Mutations in the autophagy receptor OPTN/optineurin are associated with the pathogenesis of glaucoma and amyotrophic lateral sclerosis, but the underlying molecular basis is poorly understood. The OPTN variant, M98K has been described as a risk factor for normal tension glaucoma in some ethnic groups. Here, we examined the consequence of the M98K mutation in affecting cellular functions of OPTN. Overexpression of M98K-OPTN induced death of retinal ganglion cells (RGC-5 cell line), but not of other neuronal and non-neuronal cells. Enhanced levels of the autophagy marker, LC3-II, a post-translationally modified form of LC3, in M98K-OPTN-expressing cells and the inability of an LC3-binding-defective M98K variant of OPTN to induce cell death, suggested that autophagy contributes to cell death. Knockdown of Atg5 reduced M98K-induced death of RGC-5 cells, further supporting the involvement of autophagy. Overexpression of M98K-OPTN enhanced autophagosome formation and potentiated the delivery of transferrin receptor to autophagosomes for degradation resulting in reduced cellular transferrin receptor levels. Coexpression of transferrin receptor or supplementation of media with an iron donor reduced M98K-induced cell death. OPTN complexes with RAB12, a GTPase involved in vesicle trafficking, and M98K variant shows enhanced colocalization with RAB12. Knockdown of Rab12 increased transferrin receptor level and reduced M98K-induced cell death. RAB12 is present in autophagosomes and knockdown of Rab12 resulted in reduced formation of autolysosomes during starvation-induced autophagy, implicating a role for RAB12 in autophagy. These results also show that transferrin receptor degradation and autophagy play a crucial role in RGC-5 cell death induced by M98K variant of OPTN.

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.

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 -42 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.The Src family tyrosine kinases play an important role in linking signals received by transmembrane receptors and a variety of intracellular pathways, thereby regulating diverse cellular responses such as proliferation, differentiation, and cell death (1, 2). This is achieved through their non-catalytic sequences, which enable multiple interactions with cellular proteins, and through the kinase domain, which phosphorylates substrates to alter their activity, change their subcellular location, and effect their intermolecular interactions. The hematopoietic cell kinase (Hck) 1 is a Src family member that is expressed in cells of myelomonocytic lineage, B lymphocytes, and embryonic stem cells with higher levels in differentiated cells, suggesting a role for this enzyme in signaling pathways of mature hematopoietic cells (3-5). Hck is activated by agents that induce macrophage differentiation and in response to cytokines such as interleukin-3, granulocyte-macrophage colony stimulating factor, and leukemia inhibitory factor. It is also involved in cytokine production in macrophages in response to lipopolysaccharide and viral infection (6 -9).Structurally, Hck is similar to other members of the Src family in that it has a catalytic domain at the C terminus that is preceded by a 100-amino acid SH2 domain and a 50-amino acid SH3 domain. The SH2 and SH3 domains are protein interaction modules that mediate either intramolecular or intermolecular associations. SH2 domains bind to phosphorylated tyrosine residues in a specific amino acid context, whereas SH3 domain interacts with polyproline tracts in polypept...

SummaryRab 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).

Autophagy is a quality-control mechanism that helps to maintain cellular homeostasis by removing damaged proteins and organelles through lysosomal degradation. During autophagy, signaling events lead to the formation of a cup-shaped structure called the phagophore that matures into the autophagosome. Recruitment of the autophagy-associated Atg12-5-16L1 complex to Wipi2-positive phagophores is crucial for producing microtubule-associated protein 1 light chain 3-II (LC3-II), which is required for autophagosome formation. Here, we explored the role of the autophagy receptor optineurin (Optn) in autophagosome formation. Fibroblasts from Optn knock-out mouse showed reduced LC3-II formation and a lower number of autophagosomes and autolysosomes during both basal and starvation-induced autophagy. However, the number of Wipi2-positive phagophores was not decreased in Optn-deficient cells. We also found that the number of Atg12/16L1-positive puncta and recruitment of the Atg12-5-16L1 complex to Wipi2-positive puncta are reduced in Optn-deficient cells. Of note, Optn was recruited to Atg12-5-16L1-positive puncta, and interacted with Atg5 and also with Atg12-5 conjugate. A disease-associated Optn mutant, E478G, defective in ubiquitin binding, was also defective in autophagosome formation and recruitment to the Atg12-5-16L1-positive puncta. Moreover, we noted that Optn phosphorylation at Ser-177 was required for autophagosome formation but not for Optn recruitment to the phagophore. These results suggest that Optn potentiates LC3-II production and maturation of the phagophore into the autophagosome, by facilitating the recruitment of the Atg12-5-16L1 complex to Wipi2-positive phagophores.

