Sally Shpungin

TMF/ARA160 is a BC-box-containing protein that mediates the degradation of Stat3

TMF/ARA160 is a Golgi resident protein whose cellular functions have not been conclusively revealed. Herein we show that TMF/ARA160 can direct the proteasomal degradation of the key cell growth regulator – Stat3. TMF/ARA160 was dispersed in the cytoplasm of myogenic C2C12 cells that were grown under low-serum conditions. The cytoplasmic distribution of TMF/ARA160 was accompanied by its transient association with the tyrosine kinase Fer and with Stat3, which underwent proteasomal degradation under those conditions. Moreover, serum deprivation induced the association of ubiquitinated proteins, with the TMF/ARA160 complex. However, TMF/ARA160 did not bind Stat1, whose cellular levels were increased in serum-starved C2C12 cells. Amino-acid sequence analysis identified a BC-box element in TMF/ARA160 that mediated the binding of this protein to elongin C. Ectopic expression of TMF/ARA160 in serum-starved C2C12 cells drove the ubiquitination and proteasomal degradation of Stat3, an effect that was not caused by TMF/ARA160 devoid of the BC-box motif. Thus, the Golgi apparatus harbors a novel BC-box-containing protein that can direct Stat3 to proteasomal degradation. Interestingly, the level of TMF/ARA160 was significantly decreased in malignant brain tumors, implying a suppressive role of that protein in tumor progression.

Downregulation of Fer induces PP1 activation and cell-cycle arrest in malignant cells

Fer is a nuclear and cytoplasmic intracellular tyrosine kinase. Herein we show that Fer is required for cell-cycle progression in malignant cells. Decreasing the level of Fer using the RNA interference (RNAi) approach impeded the proliferation of prostate and breast carcinoma cells and led to their arrest at the G0/G1 phase. At the molecular level, knockdown of Fer resulted in the activation of the retinoblastoma protein (pRB), and this was reflected by profound hypo-phosphorylation of pRB on both cyclin-dependent kinase CDK4 and CDK2 phosphorylation sites. Dephosphorylation of pRB was not seen upon the direct targeting of either CDK4 or CDK2 expression, and was only partially achieved by the simultaneous depletion of these two kinases. Amino-acid sequence analysis revealed two protein phosphatase 1 (PP1) binding motifs in the kinase domain of Fer and the association of Fer with the pRB phosphatase PP1α was verified using co-immunoprecipitation analysis. Downregulation of Fer potentiated the activation of PP1α and overexpression of Fer decreased the enzymatic activity of that phosphatase. Our findings portray Fer as a regulator of cell-cycle progression in malignant cells and as a potential target for cancer intervention.

TMF/ARA160: A key regulator of sperm development.

TMF/ARA160 is a Golgi-associated protein to which several cellular activities have been attributed. These include, trafficking of Golgi-derived vesicles and E3 ubiquitin ligase activity. Here we show that TMF/ARA160 is required for the onset of key processes which underlie the development of mature sperm in mammals. TMF/ARA160 is highly expressed in specific spermatogenic stages. While the protein is not detected in the spermatogenic progenitor cells - spermatogonia, it accumulates in the Golgi of spermatocytes and spermatids but then disappears and is absent from spermatozoa and epididymal sperm cells. Mice that are homozygous null for TMF develop normally are healthy and the females are fertile. However, the males are sterile and their spermatids suffer from several developmental defects. They lack homing of Golgi-derived proacrosomal vesicles to the perinuclear surface, resulting in spermatozoa and epididymal sperm cells which lack acrosome. In a later developmental stage, the cytoplasm is not properly removed, thus resulting in spermatids which bare the nucleus with tightly packed DNA, surrounded by a cytoplasm. Finally, the spermatozoa of TMF(-/-) mice also suffer from misshapen heads, tails coiling around the sperm heads, and lack of motility. Taken together our findings portray TMF/ARA160 as a key regulator which is essential for the onset of key events in the differentiation and maturation of mammalian sperm and whose absence severely compromises their ability to fertilize ova.

Reprogrammed and transmissible intestinal microbiota confer diminished susceptibility to induced colitis in TMF-/- mice.

