Regulation of WASH-dependent actin polymerization and protein trafficking by ubiquitination.

Endosomal protein trafficking is an essential cellular process that is deregulated in several diseases and targeted by pathogens. Here, we describe a role for ubiquitination in this process. We find that the E3 RING ubiquitin ligase, MAGE-L2-TRIM27, localizes to endosomes through interactions with the retromer complex. Knockdown of MAGE-L2-TRIM27 or the Ube2O E2 ubiquitin-conjugating enzyme significantly impaired retromer-mediated transport. We further demonstrate that MAGE-L2-TRIM27 ubiquitin ligase activity is required for nucleation of endosomal F-actin by the WASH regulatory complex, a known regulator of retromer-mediated transport. Mechanistic studies showed that MAGE-L2-TRIM27 facilitates K63-linked ubiquitination of WASH K220. Significantly, disruption of WASH ubiquitination impaired endosomal F-actin nucleation and retromer-dependent transport. These findings provide a cellular and molecular function for MAGE-L2-TRIM27 in retrograde transport, including an unappreciated role of K63-linked ubiquitination and identification of an activating signal of the WASH regulatory complex.

Regulation of T-cell activation by the cytoskeleton.

To become activated, T cells must efficiently recognize antigen-presenting cells or target cells through several complex cytoskeleton-dependent processes, including integrin-mediated adhesion, immunological-synapse formation, cellular polarization, receptor sequestration and signalling. The actin and microtubule systems provide the dynamic cellular framework that is required to orchestrate these processes and ultimately contol T-cell activation. Here, we discuss recent advances that have furthered our understanding of the crucial importance of the T-cell cytoskeleton in controlling these aspects of T-cell immune recognition.

Nuclear FAM21 participates in NF-κB-dependent gene regulation in pancreatic cancer cells.

The pentameric WASH complex is best known for its role in regulating receptor trafficking from retromer-rich endosomal subdomains. FAM21 functions to stabilize the WASH complex through its N-terminal head domain and localizes it to endosomes by directly binding the retromer through its extended C-terminal tail. Herein, we used affinity purification combined with mass spectrometry to identify additional FAM21-interacting proteins. Surprisingly, multiple components of the nuclear factor κB (NF-κB) pathway were identified, including the p50 and p65 (RelA) NF-κB subunits. We show that FAM21 interacts with these components and regulates NF-κB-dependent gene transcription at the level of p65 chromatin binding. We further demonstrate that FAM21 contains a functional monopartite nuclear localization signal sequence (NLS) as well as a CRM1/exportin1-dependent nuclear export signal (NES), both of which work jointly with the N-terminal head domain and C-terminal retromer recruitment domain to regulate FAM21 cytosolic and nuclear subcellular localization. Finally, our findings indicate that FAM21 depletion sensitizes pancreatic cancer cells to gemcitabine and 5-fluorouracil. Thus, FAM21 not only functions as an integral component of the cytoplasmic WASH complex, but also modulates NF-κB gene transcription in the nucleus.

Structure and control of the actin regulatory WAVE complex.

Members of the Wiskott-Aldrich syndrome protein (WASP) family control cytoskeletal dynamics by promoting actin filament nucleation with the Arp2/3 complex. The WASP relative WAVE regulates lamellipodia formation within a 400-kilodalton, hetero-pentameric WAVE regulatory complex (WRC). The WRC is inactive towards the Arp2/3 complex, but can be stimulated by the Rac GTPase, kinases and phosphatidylinositols. Here we report the 2.3-ångstrom crystal structure of the WRC and complementary mechanistic analyses. The structure shows that the activity-bearing VCA motif of WAVE is sequestered by a combination of intramolecular and intermolecular contacts within the WRC. Rac and kinases appear to destabilize a WRC element that is necessary for VCA sequestration, suggesting the way in which these signals stimulate WRC activity towards the Arp2/3 complex. The spatial proximity of the Rac binding site and the large basic surface of the WRC suggests how the GTPase and phospholipids could cooperatively recruit the complex to membranes.

