Ghanshyam Swarup

NF-κB mediates tumor necrosis factor α-induced expression of optineurin, a negative regulator of NF-κB

Optineurin is a ubiquitously expressed multifunctional cytoplasmic protein encoded by OPTN gene. The expression of optineurin is induced by various cytokines. Here we have investigated the molecular mechanisms which regulate optineurin gene expression and the relationship between optineurin and nuclear factor κB (NF-κB). We cloned and characterized human optineurin promoter. Optineurin promoter was activated upon treatment of HeLa and A549 cells with tumor necrosis factor α (TNFα). Mutation of a putative NF-κB-binding site present in the core promoter resulted in loss of basal as well as TNFα-induced activity. Overexpression of p65 subunit of NF-κB activated this promoter through NF-κB site. Oligonucleotides corresponding to this putative NF-κB-binding site showed binding to NF-κB. TNFα-induced optineurin promoter activity was inhibited by expression of inhibitor of NF-κB (IκBα) super-repressor. Blocking of NF-κB activation resulted in inhibition of TNFα-induced optineurin gene expression. Overexpressed optineurin partly inhibited TNFα-induced NF-κB activation in Hela cells. Downregulation of optineurin by shRNA resulted in an increase in TNFα-induced as well as basal NF-κB activity. These results show that optineurin promoter activity and gene expression are regulated by NF-κB pathway in response to TNFα. In addition these results suggest that there is a negative feedback loop in which TNFα-induced NF-κB activity mediates expression of optineurin, which itself functions as a negative regulator of NF-κB.

Regulation of endocytic trafficking of transferrin receptor by optineurin and its impairment by a glaucoma-associated mutant

BackgroundOptineurin is a multifunctional protein involved in several functions such as vesicular trafficking from the Golgi to the plasma membrane, NF-κB regulation, signal transduction and gene expression. Mutations in optineurin are associated with glaucoma, a neurodegenerative eye disease that causes blindness. Genetic evidence suggests that the E50K (Glu50Lys) is a dominant disease-causing mutation of optineurin. However, functional alterations caused by mutations in optineurin are not known. Here, we have analyzed the role of optineurin in endocytic recycling and the effect of E50K mutant on this process.ResultsWe show that the knockdown of optineurin impairs trafficking of transferrin receptor to the juxtanuclear region. A point mutation (D474N) in the ubiquitin-binding domain abrogates localization of optineurin to the recycling endosomes and interaction with transferrin receptor. The function of ubiquitin-binding domain of optineurin is also needed for trafficking of transferrin to the juxtanuclear region. A disease causing mutation, E50K, impairs endocytic recycling of transferrin receptor as shown by enlarged recycling endosomes, slower dynamics of E50K vesicles and decreased transferrin uptake by the E50K-expressing cells. This impaired trafficking by the E50K mutant requires the function of its ubiquitin-binding domain. Compared to wild type optineurin, the E50K optineurin shows enhanced interaction and colocalization with transferrin receptor and Rab8. The velocity of Rab8 vesicles is reduced by co-expression of the E50K mutant. These results suggest that the E50K mutant affects Rab8-mediated transferrin receptor trafficking.ConclusionsOur results suggest that optineurin regulates endocytic trafficking of transferrin receptor to the juxtanuclear region. The E50K mutant impairs trafficking at the recycling endosomes due to altered interactions with Rab8 and transferrin receptor. These results also have implications for the pathogenesis of glaucoma caused by the E50K mutation because endocytic recycling is vital for maintaining homeostasis.

