TACC3 is a microtubule plus end–tracking protein that promotes axon elongation and also regulates microtubule plus end dynamics in multiple embryonic cell types

Microtubule plus end dynamics are regulated by a conserved family of proteins called plus end–tracking proteins (+TIPs). It is unclear how various +TIPs interact with each other and with plus ends to control microtubule behavior. The centrosome-associated protein TACC3, a member of the transforming acidic coiled-coil (TACC) domain family, has been implicated in regulating several aspects of microtubule dynamics. However, TACC3 has not been shown to function as a +TIP in vertebrates. Here we show that TACC3 promotes axon outgrowth and regulates microtubule dynamics by increasing microtubule plus end velocities in vivo. We also demonstrate that TACC3 acts as a +TIP in multiple embryonic cell types and that this requires the conserved C-terminal TACC domain. Using high-resolution live-imaging data on tagged +TIPs, we show that TACC3 localizes to the extreme microtubule plus end, where it lies distal to the microtubule polymerization marker EB1 and directly overlaps with the microtubule polymerase XMAP215. TACC3 also plays a role in regulating XMAP215 stability and localizing XMAP215 to microtubule plus ends. Taken together, our results implicate TACC3 as a +TIP that functions with XMAP215 to regulate microtubule plus end dynamics.     

Cdk1 and Plk1 mediate a CLASP2 phospho-switch that stabilizes kinetochore–microtubule attachments

Accurate chromosome segregation during mitosis relies on a dynamic kinetochore (KT)–microtubule (MT) interface that switches from a labile to a stable condition in response to correct MT attachments. This transition is essential to satisfy the spindle-assembly checkpoint (SAC) and couple MT-generated force with chromosome movements, but the underlying regulatory mechanism remains unclear. In this study, we show that during mitosis the MT- and KT-associated protein CLASP2 is progressively and distinctively phosphorylated by Cdk1 and Plk1 kinases, concomitant with the establishment of KT–MT attachments. CLASP2 S1234 was phosphorylated by Cdk1, which primed CLASP2 for association with Plk1. Plk1 recruitment to KTs was enhanced by CLASP2 phosphorylation on S1234. This was specifically required to stabilize KT–MT attachments important for chromosome alignment and to coordinate KT and non-KT MT dynamics necessary to maintain spindle bipolarity. CLASP2 C-terminal phosphorylation by Plk1 was also required for chromosome alignment and timely satisfaction of the SAC. We propose that Cdk1 and Plk1 mediate a fine CLASP2 “phospho-switch” that temporally regulates KT–MT attachment stability.     

PI3K Class II α Controls Spatially Restricted Endosomal PtdIns3P and Rab11 Activation to Promote Primary Cilium Function

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EHSH1/intersectin, a protein that contains EH and SH3 domains and binds to dynamin and SNAP-25. A protein connection between exocytosis and endocytosis?

In yeast two-hybrid screens for proteins that bind to SNAP-25 and may be involved in exocytosis, we isolated a protein called EHSH1 (for EH domain/SH3 domain-containing protein). Cloning of full-length cDNAs revealed that EHSH1 is composed of an N-terminal region with two EH domains, a central region that is enriched in lysine, leucine, glutamate, arginine, and glutamine (KLERQ domain), and a C-terminal region comprised of five SH3 domains. The third SH3 domain is alternatively spliced. Data bank searches demonstrated that EHSH1 is very similar to Xenopus and human intersectins and to human SH3P17. In addition, we identified expressed sequence tags that encode a second isoform of EHSH1, called EHSH2. EHSH1 is abundantly expressed in brain and at lower levels in all other tissues tested. In binding studies, we found that the central KLERQ domain of EHSH1 binds to recombinant or native brain SNAP-25 and SNAP-23. The C-terminal SH3 domains, by contrast, quantitatively interact with dynamin, a protein involved in endocytosis. Dynamin strongly binds to the alternatively spliced central SH3 domain (SH3C) and the two C-terminal SH3 domains (SH3D and SH3E) but not to the N-terminal SH3 domains (SH3A and SH3B). Immunoprecipitations confirmed that both dynamin and SNAP-25 are complexed to EHSH1 in brain. Our data suggest that EHSH1/intersectin may be a novel adaptor protein that couples endocytic membrane traffic to exocytosis. The ability of multiple SH3 domains in EHSH1 to bind to dynamin suggests that EHSH1 can cluster several dynamin molecules in a manner that is regulated by alternative splicing.     