The cytoskeletal adaptor protein IQGAP1 regulates TCR-mediated signaling and filamentous actin dynamics.

The Ras GTPase-activating-like protein IQGAP1 is a multimodular scaffold that controls signaling and cytoskeletal regulation in fibroblasts and epithelial cells. However, the functional role of IQGAP1 in T cell development, activation, and cytoskeletal regulation has not been investigated. In this study, we show that IQGAP1 is dispensable for thymocyte development as well as microtubule organizing center polarization and cytolytic function in CD8(+) T cells. However, IQGAP1-deficient CD8(+) T cells as well as Jurkat T cells suppressed for IQGAP1 were hyperresponsive, displaying increased IL-2 and IFN-γ production, heightened LCK activation, and augmented global phosphorylation kinetics after TCR ligation. In addition, IQGAP1-deficient T cells exhibited increased TCR-mediated F-actin assembly and amplified F-actin velocities during spreading. Moreover, we found that discrete regions of IQGAP1 regulated cellular activation and F-actin accumulation. Taken together, our data suggest that IQGAP1 acts as a dual negative regulator in T cells, limiting both TCR-mediated activation kinetics and F-actin dynamics via distinct mechanisms.

Dynamic remodeling of the actin cytoskeleton by FMNL1γ is required for structural maintenance of the Golgi complex.

Formin-like 1 (FMNL1) is a member of the formin family of actin nucleators, and is one of the few formins for which in vitro activities have been well characterized. However, the functional roles of this mammalian formin remain ill-defined. In particular, it is unclear how the unique in vitro biochemical properties of FMNL1 relate to its regulation of cellular processes. Here, we demonstrate that FMNL1 depletion caused a dramatic increase in cellular F-actin content, which resulted in Golgi complex fragmentation. Moreover, increased F-actin and maintenance of Golgi structure were distinctly regulated by the gamma isoform of FMNL1, which required binding to actin. Importantly, in addition to Golgi fragmentation, increased F-actin content in the absence of FMNL1 also led to cation-independent mannose 6-phosphate receptor dispersal, lysosomal enlargement and missorting of cathepsin D. Taken together, our data support a model in which FMNL1 regulates cellular F-actin levels required to maintain structural integrity of the Golgi complex and lysosomes.

Hematopoietic lineage cell-specific protein 1 functions in concert with the Wiskott-Aldrich syndrome protein to promote podosome array organization and chemotaxis in dendritic cells.

Dendritic cells (DCs) are professional APCs that reside in peripheral tissues and survey the body for pathogens. Upon activation by inflammatory signals, DCs undergo a maturation process and migrate to lymphoid organs, where they present pathogen-derived Ags to T cells. DC migration depends on tight regulation of the actin cytoskeleton to permit rapid adaptation to environmental cues. We investigated the role of hematopoietic lineage cell-specific protein 1 (HS1), the hematopoietic homolog of cortactin, in regulating the actin cytoskeleton of murine DCs. HS1 localized to lamellipodial protrusions and podosomes, actin-rich structures associated with adhesion and migration. DCs from HS1(-/-) mice showed aberrant lamellipodial dynamics. Moreover, although these cells formed recognizable podosomes, their podosome arrays were loosely packed and improperly localized within the cell. HS1 interacts with Wiskott-Aldrich syndrome protein (WASp), another key actin-regulatory protein, through mutual binding to WASp-interacting protein. Comparative analysis of DCs deficient for HS1, WASp or both proteins revealed unique roles for these proteins in regulating podosomes with WASp being essential for podosome formation and with HS1 ensuring efficient array organization. WASp recruitment to podosome cores was independent of HS1, whereas HS1 recruitment required Src homology 3 domain-dependent interactions with the WASp/WASp-interacting protein heterodimer. In migration assays, the phenotypes of HS1- and WASp-deficient DCs were related, but distinct. WASp(-/y) DCs migrating in a chemokine gradient showed a large decrease in velocity and diminished directional persistence. In contrast, HS1(-/-) DCs migrated faster than wild-type cells, but directional persistence was significantly reduced. These studies show that HS1 functions in concert with WASp to fine-tune DC cytoarchitecture and direct cell migration.