Optineurin Is Required for CYLD-Dependent Inhibition of TNFα-Induced NF-κB Activation

The nuclear factor kappa B (NF-κB) regulates genes that function in diverse cellular processes like inflammation, immunity and cell survival. The activation of NF-κB is tightly controlled and the deubiquitinase CYLD has emerged as a key negative regulator of NF-κB signalling. Optineurin, mutated in certain glaucomas and amyotrophic lateral sclerosis, is also a negative regulator of NF-κB activation. It competes with NEMO (NF-κB essential modulator) for binding to ubiquitinated RIP (receptor interacting protein) to prevent NF-κB activation. Recently we identified CYLD as optineurin-interacting protein. Here we have analysed the functional significance of interaction of optineurin with CYLD. Our results show that a glaucoma-associated mutant of optineurin, H486R, is altered in its interaction with CYLD. Unlike wild-type optineurin, the H486R mutant did not inhibit tumour necrosis factor α (TNFα)-induced NF-κB activation. CYLD mediated inhibition of TNFα-induced NF-κB activation was abrogated by expression of the H486R mutant. Upon knockdown of optineurin, CYLD was unable to inhibit TNFα-induced NF-κB activation and showed drastically reduced interaction with ubiquitinated RIP. The level of ubiquitinated RIP was increased in optineurin knockdown cells. Deubiquitination of RIP by over-expressed CYLD was abrogated in optineurin knockdown cells. These results suggest that optineurin regulates NF-κB activation by mediating interaction of CYLD with ubiquitinated RIP thus facilitating deubiquitination of RIP.

Physical and Functional Interaction between Hck Tyrosine Kinase and Guanine Nucleotide Exchange Factor C3G Results in Apoptosis, Which Is Independent of C3G Catalytic Domain

The hematopoietic cell kinase Hck is a Src family tyrosine kinase expressed in cells of myelomonocytic lineage, B lymphocytes, and embryonic stem cells. To study its role in signaling pathways we used the Hck-SH3 domain in protein interaction cloning and identified C3G, the guanine nucleotide exchange factor for Rap1 and R-Ras, as a protein that associated with Hck. This interaction was direct and was mediated partly through the proline-rich region of C3G. C3G could be co-immunoprecipitated with Hck from Cos-1 cells transfected with Hck and C3G. C3G was phosphorylated on tyrosine 504 in cells when coexpressed with Hck but not with a catalytically inactive mutant of Hck. Phosphorylation of endogenous C3G at Tyr-504 was increased by treatment of human myelomonocytic THP-1 cells with mercuric chloride, which is known to activate Hck tyrosine kinase specifically. Coexpression of Hck with C3G induced a high level of apoptosis in many cell lines by 30–02 h of transfection. Induction of apoptosis was not dependent on Tyr-504 phosphorylation or the catalytic domain of C3G but required the catalytic activity of Hck. Using dominant negative constructs of caspases we found that caspase-1, -8, and -9 are involved in this apoptotic pathway. These results suggest that C3G and Hck interact physically and functionally in vivo to activate kinase-dependent and caspase-mediated apoptosis, which is independent of catalytic domain of C3G.

Optineurin and Its Mutants: Molecules Associated with Some Forms of Glaucoma

Aims: Optineurin is a gene (OPTN) associated with normal-tension glaucoma and primary open-angle glaucoma. The aim of our study is to understand the functions of the protein optineurin in retinal ganglion cells, and the modifications that come about in its functions when mutations occur in its sequence. Methods: We have worked with the rat retinal ganglion cell line RGC5, as well as with some others such as HeLa (human cancer cell line), COS-1 (monkey kidney cell line), IMR-32 (human neuroblastoma cell line), and D407 (human retinal pigment epithelial cell line). The cDNAs of wild-type OPTN, and of its mutants H26D, E50K, R454Q and H486R, were transfected into each of these cell lines and the effects studied. Localization of the proteins in cells was monitored by confocal microscopy and cell imaging, and their interaction with other cellular proteins by yeast two-hybrid assay and co-immunoprecipitation. Results: We found optineurin to interact with several proteins, which are involved in NF-κB regulation, vesicular trafficking at recycling endosomes, immune response and transcription regulation. Some of these proteins showed altered/reduced interactions with E50K and H486R mutants. E50K, a severe phenotype-related mutant of optineurin, was found to selectively kill RGC5 cells through oxytosis and apoptosis, while none of the other mutants did so. Conclusions: Our results indicate that several independent mechanisms are likely to be involved in the pathogenesis of glaucoma caused by mutations in optineurin.

