Upon antigen recognition, T-cell receptor (TCR/CD3) and other signaling molecules become enriched in a specialized contact site between the T cell and antigen-presenting cell, i.e. the immunological synapse (IS). Enrichment occurs via mechanisms that include polarized secretion from recycling endosomes, but the Rabs and RabGAPs that regulate this are unknown. EPI64C (TBC1D10C) is an uncharacterized candidate RabGAP we identified by mass spectrometry as abundant in human peripheral blood T cells that is preferentially expressed in hematopoietic cells. EPI64C is a Rab35-GAP based both on in vitro Rab35-specific GAP activity and findings in transfection assays. EPI64C and Rab35 dominant negative (DN) constructs each impaired transferrin export from a recycling pathway in Jurkat T-cells and induced large vacuoles marked by transferrin receptor, TCR, and SNAREs implicated in TCR-polarized secretion. Rab35 localized to the plasma membrane and to intracellular vesicles where it substantially colocalized with TfR and with TCR. Rab35 was strongly recruited to the IS. Conjugate formation was impaired by transfection with Rab35-DN or EPI64C and by EPI64C knock down. TCR enrichment at the IS was impaired by Rab35-DN. Thus, EPI64C and Rab35 regulate a recycling pathway in T cells and contribute to IS formation, most likely by participating in TCR transport to the IS. The immunological synapse (IS)3 is a specialized contact between T lymphocytes and antigen-presenting cells (APC) into which accumulate T-cell antigen receptor (TCR), coreceptors, and signaling and cytoskeletal components (1-7). Enrichment of proteins in the IS occurs in part by diffusion and retention at the contact region (i.e."mutual co-capping") (8, 9). Furthermore, submembranous flow of the actomyosin cortex contributes to molecular concentration in the IS (10, 11). Recent data indicate that polarized exocytosis from a rapid recycling pathway is particularly important for the enrichment of some synapse components, including TCR (12-16).Rab proteins are major intracellular transport regulators in eukaryotes; they function in vesicle formation, motility, docking, and fusion (17, 18). Over 60 mammalian Rab proteins have been identified, and each is thought to regulate distinct intracellular transport steps through its temporal and spatial association with various interacting proteins. Guanine exchange factors and GTPase-activating proteins (GAPs) control the switch between active GTP-bound and inactive GDP-bound Rab proteins. Although the TBC (Tre/Bub2/Cdc16) domain is a hallmark of RabGAPs (19), few of the more than 50 putative TBC domain-containing proteins present in the human genome have been paired with their target Rab. One recent notable success was identification of EPI64 (EBP50-PDZ interactor of 64kD) as a GAP specific for Rab27a (20).EPI64 is a broadly expressed TBC domain-containing protein first identified in placental microvilli (21). Recent studies have implicated it in regulating microvillus architecture (22). EPI64 has two paralogs in mouse and...