WASH and WAVE actin regulators of the Wiskott-Aldrich syndrome protein (WASP) family are controlled by analogous structurally related complexes.

We recently showed that the Wiskott-Aldrich syndrome protein (WASP) family member, WASH, localizes to endosomal subdomains and regulates endocytic vesicle scission in an Arp2/3-dependent manner. Mechanisms regulating WASH activity are unknown. Here we show that WASH functions in cells within a 500 kDa core complex containing Strumpellin, FAM21, KIAA1033 (SWIP), and CCDC53. Although recombinant WASH is constitutively active toward the Arp2/3 complex, the reconstituted core assembly is inhibited, suggesting that it functions in cells to regulate actin dynamics through WASH. FAM21 interacts directly with CAPZ and inhibits its actin-capping activity. Four of the five core components show distant (approximately 15% amino acid sequence identify) but significant structural homology to components of a complex that negatively regulates the WASP family member, WAVE. Moreover, biochemical and electron microscopic analyses show that the WASH and WAVE complexes are structurally similar. Thus, these two distantly related WASP family members are controlled by analogous structurally related mechanisms. Strumpellin is mutated in the human disease hereditary spastic paraplegia, and its link to WASH suggests that misregulation of actin dynamics on endosomes may play a role in this disorder.

Protein kinase Cbeta modulates ligand-induced cell surface death receptor accumulation: a mechanistic basis for enzastaurin-death ligand synergy.

Although treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) is known to protect a subset of cells from induction of apoptosis by death ligands such as Fas ligand and tumor necrosis factor-alpha-related apoptosis-inducing ligand, the mechanism of this protection is unknown. This study demonstrated that protection in short term apoptosis assays and long term proliferation assays was maximal when Jurkat or HL-60 human leukemia cells were treated with 2-5 nm PMA. Immunoblotting demonstrated that multiple PKC isoforms, including PKCalpha, PKCbeta, PKCepsilon, and PKC, translocated from the cytosol to a membrane-bound fraction at these PMA concentrations. When the ability of short hairpin RNA (shRNA) constructs that specifically down-regulated each of these isoforms was examined, PKCbeta shRNA uniquely reversed PMA-induced protection against cell death. The PKCbeta-selective small molecule inhibitor enzastaurin had a similar effect. Although mass spectrometry suggested that Fas is phosphorylated on a number of serines and threonines, mutation of these sites individually or collectively had no effect on Fas-mediated death signaling or PMA protection. Further experiments demonstrated that PMA diminished ligand-induced cell surface accumulation of Fas and DR5, and PKCbeta shRNA or enzastaurin reversed this effect. Moreover, enzastaurin sensitized a variety of human tumor cell lines and clinical acute myelogenous leukemia isolates, which express abundant PKCbeta, to tumor necrosis factor-alpha related apoptosis-inducing ligand-induced death in the absence of PMA. Collectively, these results identify a specific PKC isoform that modulates death receptor-mediated cytotoxicity as well as a small molecule inhibitor that mitigates the inhibitory effects of PKC activation on ligand-induced death receptor trafficking and cell death.

Hematopoietic lineage cell-specific protein 1 is recruited to the immunological synapse by IL-2-inducible T cell kinase and regulates phospholipase Cgamma1 Microcluster dynamics during T cell spreading.