Significance Our data demonstrate that a knockout of a single gene (tmf1) leads to the beneficial reprogramming of the gut resident microbiota. This reprogramming results in a diminished susceptibility of the genetically modified animals to induced colitis. Notably, the reprogrammed bacterial profile is transmissible, thereby conferring altered microbiome and reduced susceptibility to induced colitis in wild-type mice, when cohoused. Our findings open previously unreported avenues for unraveling regulatory factors that affect the gut homeostasis and mammalian sensitivity to the onset of inflammatory bowel diseases. Tata Element Modulatory Factor (TMF/ARA160) is a multifunctional Golgi-associated protein, which accumulates in colonic enterocytes and goblet cells. Mice lacking TMF/ARA160 (TMF−/−) produce thick and uniform colonic mucus that resists adherent bacterial colonization and diminishes susceptibility of these mice to induced acute colitis, through a mechanism that is not fully understood. Here, we show that mucus secretion by goblet cells is altered in the colon of TMF−/− mice, resulting in the formation of a highly oligomerized colonic gel-forming mucin, MUC2. Microbiome analysis revealed a shift in the microbiota of TMF−/− mice leading to predominance of the Firmicutes phylum and a significantly higher abundance of probiotic beneficial bacterial species. Notably, this trait was transmissible, and when cohoused with wild-type animals, TMF−/− mice influenced the microbiota and diminished the susceptibility of wild-type mice to chemically induced dextran sulfate sodium colitis. Thus, altered mucus secretion in TMF−/− mouse colons is accompanied by a reprogrammed intestinal microbiota, leading to a transmissible reduced sensitivity to induced colitis.

Association of yeast SAP1, a novel member of the ‘AAA’ ATPase family of proteins, with the chromatin protein SIN1

The yeast SIN1 protein is a nuclear protein that together with other proteins behaves as a transcriptional repressor of a family of genes. In addition, sin1 mutants are defective in proper mitotic chromosome segregation. In an effort to understand the basis for these phenotypes, we employed the yeast two‐hybrid system to identify proteins that interact with SIN1 in vivo. Here, we demonstrate that SAP1, a novel protein belonging to the ‘AAA’ family of ATPases, is able to directly interact with SIN1. Furthermore, we show, using recombinant molecules in vitro, that a short 27 amino acid sequence near the N‐terminal of SIN1 is sufficient to bind SAP1. Previous experiments defined different domains of SIN that interact with other proteins and with DNA. The C‐terminal domain of SIN1 was shown to be responsible for interaction with a protein that binds the regulatory region of HO, a gene whose transcription is repressed by SIN1. The central ‘HMG1‐like region’ of SIN1 binds DNA, while the N‐terminal of SIN1 can bind CDC23, a protein that regulates chromosome segregation. These data, taken together with the results presented here, suggest that SIN1 is a multifunctional chromatin protein that can interact with a number of different proteins that are involved in several different cellular functions.

TMF/ARA160 downregulates proangiogenic genes and attenuates the progression of PC3 xenografts.

TMF/ARA160 is a Golgi‐associated protein whose level is downregulated in solid tumors. TMF changes its subcellular localization on exposure of cells to stress cues, thereby, directing proteins, such as the key transcription factor, Stat3, to proteasomal degradation. Here, we show that enforced ectopic expression of HA‐TMF in PC3 prostate carcinoma cells, which do not express Stat3, significantly attenuated the development and growth of xenograft tumors elicited by these cells in athymic mice. Immunohistochemical analysis revealed impaired angiogenesis and accelerated onset of apoptosis in the HA‐TMF‐expressing tumors. RNA expression profiling revealed the downregulation of several proangiogenic genes in HA‐TMF‐expressing xenografts. Among these were the interleukin‐8 and interleukin‐1β genes, whose expression is controlled by nuclear factor‐kB. The level of the nuclear factor‐kB component, p65/RelA, was decreased in HA‐TMF‐expressing xenografts, and TMF was found to direct the ubiquitination and proteasomal degradation of p65/RelA in metabolically stressed PC3 clones. Taken together, our findings indicate that TMF/ARA160 is a regulator of key transcription factors under metabolic constraints, thereby affecting angiogenesis and progression of solid tumors, which are subjected to metabolic stress.

Down-regulation of Fer induces ROS levels accompanied by ATM and p53 activation in colon carcinoma cells.

