TAK1 Is Recruited to the Tumor Necrosis Factor-α (TNF-α) Receptor 1 Complex in a Receptor-interacting Protein (RIP)-dependent Manner and Cooperates with MEKK3 Leading to NF-κB Activation

Receptor-interacting protein (RIP) plays a critical role in tumor necrosis factor-α (TNF-α)-induced IκB kinase (IKK) activation and subsequent activation of transcription factor NF-κB. However, the molecular mechanism by which RIP mediates TNF-α-induced NF-κB activation is not completely defined. In this study, we have found that TAK1 is recruited to the TNF-α receptor complex in a RIP-dependent manner following the stimulation of TNF-α receptor 1 (TNF-R1). Moreover, a forced recruitment of TAK1 to TNF-R1 in the absence of RIP is sufficient to mediate TNF-α-induced NF-κB activation, indicating that the major function of RIP is to recruit its downstream kinases to the TNF-R1 complex. Interestingly, we also find that TAK1 and MEKK3 form a functional complex, in which TAK1 regulates autophosphorylation of MEKK3. The TAK1-mediated regulation of MEKK3 phosphorylation is dependent on the kinase activity of TAK1. Although TAK1-MEKK3 interaction is not affected by overexpressed TAB1, TAB1 is required for TAK1 activation and subsequent MEKK3 phosphorylation. Together, we conclude that TAK1 is recruited to the TNF-R1 complex via RIP and likely cooperates with MEKK3 to activate NF-κB in TNF-α signaling

Phosphorylation and ubiquitination of the IκB kinase complex by two distinct signaling pathways

The IκB kinase (IKK) complex serves as the master regulator for the activation of NF-κB by various stimuli. It contains two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, IKKγ/NEMO. The activation of IKK complex is dependent on the phosphorylation of IKKα/β at its activation loop and the K63-linked ubiquitination of NEMO. However, the molecular mechanism by which these inducible modifications occur remains undefined. Here, we demonstrate that CARMA1, a key scaffold molecule, is essential to regulate NEMO ubiquitination upon T-cell receptor (TCR) stimulation. However, the phosphorylation of IKKα/β activation loop is independent of CARMA1 or NEMO ubiquitination. Further, we provide evidence that TAK1 is activated and recruited to the synapses in a CARMA1-independent manner and mediate IKKα/β phosphorylation. Thus, our study provides the biochemical and genetic evidence that phosphorylation of IKKα/β and ubiquitination of NEMO are regulated by two distinct pathways upon TCR stimulation

Tmem203 null male mice are sterile.

<p>Uninterrupted mating was performed between sexually mature (aged 8 weeks old) <i>Tmem203</i> null (tmem203 ^-/-), heterozygous (tmem203 ^-/+) and wild type (tmem203 ^+/+) mice for 17 weeks. All litters and litter sizes were recorded for each mating pair.</p><p>Tmem203 null male mice are sterile.</p

Gene expression profiling in Tmem203 null mouse testes indicates aberrant expression of key calcium channels and pumps.

<p>(A) A spotfire based visualization of differential gene expression displaying fold changes in gene expression versus FDR corrected P value obtained from a microarray based RNA expression analysis from a set of five <i>Tmem203</i> null and wild type mice. (B) Ingenuity based pathways enrichment of differentially expressed genes in <i>Tmem203</i> null mouse testes. For the analysis the pathways showing a significant change with a p value of >2 were considered. (C) Quantitative real time PCR analysis of RNA obtained from WT and <i>Tmem203</i> null mice testes for genes differentially expressed in the calcium signaling pathway selected from (B). Expression level of mentioned genes in <i>Tmem203</i> null mice testes relative to WT testes expression level after normalization to <i>Gapdh</i>. Data represents 4 replicates (+/- Std Dev) and validated in 2 or more RNA preparations from <i>Tmem203</i> null and WT testes. *—Transcript not detected in <i>Tmem203</i> null mice testes.</p

Intracellular store calcium flux and store operated calcium entry kinetics in testicular cells from WT and <i>Tmem203</i> null mice.

<p>Flou3 and Fura red loaded testicular cells prepared from WT and <i>Tmem203</i> null mice were analyzed by flow cytometry to follow cytosolic calcium kinetics in the gated predominately round spermatids population. Intracellular store calcium flux was measured by recording Flou3 calcium bound to Fura red calcium free ratio in the presence of 1mM EGTA in response to SERCA inhibitor- Thapsigargin (A); Calcium ionophore—Ionomycin (B); Similarly the store operated calcium entry kinetics was followed in WT and <i>Tmem203</i> null testicular round spermatids gated population by depleting the stores by Thapsigargin (C) or Ionomycin (D) followed with addition of 2mM CaCl₂.</p

TMEM203 expression drives calcineurin dependent transcription factor activation by elevating the basal cytosolic calcium levels in HeLa cells.