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PTP-S2/TC45 is a nuclear protein tyrosine phosphatase, which induces p53-dependent apoptosis. Here we show that the p53 protein level increased in MCF-7 cells in response to PTP-S2 overexpression. PTP-S2-induced p53 protein was transcriptionally active and it could activate caspase-1 gene expression from endogenous as well as ectopic promoter. Coexpression of an active site mutant of procaspase-1 strongly inhibited PTP-S2-induced apoptosis. Mutant procaspase-1 also inhibited apoptosis induced by p53 overexpression or doxorubicin treatment, which induce caspase-1 gene expression. In contrast, apoptosis induced by staurosporine or cycloheximide, which do not increase caspase-1 gene expression, was not a¡ected by mutant procaspase-1. These results suggest that caspase-1 may be one of the mediators of p53-dependent apoptosis in human cells.

Tumor necrosis factor-a (TNF-a) is a multifunctional cytokine that plays an important role in the immune response, inflammation, control of cell death and cell proliferation. The biological effects of TNF-a are mediated mostly through tumor necrosis factor receptor-1 (TNF-R1), a cell-surface receptor. TNF-R1 is a type 1 transmembrane protein that contains four cysteine-rich repeats in the extracellular domain. The distal cysteinerich domain mediates homophilic interaction of the receptor molecules, thereby keeping the receptors in a silent, homomultimerized state [1]. Binding of the trimeric TNF-a ligand results in the re-organization of pre-assembled TNF-R1 complexes. These events signal the recruitment of tumor necrosis factor-a receptor associated death domain to the intracellular death domain of TNF-R1. TNF-R1-bound tumor necrosis factor-a receptor associated death domain serves as platform for the binding of TNF receptor-associated Tumour necrosis factor-a (TNF-a) is a cytokine that is involved in many functions, including the inflammatory response, immunity and apoptosis. Some of the responses of TNF-a are mediated by caspase-1, which is involved in the production of the pro-inflammatory cytokines interleukin1b, interleukin-18 and interleukin-33. The molecular mechanisms involved in TNF-a-induced caspase-1 gene expression remain poorly defined, despite the fact that signaling by TNF-a has been well studied. The present study was undertaken to investigate the mechanisms involved in the induction of caspase-1 gene expression by TNF-a. Treatment of A549 cells with TNF-a resulted in an increase in caspase-1 mRNA and protein expression, which was preceded by an increase in interferon regulatory factor-1 and p73 protein levels. Caspase-1 promoter reporter was activated by the treatment of cells with TNF-a. Mutation of the interferon regulatory factor-1 binding site resulted in the almost complete loss of basal as well as of TNF-ainduced caspase-1 promoter activity. Mutation of the p53 ⁄ p73 responsive site resulted in reduced TNF-a-induced promoter activity. Blocking of p73 function by a dominant negative mutant or by a p73-directed small hairpin RNA reduced basal as well as TNF-a-induced caspase-1 promoter activity. TNF-a-induced caspase-1 mRNA and protein levels were reduced when p73 mRNA was down-regulated by small hairpin RNA. Caspase-5 gene expression was induced by TNF-a, which was inhibited by the small hairpin RNA-mediated down-regulation of p73. Our results show that TNF-a induces p73 gene expression, which, together with interferon regulatory factor-1, plays an important role in mediating caspase-1 promoter activation by TNF-a.Abbreviations CAT, chloramphenicol acetyltransferase; Cdk-2, cyclin dependent kinase 2; CMV, cytomegalovirus; Ets-1, E26 transformation-specific sequence 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IFN, interferon; IRF-1, interferon regulatory factor-1; NF-jB, nuclear factor-jB; shRNA, short hairpin RNA; TNF-a, tumor necrosis factor-a; TNF-R1, tumor necrosis factor recept...