The proteins STIM1 and Orai1 are the long sought components of the store-operated channels required in T-cell activation. However, little is known about the interaction of these proteins in T-cells after engagement of the T-cell receptor. We found that T-cell receptor engagement caused STIM1 and Orai1 to colocalize in puncta near the site of stimulation and accumulate in a dense structure on the opposite side of the T-cell. FRET measurements showed a close interaction between STIM1 and Orai1 both in the puncta and in the dense cap-like structure. The formation of cap-like structures did not entail rearrangement of the entire endoplasmic reticulum. Cap formation depended on TCR engagement and tyrosine phosphorylation, but not on channel activity or Ca 2؉ influx. These caps were very dynamic in T-cells activated by contact with superantigen pulsed B-cells and could move from the distal pole to an existing or a newly forming immunological synapse. One function of this cap may be to provide preassembled Ca 2؉ channel components to existing and newly forming immunological synapses. INTRODUCTIONT-cell activation in response to antigen induces and requires the elevation of intracellular Ca 2ϩ (Hogan et al., 2003;Quintana et al., 2005;Feske, 2007). T-cell receptor (TCR) engagement leads to activation of well-documented signal transduction pathways that cause a rapid release of Ca 2ϩ from the endoplasmic reticulum (ER; Samelson, 2002;Panyi et al., 2004;Gwack et al., 2007a). The depletion of Ca 2ϩ from this internal store then leads to the opening of ion channels in the plasma membrane (PM) allowing an influx of Ca 2ϩ from outside the cell. The store-operated channel responsible for Ca 2ϩ influx in T-cells is known as the Ca 2ϩ release-activated Ca 2ϩ (CRAC) channel, which has been studied by electrophysiological techniques for many years (Lewis, 2001;Prakriya and Lewis, 2003;Cahalan et al., 2007;Hewavitharana et al., 2007;Hogan and Rao, 2007;Putney, 2007a).Sustained elevation of cytosolic Ca 2ϩ is required for complete T-cell activation (Iezzi et al., 1998;Lewis, 2001;Hogan et al., 2003;Feske, 2007). High levels of intracellular Ca 2ϩ are necessary to maintain the interaction between a T-cell and antigen-presenting cell (APC) that leads to formation of the specialized contact surface known as the immunological synapse (IS). Increased Ca 2ϩ levels are also required for the activation of transcription factors. In particular, elevated Ca 2ϩ maintains prolonged nuclear accumulation of nuclear factor of activated T-cells (NFAT) by activating the phosphatase calcineurin that dephosphorylates NFAT, allowing it to translocate to the nucleus and activate genes such as IL2 (Hogan et al., 2003;Macian, 2005). Several hours of Ca 2ϩ influx are required to complete the T-cell activation program, which involves expression of a large number of activation-associated genes (Lewis, 2001;Macian et al., 2002;Hogan et al., 2003).Although sustained Ca 2ϩ influx in T-cells has been studied for decades, the proteins involved have only been ident...

T-cell antigen receptor engagement causes the rapid assembly of signaling complexes. The adapter protein SLP-76, detected as SLP-yellow fluorescent protein, initially clustered with the TCR and other proteins, then translocated medially on microtubules. As shown by total internal reflection fluorescence microscopy and the inhibition of SLP-76 movement at 168C, this movement required endocytosis. Immunoelectron microscopy showed SLP-76 staining of smooth pits and tubules. Cholesterol depletion decreased the movement of SLP-76 clusters, as did coexpression of the ubiquitin-interacting motif domain from eps15. These data are consistent with the internalization of SLP-76 via a lipid raft-dependent pathway that requires interaction of the endocytic machinery with ubiquitinylated proteins. The endocytosed SLP-76 clusters contained phosphorylated SLP-76 and phosphorylated LAT. The raft-associated, transmembrane protein LAT likely targets SLP-76 to endocytic vesicles. The endocytosis of active SLP-76 and LAT complexes suggests a possible mechanism for downregulation of signaling complexes induced by TCR activation.

The morphogenesis of skin epithelia and adult hair follicle cycling both require integrated signaling between the epithelium and underlying mesenchyme. Because of their unique regulation, keratin intermediate filaments represent useful markers for the analysis of determination and differentiation processes in complex epithelia, such as the skin. In this study, we analyzed the distribution of mouse type I keratin 16 during skin morphogenesis, in the adult hair cycle, and in challenged epidermis. In mature hair follicles, we find keratin 16 along with its type II keratin partner keratin 6 in the companion layer of the outer root sheath during anagen and in the club hair sheath during catagen and telogen. During embryonic development, the distribution of keratin 16 is uncoupled from its presumed polymerization partner, keratin 6. Keratin 16 initially localizes within early hair germs, but rapidly shifts to a subset of cells at the interface of basal and suprabasal cells above and around the hair germ. The presence of keratin 16 at the transition between mitotically active and differentiating cells is recapitulated in primary keratinocytes cultured in vitro and in phorbol 12-myristate 13-acetate-treated back skin in vivo. We propose that keratin 16 marks cells in an intermediate state of cellular properties in which keratinocytes retain the flexibility required for activities such as cell migration and even mitosis but are resilient enough to provide the structural integrity required of the early suprabasal layers in the context of development, adult hair cycling, and wound repair.