<p>(A) Stably expressed CRTC1-GFP localization was visualized using fluorescent microscope in HeLa-CRTC1-GFP cell line transiently expressing TMEM203–FLAG for 48 hrs. CRTC1-GFP (green) nuclear translocation was induced in cells co-expressing TMEM203-Flag (red). Nuclei (blue) were visualized with Hoechst. Nuclear translocation was inhibited by treatment with 5nM Cyclosporine A or 10nM FK506 for 2 hour prior to fixing the cells. Scale bars = 15 μm. (B) HeLa cells were co-transfected with NFAT2 (1–402)-GFP and TMEM203-FLAG or empty vector. 48 hours later the cells were visualized using fluorescent microscope. Scale bars = 15 μm. (C) HeLa cells were co-transfected with NFAT2 (1–402)-GFP and TMEM203-FLAG or empty vector as indicated. 48 hours later the cells were treated with 5nM Cyclosporine A (CsA) or 10nM FK506 for 2 hours and total cell lysates were prepared. The lysates were subjected to immunoblotting with indicated antibodies. (D) TMEM203-mcherry or mcherry transfected HeLa cells were seeded onto coverslips and single cell Fura-2 fluorescence based calcium measurements were performed. The measurements showed elevated basal calcium levels in TMEM203-mcherry expressing cells. (Mean; +/- SE; n = 64 cells (mcherry); 55 cells (TMEM203-mcherry) from multiple coverslips; p value = 4.06719E-30).</p

TMEM203 interacts with regulators of ER calcium stores and overexpression depletes ER calcium stores.

<p>(A) Confocal analysis of HeLa cells transiently expressing TMEM203-GFP with organelle specific markers for ER (top:Calreticulin-RFP), Mitochondria (middle:BDHA-RFP) or plasma membrane (bottom:LCK-RFP). Separation or colocalization of TMEM203-GFP and organelle marker(s) were visualized by the linescan function of MetaMorph: the fluorescence intensity of each pixel of the line of interest (white lines ~ 75 μm) is shown as a xy-graph for the corresponding green and red channels. The line scan shows that TMEM203-GFP predominately overlapped with the ER marker. (Representative of ~ 50 cells from 2 independent experiments). Note, we cannot rule out that TMEM203 is completely absent from the the mitochondria. (B) Western analysis of complexes immune-precipitated TMEM203-Flag from HEK293 cells with indicated antibodies shows specific interaction with endogenous STIM1, IP3R and SERCA2. (Representative of atleast 2 independent experiments). (C) pTUNE-TMEM203-293cells were treated with the indicated dose of IPTG for 48 hrs to induce TMEM203 expression. Levels of TMEM203-Flag protein were detected by western blot. (D) These IPTG induced cells were subjected to Indo-1 based calcium flux measurements by flow cytometry by first treating with thapsigargin (TG) and EGTA. (E) As in (D) but the cells were treated with Ionomycin. (F) As in (D) but following TG treatment CaCl₂ was added to record SOCE.</p

<i>Tmem203</i> null mice completely lack mature spermatozoa in epididymis.

<p>(A) Computer assisted sperm analyzer based analysis of epididymis preparations from wild type and <i>Tmem203</i> null mice showed complete absence (*) of mature spermatozoa in <i>Tmem203</i> null mice. Data is representative of two independent experiments. (B) Representative photomicrographs illustrating hematoxylin and eosin (H&E)-stained sections of proximal epididymis (caput; upper left and right panels) and distal epididymis (cauda; lower left and right panels) from a 48-week-old wild type mouse (upper and lower left panels) and from a 48-week-old <i>Tmem203</i> null mice (upper and lower right panels). Note the complete absence of mature spermatozoa in the epididymis of the <i>Tmem203</i> null mice compared to the wild type mice in which numerous mature spermatozoa are observed; tubular lumina of the epididymis from <i>Tmem203</i> null mice contains eosinophilic proteinaceous material mixed with cellular debris. Scale bars = 50 μm.</p

<i>Tmem203</i> null mice exhibit a disruption of spermiogenesis.

<p>(A-B) Propidium iodide based DNA flow cytometry analysis of testicular cell suspensions from wild-type (red tracer) and <i>Tmem</i>203 null mice (green tracer) at 35 day (A) or 30 week (B) (<i>n =</i> 2 or 3 for each genotype). Arrows highlight the differences between the wild-type and <i>Tmem203</i> null samples. Abbreviations: haploid-condensed (1n-C)-elongated spermatids; haploid (1n) round spermatids; diploid (2n)—Sertoli cells, spermatogonia; S-ph, spermatogonia synthesizing DNA and the tetraploid (4n)—pachytene spermatocytes and G2 spermatogonia (C) Representative photomicrographs illustrating hematoxylin and eosin (H&E) stained sections of Stage VII seminiferous tubules from a 48-week-old wild type mouse (left panels) and from a 48-week-old cMAC knockout mouse (right panels). Compared to the seminiferous tubule from the wild type mouse (left panels) the predominant morphological changes observed in the seminiferous tubule of the cMAC knockout mouse (right panels) are characterized by an overall subtle, relative reduction in numbers of late stage post-meiotic spermatids (steps 9–16), degenerative intracytoplasmic vacuolar changes most prominent in step 16 spermatids and complete lack of spermiation (disengagement of step 16 spermatozoa from the Sertoli cell and release into the tubular lumen). Lower panels illustrate higher magnification of areas enclosed by square boxes in the upper left and right panels. Scale bars = 50 μm (upper panels) and 25 μm (lower panels). (D-E) Representative transmission electron micrographs of Stage VII seminiferous tubules from a 32-week-old wild type mouse (left panels) and from a 32-week-old <i>Tmem203</i> null mouse (right panels). Labeled are step 7 spermatocytes (7), residual bodies (rb), endoplasmic reticulum (er) and degenerate, misshapen spermatid heads (asterisks) mitochondrial sheath (ms) surrounding the outer dense fibers, axoneme and axoneme complex of microtubules. Phagocytosis by Sertoli cells of degenerate spermatids is illustrated in both the top and bottom panel on the right for the <i>Tmem203</i> null mouse. Residual bodies (rb) contain dense aggregations of RNA, lipid, clear vesicles, multivesicular bodies and other organelles. Scale bars = 5.0 μm (for D),2.0 μm (for E).</p