Calpain-7 binds to CHMP1B at its second α-helical region and forms a ternary complex with IST1

Some intracellular proteins involved in the endosomal sorting complex required for transport (ESCRT) system have microtubule interacting and transport (MIT) domains and bind to ESCRT-III protein family members named charged multivesicular body proteins (CHMPs) at their C-terminal regions containing MIT-interacting motifs (MIMs). While two types of MIMs (MIM1 and MIM2) have been reported, CHMP1B has MIM1 and IST1 has both MIM1 and MIM2. Previously, we demonstrated that CHMP1B and IST1 directly interacted with a tandem repeat of MIT domains of calpain-7 (CL7MIT) and that autolytic activity of calpain-7 was enhanced by IST1 in vitro but not by overexpression of IST1 in HEK293T cells. In this study, we detected enhancement of autolysis of mGFP-fused calpain-7 by coexpression with CHMP1B and observed further activation by additional coexpression of IST1 in HEK293T cells. We found that CL7MIT interacted with the second α-helical region of CHMP1B but not with the canonical C-terminal region containing MIM1 in vitro. Co-immunoprecipitation assays demonstrated that the interaction between CL7MIT and CHMP1B and between CL7MIT and IST1 became stronger when IST1 or CHMP1B was additionally coexpressed, suggesting formation of ternary complex of calpain-7, IST1 and CHMP1B. Moreover, subcellular fractionation analyses revealed increase of calpain-7 in membrane/organelle fractions by concomitant overexpression of these ESCRT-III family member proteins.     

The 3′ end of Turnip crinkle virus contains a highly interactive structure including a translational enhancer that is disrupted by binding to the RNA-dependent RNA polymerase

Precise temporal control is needed for RNA viral genomes to translate sufficient replication-required products before clearing ribosomes and initiating replication. A 3′ translational enhancer in Turnip crinkle virus (TCV) overlaps an internal T-shaped structure (TSS) that binds to 60S ribosomal subunits. The higher-order structure in the region was examined through alteration of critical sequences revealing novel interactions between an H-type pseudoknot and upstream residues, and between the TSS and internal and terminal loops of an upstream hairpin. Our results suggest that the TSS forms a stable scaffold that allows for simultaneous interactions with external sequences through base pairings on both sides of its large internal symmetrical loop. Binding of TCV RNA-dependent RNA polymerase (RdRp) to the region potentiates a widespread conformational shift with substantial rearrangement of the TSS region, including the element required for efficient ribosome binding. Degrading the RdRp caused the RNA to resume its original conformation, suggesting that the initial conformation is thermodynamically favored. These results suggest that the 3′ end of TCV folds into a compact, highly interactive structure allowing RdRp access to multiple elements including the 3′ end, which causes structural changes that potentiate the shift between translation and replication.     

Inhibition of HIV-1 infection of peripheral blood mononuclear cells by a monoclonal antibody that binds to phosphoinositides and induces secretion of β-chemokines

A murine IgG mAb, WR321, selected for the ability to bind to phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate, but an inability to bind to any of 17 other lipids, including phosphatidylinositol, was examined as a probe for studying interactions of HIV-1 with primary human peripheral blood mononuclear cells. The WR321 mAb broadly neutralized CCR5-tropic strains of HIV-1 to prevent infection of the cells. The mAb also exhibited direct interaction with cells in the culture, resulting in secretion of chemokines that interfered with the interaction of HIV-1 virions with CCR5, the coreceptor for HIV-1 on the susceptible cells, leading to inhibition of infection by HIV-1. Phosphoinositides that are recognized by WR321 do not exist on the external surface of cells, but are concentrated on the inner surface (cytoplasmic leaflet) of the plasma membrane. Murine anti-phosphoinositide mAbs similar to WR321 have previously been directly microinjected into a variety of cultured cells, resulting in important changes in the functions of the cells. The present results suggest that binding of a mAb to phosphoinositides, resulting in secretion of β-chemokines into the culture medium and neutralization of infection by CCR5-tropic HIV-1 of nearby susceptible cells, occurred by uptake and binding of the mAb at an intracellular location in the cultured cells that then led to secretion of HIV-1-inhibitory β-chemokines.     