Caspase-1 activator Ipaf is a p53-inducible gene involved in apoptosis

The tumor suppressor protein p53 regulates transcription of many genes that mediate cell cycle arrest, apoptosis, DNA repair and other cellular responses. Here we show that Ipaf, a human CED-4 homologue and an activator of caspase-1, is induced by p53. Overexpression of p53 by transfection in U2OS and A549 cells increased Ipaf mRNA levels. Treatment of p53-positive cell lines U2OS and MCF-7 with the DNA damaging drug, doxorubicin, which increases p53 protein level, induced expression of Ipaf mRNA but similar treatment of MCF-7-mp53 (a clone of MCF-7 cells expressing mutant p53) and p53-negative K562 cells showed much less induction of Ipaf gene expression. Expression analysis for Ipaf mRNA in doxorubicin-treated human tumor cell lines suggests that p53-dependent as well as p53-independent mechanisms are involved in the regulation of Ipaf gene expression in a cell-type-specific manner. The Ipaf promoter was activated by normal p53 but not by His273 mutant of p53. A functional p53-binding site was identified in the Ipaf promoter. A dominant-negative mutant of Ipaf inhibited p53-induced and doxorubicin-induced apoptosis by about 50%. Ipaf-directed small hairpin RNA downregulated p53-induced Ipaf gene expression and also reduced p53-induced apoptosis. Doxorubicin-induced apoptosis was also inhibited by Ipaf-directed small hairpin RNA. Our results show that p53 can directly induce Ipaf gene transcription, which contributes to p53-dependent apoptosis in at least some human cells.

Phosphorylated guanine nucleotide exchange factor C3G, induced by pervanadate and Src family kinases localizes to the Golgi and subcortical actin cytoskeleton

BackgroundThe guanine nucleotide exchange factor C3G (RapGEF1) along with its effector proteins participates in signaling pathways that regulate eukaryotic cell proliferation, adhesion, apoptosis and embryonic development. It activates Rap1, Rap2 and R-Ras members of the Ras family of GTPases. C3G is activated upon phosphorylation at tyrosine 504 and therefore, determining the localization of phosphorylated C3G would provide an insight into its site of action in the cellular context.ResultsC3G is phosphorylated in vivo on Y504 upon coexpression with Src or Hck, two members of the Src family tyrosine kinases. Here we have determined the subcellular localization of this protein using antibodies specific to C3G and Tyr 504 phosphorylated C3G (pY504 C3G). While exogenously expressed C3G was present mostly in the cytosol, pY504 C3G formed upon Hck or Src coexpression localized predominantly at the cell membrane and the Golgi complex. Tyrosine 504-phosphorylated C3G showed colocalization with Hck and Src. Treatment of Hck and C3G transfected cells with pervanadate showed an increase in the cytosolic staining of pY504 C3G suggesting that tyrosine phosphatases may be involved in dephosphorylating cytosolic phospho-C3G. Expression of Src family kinases or treatment of cells with pervanadate resulted in an increase in endogenous pY504 C3G, which was localized predominantly at the Golgi and the cell periphery. Endogenous pY504 C3G at the cell periphery colocalized with F-actin suggesting its presence at the subcortical actin cytoskeleton. Disruption of actin cytoskeleton by cytochalasin D abolished phospho-C3G staining at the periphery of the cell without affecting its Golgi localization.ConclusionsThese findings show that tyrosine kinases involved in phosphorylation of C3G are responsible for regulation of its localization in a cellular context. We have demonstrated the localization of endogenous C3G modified by tyrosine phosphorylation to defined subcellular domains where it may be responsible for restricted activation of signaling pathways.

Molecular cloning and expresion of a protein-tyrosine phosphatase showing homology with transcription factors Fos and Jun

A cDNA clone coding for a protein‐tyrosine phosphatase (PTPase) was isolated from a rat spleen cDNA library. Nucleotide sequence of the clone showed an open reading frame coding for a polypeptide of 363 amino acids. Expression of this clone in E. coli in an expression vector showed PTPase activity. The non‐catalytic region of this PTPase located at the carboxy terminus shows homology with the basic domains of transcription factors Fos and Jun. Northern blot analysis showed that a 1.7 kb transcript was present in many tissues and cells, the highest level being in macrophages. This PTPase is a rat homolog of human T‐cell PTPase although it shows 3 large deletions in the carboxy terminal non‐catalytic region.

