of the TGF-β receptors and consequently, results in the high TGF-β signaling activity (Fig. 9). This is the first report that directly involves Ras protein in the up-regultion of the TGF-β signaling.
This work was partially supported by Australian Commonwealth Government National Health and Medical Research Council (NHMRC) funding to H-JZ (#433618&433619).
Availability of data and materials
Availability in supplementary data.
SL and JI conducted all the experiments, RJS and H-JZ initiated the work, H-JZ designed all experiments in consultation with SL, JI&RJS. SL&H-JZ drafted the manuscript and JI&RJS made modifications. All authors read and approved the final manuscript.
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Gene manipulations were conducted in according with University of Melbourne Gene Technology and Biosafety Committee (IBC No 301) approval 2014/008.
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