Evidence that a polymorphism within the 3′UTR of glutathione peroxidase 4 is functional and is associated with susceptibility to colorectal cancer

Low selenium (Se) status has been associated with increased risk of colorectal cancer (CRC). Se is present as the amino acid selenocysteine in selenoproteins, such as the glutathione peroxidases. Se incorporation requires specific RNA structures in the 3' untranslated region (3'UTR) of the selenoprotein mRNAs. A single nucleotide polymorphism (SNP) occurs at nucleotide 718 (within the 3'UTR) in the glutathione peroxidase 4 gene. In the present study, Caco-2 cells were transfected with constructs in which type 1 iodothyronine deiodinase coding region was linked to the GPx4 3'UTR with either C or T variant at position 718. Higher reporter activity was observed in cells expressing the C variant compared to those expressing the T variant, under either Se-adequate or Se-deficient conditions. In addition, a disease association study was carried out in cohorts of patients with either adenomatous polyps, colorectal adenocarcinomas and in healthy controls. A higher proportion of individuals with CC genotype at the GPx4 T/C 718 SNP was present in the cancer group, but not in the polyp group, compared with the control group (P < 0.05). The present data demonstrate the functionality of the GPx4 T/C 718 SNP and suggest that T genotype is associated with lower risk of CRC.     

Membrane transport of WAVE2 and lamellipodia formation require Pak1 that mediates phosphorylation and recruitment of stathmin/Op18 to Pak1–WAVE2–kinesin complex

Membrane transport of WAVE2 that leads to lamellipodia formation requires a small GTPase Rac1, the motor protein kinesin, and microtubules. Here we explore the possibility of whether the Rac1-dependent and kinesin-mediated WAVE2 transport along microtubules is regulated by a p21-activated kinase Pak as a downstream effector of Rac1. We find that Pak1 constitutively binds to WAVE2 and is transported with WAVE2 to the leading edge by stimulation with hepatocyte growth factor (HGF). Concomitantly, phosphorylation of tubulin-bound stathmin/Op18 at serine 25 (Ser25) and Ser38, microtubule growth, and stathmin/Op18 binding to kinesin–WAVE2 complex were induced. The HGF-induced WAVE2 transport, lamellipodia formation, stathmin/Op18 phosphorylation at Ser38 and binding to kinesin–WAVE2 complex, but not stathmin/Op18 phosphorylation at Ser25 and microtubule growth, were abrogated by Pak1 inhibitor IPA-3 and Pak1 depletion with small interfering RNA (siRNA). Moreover, stathmin/Op18 depletion with siRNA caused significant inhibition of HGF-induced WAVE2 transport and lamellipodia formation, with HGF-independent promotion of microtubule growth. Collectively, it is suggested that Pak1 plays a critical role in HGF-induced WAVE2 transport and lamellipodia formation by directing Pak1–WAVE2–kinesin complex toward the ends of growing microtubules through phosphorylation and recruitment of tubulin-bound stathmin/Op18 to the complex.     

Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7–RILP–p150Glued and late endosome positioning

Late endosomes (LEs) have characteristic intracellular distributions determined by their interactions with various motor proteins. Motor proteins associated to the dynactin subunit p150^Glued bind to LEs via the Rab7 effector Rab7-interacting lysosomal protein (RILP) in association with the oxysterol-binding protein ORP1L. We found that cholesterol levels in LEs are sensed by ORP1L and are lower in peripheral vesicles. Under low cholesterol conditions, ORP1L conformation induces the formation of endoplasmic reticulum (ER)–LE membrane contact sites. At these sites, the ER protein VAP (VAMP [vesicle-associated membrane protein]-associated ER protein) can interact in trans with the Rab7–RILP complex to remove p150^Glued and associated motors. LEs then move to the microtubule plus end. Under high cholesterol conditions, as in Niemann-Pick type C disease, this process is prevented, and LEs accumulate at the microtubule minus end as the result of dynein motor activity. These data explain how the ER and cholesterol control the association of LEs with motor proteins and their positioning in cells.     