The MLL-partial tandem duplication (PTD) associates with high-risk cytogenetically normal acute myeloid leukemia (AML). Concurrent presence of FLT3-internal tandem duplication (ITD) is observed in 25% of patients with MLL-PTD AML. However, mice expressing either Mll-PTD or Flt3-ITD do not develop AML, suggesting that 2 mutations are necessary for the AML phenotype. Thus, we generated a mouse expressing both Mll-PTD and IntroductionAcute myeloid leukemia (AML) is a genetically heterogeneous disease. Recurrent cytogenetic and molecular gene aberrations have been used to classify AML patients into distinct subsets that differ in biologic, clinical, and prognostic characteristics. The MLL gene, located at chromosome band 11q23, encodes for a protein involved in epigenetic regulation of gene expression. 1 In AML, this gene is frequently involved in chromosome translocations at 11q23 and, at the molecular level, is fused with one of more than 50 different partners. 2 We first reported an internal duplication of MLL, an in-frame repeat producing an elongated protein retaining all functional domains, in cytogenetically normal (CN)-AML. 3 Approximately 5% to 7% of CN-AML patients have a MLL-partial tandem duplication (PTD) mutation, which is associated with unfavorable prognosis, 3-5 but if and how it contributes to myeloid leukemogenesis remain to be elucidated. Approximately 25% of CN-AML patients with the MLL-PTD had constitutive activation of FLT3, a tyrosine kinase receptor regulating proliferation and survival of hematopoietic cells, via an internal tandem duplication (FLT3-ITD), and have a very poor prognosis. 6 This suggests that both MLL-PTD and FLT3-ITD mutations are necessary for an AML phenotype, as supported by the broadly accepted 2-hit model. 7 Indeed, a Mll-PTD mouse, created by knocking in exons 5 to 11 in-frame and driven off of the endogenous Mll promoter, did not develop AML. 8 8,9 This model, which develops AML, is the first that requires the MLL-PTD. In this model, the 2 mutated genes are regulated by their respective endogenous promoters to recapitulate the Mll PTD/WT :Flt3 ITD/WT AML found in humans. This differs from some other doublemutant mouse models of human AML that carry one mutation driven by the endogenous promoter and the other mutation driven by transgenes, 10 or introduced via viral transduction, 11 or 2 mutations driven off 2 endogenous promoters but requiring BM transplantation. 12 Methods Mouse strainsThe Mll PTD/WT , Flt3 ITD/WT , and Flt3 ITD/ITD mice were generated and genotyped as previously described. 8,9 Male Flt3 ITD/WT Balb/c mice were backcrossed onto the C57Bl/6J strain to purity and then bred with Mll PTD/WT mice to generate Mll PTD/WT :Flt3 ITD/WT double knock-in offspring. Genotyping was performed as previously described. 8,9 All animals studied were compared within litters and/or age-and sex-matched. All experiments were conducted under an approved The Ohio State University Institutional Submitted March 2, 2012; accepted May 17, 2012. Prepublished online as Blood Firs...