Productive T cell activation requires efficient reorganization of the actin cytoskeleton. We showed previously that the actin-regulatory protein, hematopoietic lineage cell-specific protein 1 (HS1), is required for the stabilization of F-actin and Vav1 at the immunological synapse and for efficient calcium responses. The Tec family kinase IL-2-inducible T cell kinase (Itk) regulates similar aspects of T cell activation, suggesting that these proteins act in the same pathway. Using video microscopy, we show that T cells lacking Itk or HS1 exhibited similar defects in actin responses, extending unstable lamellipodial protrusions upon TCR stimulation. HS1 and Itk could be coimmunoprecipitated from T cell lysates, and GST-pulldown studies showed that Itk's Src homology 2 domain binds directly to two phosphotyrosines in HS1. In the absence of Itk, or in T cells overexpressing an Itk Src homology 2 domain mutant, HS1 failed to localize to the immunological synapse, indicating that Itk serves to recruit HS1 to sites of TCR engagement. Because Itk is required for phospholipase C (PLC)gamma1 phosphorylation and calcium store release, we examined the calcium signaling pathway in HS1(-/-) T cells in greater detail. In response to TCR engagement, T cells lacking HS1 exhibited diminished calcium store release, but TCR-dependent PLCgamma1 phosphorylation was intact, indicating that HS1's role in calcium signaling is distinct from that of Itk. HS1-deficient T cells exhibited defective cytoskeletal association of PLCgamma1 and altered formation of PLCgamma1 microclusters. We conclude that HS1 functions as an effector of Itk in the T cell actin-regulatory pathway, and directs the spatial organization of PLCgamma1 signaling complexes.

Differential regulation of TCR-mediated gene transcription by Vav family members.

Although all three Vav family members are expressed in T lymphocytes, the role that Vav3 plays in T cell activation is poorly defined. Here we show that, like Vav1, Vav3 undergoes rapid tyrosine phosphorylation after T cell receptor (TCR) cross-linkage and interacts with the adaptor molecules SLP76 and 3BP2 in a SH2-dependent manner. However, depletion of Vav1 but not Vav3 protein by RNA interference affects TCR-mediated IL-2 promoter activity. In contrast, Vav3 function is specifically required for coupling TCR stimulation to serum response element-mediated gene transcription. These data indicate that, although both Vav proteins are biochemically coupled to the TCR, they regulate distinct molecular pathways leading to defined gene transcriptional events.

The Ezh2 methyltransferase complex: actin up in the cytosol.

Ezh2, a polycomb group protein, is known to function in histone methylation, thereby regulating gene expression. However, in a recent study by Su et al., the Ezh2-containing complex has been given an additional role in cellular regulation. Cytosolic Ezh2 methyltransferase complexes were shown to associate with Vav1 and control receptor-induced actin polymerization and proliferation in a methylation-dependent manner. Overall, these findings implicate lysine methylation as a posttranslational modification crucial for receptor-mediated signal transduction events.

T cell activation and the cytoskeleton: you can't have one without the other.

More than a quarter of a century has passed since the observation that T cells rapidly polarize their actin and microtubule cytoskeletal systems toward antigen-presenting cells during activation. Since this initial discovery, several receptors on T cells (e.g., T cell receptor [TCR], co-receptors, integrins, and chemokine receptors) have been identified to regulate these two cytoskeletal networks through complex signaling pathways, which are still being elucidated. There is now an undeniable body of biochemical, pharmacological, and genetic evidence indicating that regulators of actin and microtubule dynamics are crucial for T cell activation and effector functions. In fact, the actin cytoskeleton participates in the initial clustering of TCR-major histocompatibility complex or peptide complexes, formation and stabilization of the immune synapse, integrin-mediated adhesion, and receptor sequestration, whereas both the actin and microtubule cytoskeletons regulate the establishment of cell polarity, cell migration, and directed secretion of cytokines and cytolytic granules. Over the past several years, we have begun to more thoroughly understand the contributions of specific actin-regulatory and actin-nucleating proteins that govern these processes. Herein, we discuss our current understanding of how activating receptors on T lymphocytes regulate the actin and microtubule cytoskeletons, and how in turn, these distinct but integrated cytoskeletal networks coordinate T cell immune responses.