PI3K is involved in β1 integrin clustering by PSGL-1 and promotes β1 integrin-mediated Jurkat cell adhesion to fibronectin

P-selectin glycoprotein ligand-1 (PSGL-1) is involved in the initial step of lymphocyte homing by interacting with P- or E-selectins expressed on activated endothelium cells. Besides, it also functions as a receptor to induce signals that increase integrin affinity to ligands and mediate cell adhesion to endothelium. Integrin is required for the second step of lymphocyte homing, whose activation has been reported tightly regulated by inside-out signals triggered by chemokines or the shear-stress generated during lymphocyte rolling on endothelium. However, the relationship between PSGL-1-triggered signals and integrin activation is not clear. In this study, we demonstrated that PSGL-1 ligation induces β1 integrin-mediated adhesion to fibronectin via regulation of both β1 subunit clustering and conformation changes. Phosphoinositide 3-kinase (PI3K) is required for PSGL-1-induced β1 integrin clustering which ultimately regulates β1 integrin-mediated Jurkat cell adhesion to fibronectin. However, PI3K is not involved in the conformation changes or increases in the total expression of β1 integrin. Taken together, we found a novel signal pathway, PSGL-1-PI3K-β1 integrin, demonstrating the cooperation between initial adhesion and subsequent arrest and stable adhesion.     

Trafficking of the Menkes copper transporter ATP7A is regulated by clathrin-, AP-2–, AP-1–, and Rab22-dependent steps

The transporter ATP7A mediates systemic copper absorption and provides cuproenzymes in the trans-Golgi network (TGN) with copper. To regulate metal homeostasis, ATP7A constitutively cycles between the TGN and plasma membrane (PM). ATP7A trafficking to the PM is elevated in response to increased copper load and is reversed when copper concentrations are lowered. Molecular mechanisms underlying this trafficking are poorly understood. We assess the role of clathrin, adaptor complexes, lipid rafts, and Rab22a in an attempt to decipher the regulatory proteins involved in ATP7A cycling. While RNA interference (RNAi)–mediated depletion of caveolin 1/2 or flotillin had no effect on ATP7A localization, clathrin heavy chain depletion or expression of AP180 dominant-negative mutant not only disrupted clathrin-regulated pathways, but also blocked PM-to-TGN internalization of ATP7A. Depletion of the μ subunits of either adaptor protein-2 (AP-2) or AP-1 using RNAi further provides evidence that both clathrin adaptors are important for trafficking of ATP7A from the PM to the TGN. Expression of the GTP-locked Rab22a_Q64L mutant caused fragmentation of TGN membrane domains enriched for ATP7A. These appear to be a subdomain of the mammalian TGN, showing only partial overlap with the TGN marker golgin-97. Of importance, ATP7A remained in the Rab22a_Q64L-generated structures after copper treatment and washout, suggesting that forward trafficking out of this compartment was blocked. This study provides evidence that multiple membrane-associated factors, including clathrin, AP-2, AP-1, and Rab22, are regulators of ATP7A trafficking.     

3β-taraxerol of Mangifera indica, a PI3K dependent dual activator of glucose transport and glycogen synthesis in 3T3-L1 adipocytes

Background

The present study focuses on identifying and developing an anti-diabetic molecule from plant sources that would effectively combat insulin resistance through proper channeling of glucose metabolism involving glucose transport and storage.

Methods

Insulin-stimulated glucose uptake formed the basis for isolation of a bioactive molecule through column chromatography followed by its characterization using NMR and mass spectroscopic analysis. Mechanism of glucose transport and storage was evaluated based on the expression profiling of signaling molecules involved in the process.