Fer is an intracellular tyrosine kinase which resides in both the cytoplasm and nucleus of mammalian cells. This kinase was also found in all malignant cell-lines analyzed and was shown to support cell-cycle progression in cancer cells. Herein we show that knock-down of Fer, both, impairs cell-cycle progression and imposes programmed cell death in colon carcinoma (CC) cells. The cell-cycle arrest and apoptotic death invoked by the depletion of Fer were found to depend on the activity of p53. Accordingly, down regulation of Fer led to the activation of the Ataxia Telangiectasia Mutated protein (ATM) and its down-stream effector-p53. Knock-down of Fer also increased the level of Reactive-Oxygen Species (ROS) in CC cells, and subjection of Fer depleted cells to ROS neutralizing scavengers significantly decreased the induced phosphorylation and activation of ATM and p53. Notably, over-expression of Fer opposed the Doxorubicin driven activation of ATM and p53, which can be mediated by ROS. Collectively, our findings imply that Fer sustains low ROS levels in CC cells, thereby restraining the activation of ATM and p53 in these cells.

Hsp90 and a tyrosine embedded in the Hsp90 recognition loop are required for the Fer tyrosine kinase activity

Hsp90 is a key regulator of tyrosine kinases activity and is therefore considered as a promising target for intervention with deregulated signaling pathways in malignant cells. Here we describe a novel Hsp90 client - the intracellular tyrosine kinase, Fer, which is subjected to a unique regulatory regime by this chaperone. Inhibition of Hsp90 activity led to proteasomal degradation of the Fer enzyme. However, circumventing the dependence of Fer accumulation on Hsp90, revealed the dependence of the Fer kinase activity and its ability to phosphorylate Stat3 on the chaperone, expressing the necessity of Hsp90 for its function. Mutation analysis unveiled a tyrosine (Tyr(616)) embedded in the Hsp90 recognition loop, which is required for the kinase activity of Fer. Replacement of this tyrosine by phenylalanine (Y616F) disabled the auto-phosphorylation activity of Fer and abolished its ability to phosphorylate Stat3. Notably, surrounding the replaced Y616F with subtle mutations restored the auto and trans-phosphorylation activities of Fer suggesting that Y(616) is not itself an essential auto-phosphorylation site of the kinase. Taken together, our results portray Hsp90 and its recognition loop as novel positive regulators of the Fer tyrosine kinase stability and activity.

Testosterone deficiency accompanied by testicular and epididymal abnormalities in TMF−/− mice

TMF/ARA160 is a Golgi-associated protein, which is essential for spermiogenesis. In this study, we show that lack of TMF/ARA160 leads to defects in both the testis and the epididymis. In the testis, spermatid retention and extensive proliferation of Leydig cells were observed. Concomitantly, the serum levels of luteinizing hormone (LH), a stimulator of Leydig cell proliferation, were significantly increased in TMF(-/-) mice. Structural and functional defects were also seen in the epididymis. These included apoptosis of epithelial epididymal cells and sperm stasis in the cauda. Notably, the serum testosterone levels of TMF(-/-) mice were significantly lower than those of wt mice, and external testosterone administration decreased the number of apoptotic epithelial epididymal cells in TMF(-/-) animals. In summary, we show here for the first time that TMF/ARA160 participates in the control of serum testosterone levels in males, and its absence results in major testicular and epididymal defects.

Intronic Promoter Drives the BORIS-regulated Expression of FerT in Colon Carcinoma Cells

Background: Identification of cancer testis antigens (CTA) provides new tools for cancer diagnosis and therapy. Results: Expression of the spermatogenic protein FerT recurred in colon carcinoma (CC) cells via DNA demethylation and activation of an intronic promoter. Conclusion: FerT is a new CTA whose expression is regulated by a novel mechanism. Significance: FerT may serve as a new target for CC diagnosis and therapy. Fer is an intracellular tyrosine kinase that accumulates in most mammalian tissues. A truncated variant of Fer, FerT, is uniquely detected in spermatogenic cells and is absent from normal somatic tissues. Here, we show that in addition to Fer, FerT also accumulates in CC cells and in metastases derived from colorectal tumors, but not in normal human cells. Thus, FerT is a new member of the CTA protein family. Transcription of the ferT gene in CC cells was found to be driven by an intronic promoter residing in intron 10 of the fer gene and to be regulated by another CTA, the Brother of the Regulator of Imprinted Sites (BORIS) transcription factor. BORIS binds to the ferT promoter and down-regulation of BORIS significantly decreases the expression of ferT in CC cells. Accumulation of the ferT RNA was also regulated by the DNA methylation status and paralleled the expression profile of the boris transcript. Accordingly, the intronic ferT promoter was found to be hypomethylated in cancer cells expressing the FerT protein, by comparison with non-expressers. Collectively, we show here that FerT is a new CTA whose accumulation in CC cells, commonly considered low CTA expressers, is controlled by a novel transcription regulatory mechanism.