Optineurin mediates a negative regulation of Rab8 by the GTPase-activating protein TBC1D17

Summary Rab GTPases regulate various membrane trafficking pathways but the mechanisms by which GTPase-activating proteins recognise specific Rabs are not clear. Rab8 is involved in controlling several trafficking processes, including the trafficking of transferrin receptor from the early endosome to the recycling endosome. Here, we provide evidence to show that TBC1D17, a Rab GTPase-activating protein, through its catalytic activity, regulates Rab8-mediated endocytic trafficking of transferrin receptor. Optineurin, a Rab8-binding effector protein, mediates the interaction and colocalisation of TBC1D17 with Rab8. A non-catalytic region of TBC1D17 is required for direct interaction with optineurin. Co-expression of Rab8, but not other Rabs tested, rescues the inhibition of transferrin receptor trafficking by TBC1D17. The activated GTP-bound form of Rab8 is localised to the tubules emanating from the endocytic recycling compartment. Through its catalytic activity, TBC1D17 inhibits recruitment of Rab8 to the tubules and reduces colocalisation of transferrin receptor and Rab8. Knockdown of optineurin or TBC1D17 results in enhanced recruitment of Rab8 to the tubules. A glaucoma-associated mutant of optineurin, E50K, causes enhanced inhibition of Rab8 by TBC1D17, resulting in defective endocytic recycling of transferrin receptor. Our results show that TBC1D17, through its interaction with optineurin, regulates Rab8-mediated endocytic recycling of transferrin receptor and recruitment of Rab8 to the endocytic recycling tubules. We describe a mechanism of regulating a Rab GTPase by an effector protein (optineurin) that acts as an adaptor to bring together a Rab (Rab8) and its GTPase-activating protein (TBC1D17).

Regulation of p73 by Hck through kinase-dependent and independent mechanisms.

Backgroundp73, a p53 family member is a transcription factor that plays a role in cell cycle, differentiation and apoptosis. p73 is regulated through post translational modifications and protein interactions. c-Abl is the only known tyrosine kinase that phosphorylates and activates p73. Here we have analyzed the role of Src family kinases, which are involved in diverse signaling pathways, in regulating p73.ResultsExogenously expressed as well as cellular Hck and p73 interact in vivo. In vitro binding assays show that SH3 domain of Hck interacts with p73. Co-expression of p73 with Hck or c-Src in mammalian cells resulted in tyrosine phosphorylation of p73. Using site directed mutational analysis, we determined that Tyr-28 was the major site of phosphorylation by Hck and c-Src, unlike c-Abl which phosphorylates Tyr-99. In a kinase dependent manner, Hck co-expression resulted in stabilization of p73 protein in the cytoplasm. Activation of Hck in HL-60 cells resulted in tyrosine phosphorylation of endogenous p73. Both exogenous and endogenous Hck localize to the nuclear as well as cytoplasmic compartment, just as does p73. Ectopically expressed Hck repressed the transcriptional activity of p73 as determined by promoter assays and semi-quantitative RT-PCR analysis of the p73 target, Ipaf and MDM2. SH3 domain- dependent function of Hck was required for its effect on p73 activity, which was also reflected in its ability to inhibit p73-mediated apoptosis. We also show that Hck interacts with Yes associated protein (YAP), a transcriptional co-activator of p73, and shRNA mediated knockdown of YAP protein reduces p73 induced Ipaf promoter activation.ConclusionWe have identified p73 as a novel substrate and interacting partner of Hck and show that it regulates p73 through mechanisms that are dependent on either catalytic activity or protein interaction domains. Hck-SH3 domain-mediated interactions play an important role in the inhibition of p73-dependent transcriptional activation of a target gene, Ipaf, as well as apoptosis.