Dynamin 2 regulates granule exocytosis during NK cell-mediated cytotoxicity.

NK cells are innate immune cells that can eliminate their targets through granule release. In this study, we describe a specialized role for the large GTPase Dynamin 2 (Dyn2) in the regulation of these secretory events leading to cell-mediated cytotoxicity. By modulating the expression of Dyn2 using small interfering RNA or by inhibiting its activity using a pharmacological agent, we determined that Dyn2 does not regulate conjugate formation, proximal signaling, or granule polarization. In contrast, during cell-mediated killing, Dyn2 localizes with lytic granules and polarizes to the NK cell-target interface where it regulates the final fusion of lytic granules with the plasma membrane. These findings identify a novel role for Dyn2 in the exocytic events required for effective NK cell-mediated cytotoxicity.

The WAVE2 complex regulates actin cytoskeletal reorganization and CRAC-mediated calcium entry during T cell activation.

BACKGROUND: The engagement of the T cell receptor results in actin cytoskeletal reorganization at the immune synapse (IS) and the triggering of biochemical signaling cascades leading to gene regulation and, ultimately, cellular activation. Recent studies have identified the WAVE family of proteins as critical mediators of Rac1-induced actin reorganization in other cell types. However, whether these proteins participate in actin reorganization at the IS or signaling pathways in T cells has not been investigated. RESULTS: By using a combination of biochemical, genetic, and cell biology approaches, we provide evidence that WAVE2 is recruited to the IS, is biochemically modified, and is required for actin reorganization and beta-integrin-mediated adhesion after TCR crosslinking. Moreover, we show that WAVE2 regulates calcium entry at a point distal to PLCgamma1 activation and IP(3)-mediated store release. CONCLUSIONS: These data reveal a role for WAVE2 in regulating multiple pathways leading to T cell activation. In particular, this work shows that WAVE2 is a key component of the actin regulatory machinery in T cells and that it also participates in linking intracellular calcium store depletion to calcium release-activated calcium (CRAC) channel activation.

Vpr R77Q is associated with long-term nonprogressive HIV infection and impaired induction of apoptosis.

The absence of immune defects that occurs in the syndrome of long-term nonprogressive (LTNP) HIV infection offers insights into the pathophysiology of HIV-induced immune disease. The (H[F/S]RIG)(2) domain of viral protein R (Vpr) induces apoptosis and may contribute to HIV-induced T cell depletion. We demonstrate a higher frequency of R77Q Vpr mutations in patients with LTNP than in patients with progressive disease. In addition, T cell infections using vesicular stomatitis virus G (VSV-G) pseudotyped HIV-1 Vpr R77Q result in less (P = 0.01) T cell death than infections using wild-type Vpr, despite similar levels of viral replication. Wild-type Vpr-associated events, including procaspase-8 and -3 cleavage, loss of mitochondrial transmembrane potential (deltapsi(m)), and DNA fragmentation factor activation are attenuated by R77Q Vpr. These data highlight the pathophysiologic role of Vpr in HIV-induced immune disease and suggest a novel mechanism of LTNP.

Structural and mechanistic insights into regulation of the retromer coat by TBC1d5.

Retromer is a membrane coat complex that is recruited to endosomes by the small GTPase Rab7 and sorting nexin 3. The timing of this interaction and consequent endosomal dynamics are thought to be regulated by the guanine nucleotide cycle of Rab7. Here we demonstrate that TBC1d5, a GTPase-activating protein (GAP) for Rab7, is a high-affinity ligand of the retromer cargo selective complex VPS26/VPS29/VPS35. The crystal structure of the TBC1d5 GAP domain bound to VPS29 and complementary biochemical and cellular data show that a loop from TBC1d5 binds to a conserved hydrophobic pocket on VPS29 opposite the VPS29-VPS35 interface. Additional data suggest that a distinct loop of the GAP domain may contact VPS35. Loss of TBC1d5 causes defective retromer-dependent trafficking of receptors. Our findings illustrate how retromer recruits a GAP, which is likely to be involved in the timing of Rab7 inactivation leading to membrane uncoating, with important consequences for receptor trafficking.