Linker for activation of T cells (LAT) plays a central role in T-cell activation by nucleating signaling complexes that are critical for the propagation of T-cell signals from the plasma membrane to the cellular interior. The role of phosphorylation and palmitoylation in LAT function has been well studied, but not much is known about other strategies by which the cell modulates LAT activity. We have focused on LAT ubiquitylation and have mapped the sites on which LAT is ubiquitylated. To elucidate the biological role of this process, we substituted LAT lysines with arginines. This resulted in a dramatic decrease in overall LAT ubiquitylation. Ubiquitylationresistant mutants of LAT were internalized at rates comparable to wild-type LAT in a mechanism that required Cbl family proteins. However, these mutants displayed a defect in protein turnover rates. T-cell signaling was elevated in cells reconstituted with LAT mutants resistant to ubiquitylation, indicating that inhibition of LAT ubiquitylation enhances T-cell potency. These results support LAT ubiquitylation as a molecular checkpoint for attenuation of T-cell signaling.endocytosis | protein degradation | ubiquitin A ntigen activation of T cells leads to major cellular changes essential to the onset of a productive immune response. The earliest events occur upon T-cell antigen receptor (TCR) engagement and include activation of protein tyrosine kinases (PTKs) and phosphorylation of multiple protein substrates. One critical PTK substrate is the integral membrane adapter molecule linker for activation of T cells (LAT), which upon phosphorylation, serves as a nucleation site for the formation of protein complexes composed of itself, other adapter molecules, and essential signaling enzymes activated at this site (1). A large number of studies have revealed that LAT-based complexes catalyze critical TCR-mediated signaling reactions and lead to the induction of multiple downstream pathways that direct almost all TCRinitiated cellular responses (2).T-cell signaling must be tightly regulated, otherwise autoimmunity or a compromised immune response might ensue. Clearly, signal attenuation occurs at various stages during signal propagation. Proximally, the number and type of inhibitory coreceptors that are engaged on the T cell following TCR engagement greatly influence signal strength (3, 4). In addition, activation of the TCR also triggers mechanisms within the TCR signaling machinery that can decrease TCR-mediated signaling, including dephosphorylation of substrates by phosphatases, as well as phosphorylation on inhibitory residues by kinases (5). Finally, ubiquitylation and activationinduced endocytosis of proteins from the plasma membrane and subsequent degradation may eliminate activated signaling molecules (6, 7). Thus, tuning the excitation threshold of a T cell requires the coordinated regulation of multiple negative regulators.A growing body of evidence supports the concept that clustering of the TCR and downstream signaling proteins is associated with and is ...

• Echinomycin can selectively kill the leukemia-initiating cell in relapsed AML without normal stem cell toxicity.• In vivo delivery of echinomycin can induce long-term complete remission in a murine model of relapsed AML.Acute myeloid leukemia (AML) often relapses following chemotherapy-induced remission and is generally chemo-resistant. Given the potential role for cancer stem cells in relapse, targeting of the leukemia-initiating cell (LIC) in AML may provide improved outcome following remission induction. However, due to overlap in their self-renewal program with normal hematopoietic stem cells (HSCs), therapeutic targeting of the LIC may have an adverse effect on long-term hematopoietic recovery.Here we used a mouse model of relapsed AML to explore whether the hypoxiainducible factor (HIF)1a inhibitor echinomycin can be used to treat relapsed AML without affecting host HSCs. We show that echinomycin cured 40% to 60% of mice transplanted with relapsed AML. Bone marrow cells from the cured mice displayed normal composition of HSCs and their progenitors and were as competent as those isolated from nonleukemic mice in competitive repopulation assays. Importantly, in mice with complete remission, echinomycin appeared to completely eliminate LICs because no leukemia could be propagated in vivo following serial transplantation. Taken together, our data demonstrate that in a mouse model of relapsed AML, low-dose echinomycin selectively targets LICs and spares normal hematopoiesis. (Blood. 2014; 124(7):1127-1135

Intermediate filaments (IFs) are fibrous polymers encoded by a large family of differentially expressed genes that provide crucial structural support in the cytoplasm and nucleus in higher eukaryotes. The mechanisms involved in bringing together ∼16 elongated coiled-coil dimers to form an IF are poorly defined. Available evidence suggests that tetramer subunits play a key role during IF assembly and regulation. Through molecular modeling and site-directed mutagenesis, we document a hitherto unnoticed hydrophobic stripe exposed at the surface of coiled-coil keratin heterodimers that contributes to the extraordinary stability of heterotetramers. The inability of K16 to form urea-stable tetramers in vitro correlates with an increase in its turnover rate in vivo. The data presented support a specific conformation for the assembly competent IF tetramer, provide a molecular basis for their differential stability in vitro, and point to the physiological relevance associated with this property in vivo.