COMMD1 is linked to the WASH complex and regulates endosomal trafficking of the copper transporter ATP7A.

COMMD1 deficiency results in defective copper homeostasis, but the mechanism for this has remained elusive. Here we report that COMMD1 is directly linked to early endosomes through its interaction with a protein complex containing CCDC22, CCDC93, and C16orf62. This COMMD/CCDC22/CCDC93 (CCC) complex interacts with the multisubunit WASH complex, an evolutionarily conserved system, which is required for endosomal deposition of F-actin and cargo trafficking in conjunction with the retromer. Interactions between the WASH complex subunit FAM21, and the carboxyl-terminal ends of CCDC22 and CCDC93 are responsible for CCC complex recruitment to endosomes. We show that depletion of CCC complex components leads to lack of copper-dependent movement of the copper transporter ATP7A from endosomes, resulting in intracellular copper accumulation and modest alterations in copper homeostasis in humans with CCDC22 mutations. This work provides a mechanistic explanation for the role of COMMD1 in copper homeostasis and uncovers additional genes involved in the regulation of copper transporter recycling.

Dendritic cells utilize the evolutionarily conserved WASH and retromer complexes to promote MHCII recycling and helper T cell priming.

Immature dendritic cells (DCs) maintain a highly dynamic pool of recycling MHCII that promotes sampling of environmental antigens for presentation to T helper cells. However, the molecular basis of MHCII recycling and the cellular machinery that orchestrates MHCII trafficking are incompletely understood. Using a mouse model we show that WASH, an actin regulatory protein that facilitates retromer function, is essential for MHCII recycling and efficient priming of T helper cells. We further demonstrate that WASH deficiency results in impaired MHCII surface levels, recycling, and an accumulation of polyubiquitinated MHCII complexes, which are subsequently slated for premature lysosomal degradation. Consequently, conditional deletion of the Wash gene in DCs impairs priming of both conventional and autoimmune T helper cells in vivo and attenuates disease progression in a model of experimental autoimmune encephalitis (EAE). Thus, we identify a novel mechanism in which DCs employ the evolutionarily conserved WASH and retromer complex for MHCII recycling in order to regulate T helper cell priming.

WASH knockout T cells demonstrate defective receptor trafficking, proliferation, and effector function.

WASH is an Arp2/3 activator of the Wiskott-Aldrich syndrome protein superfamily that functions during endosomal trafficking processes in collaboration with the retromer and sorting nexins, but its in vivo function has not been examined. To elucidate the physiological role of WASH in T cells, we generated a WASH conditional knockout (WASHout) mouse model. Using CD4(Cre) deletion, we found that thymocyte development and naive T cell activation are unaltered in the absence of WASH. Surprisingly, despite normal T cell receptor (TCR) signaling and interleukin-2 production, WASHout T cells demonstrate significantly reduced proliferative potential and fail to effectively induce experimental autoimmune encephalomyelitis. Interestingly, after activation, WASHout T cells fail to maintain surface levels of TCR, CD28, and LFA-1. Moreover, the levels of the glucose transporter, GLUT1, are also reduced compared to wild-type T cells. We further demonstrate that the loss of surface expression of these receptors in WASHout cells results from aberrant accumulation within the collapsed endosomal compartment, ultimately leading to degradation within the lysosome. Subsequently, activated WASHout T cells experience reduced glucose uptake and metabolic output. Thus, we found that WASH is a newly recognized regulator of TCR, CD28, LFA-1, and GLUT1 endosome-to-membrane recycling. Aberrant trafficking of these key T cell proteins may potentially lead to attenuated proliferation and effector function.