Results

The study reports (i) the isolation of a bioactive compound 3β-taraxerol from the ethyl acetate extract (EAE) of the leaves of Mangifera indica (ii) the bioactive compound exhibited insulin-stimulated glucose uptake through translocation and activation of the glucose transporter (GLUT4) in an IRTK and PI3K dependent fashion. (iii) the fate of glucose following insulin-stimulated glucose uptake was ascertained through glycogen synthesis assay that involved the activation of PKB and suppression of GSK3β.

General significance

This study demonstrates the dual activity of 3β-taraxerol and the ethyl acetate extract of Mangifera indica as a glucose transport activator and stimulator of glycogen synthesis. 3β-taraxerol can be validated as a potent candidate for managing the hyperglycemic state.     

Ethylene-induced stomatal closure is mediated via MKK1/3–MPK3/6 cascade to EIN2 and EIN3

Mitogen-activated protein kinases (MPKs) play essential roles in guard cell signaling, but whether MPK cascades participate in guard cell ethylene signaling and interact with hydrogen peroxide (H₂O₂), nitric oxide (NO), and ethylene-signaling components remain unclear. Here, we report that ethylene activated MPK3 and MPK6 in the leaves of wild-type Arabidopsis thaliana as well as ethylene insensitive2 (ein2), ein3, nitrate reductase1 (nia1), and nia2 mutants, but this effect was impaired in ethylene response1 (etr1), nicotinamide adenine dinucleotide phosphate oxidase AtrbohF, mpk kinase1 (mkk1), and mkk3 mutants. By contrast, the constitutive triple response1 (ctr1) mutant had constitutively active MPK3 and MPK6. Yeast two-hybrid, bimolecular fluorescence complementation, and pull-down assays indicated that MPK3 and MPK6 physically interacted with MKK1, MKK3, and the C-terminal region of EIN2 (EIN2 CEND). mkk1, mkk3, mpk3, and mpk6 mutants had typical levels of ethylene-induced H₂O₂ generation but impaired ethylene-induced EIN2 CEND cleavage and nuclear translocation, EIN3 protein accumulation, NO production in guard cells, and stomatal closure. These results show that the MKK1/3–MPK3/6 cascade mediates ethylene-induced stomatal closure by functioning downstream of ETR1, CTR1, and H₂O₂ to interact with EIN2, thereby promoting EIN3 accumulation and EIN3-dependent NO production in guard cells.     

The apical Na+–HCO3− cotransporter Slc4a7 (NBCn1) does not contribute to bicarbonate transport by mouse salivary gland ducts

The HCO₃ ⁻ secretion mechanism in salivary glands is unclear but is thought to rely on the co-ordinated activity of multiple ion transport proteins including members of the Slc4 family of bicarbonate transporters. Slc4a7 was immunolocalized to the apical membrane of mouse submandibular duct cells. In contrast, Slc4a7 was not detected in acinar cells, and correspondingly, Slc4a7 disruption did not affect fluid secretion in response to cholinergic or β-adrenergic stimulation in the submandibular gland (SMG). Much of the Na ⁺-dependent intracellular pH (pH _i) regulation in SMG duct cells was insensitive to 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, S0859, and to the removal of extracellular HCO ₃ ⁻. Consistent with these latter observations, the Slc4a7 null mutation had no impact on HCO ₃ ⁻ secretion nor on pH _i regulation in duct cells. Taken together, our results revealed that Slc4a7 targets to the apical membrane of mouse SMG duct cells where it contributes little if any to pH _i regulation or stimulated HCO ₃ ⁻ secretion.     

Identification of New Regions in HIV-1 gp120 Variable 2 and 3 Loops that Bind to α4β7 Integrin Receptor

Background

The gut mucosal homing integrin receptor α4β7 present on activated CD4⁺ T cells interacts with the HIV-1 gp120 second variable loop (V2). Case control analysis of the RV144 phase III vaccine trial demonstrated that plasma IgG binding antibodies specific to scaffolded proteins expressing the first and second variable regions (V1V2) of HIV envelope protein gp120 containing the α4β7 binding motif correlated inversely with risk of infection. Subsequently antibodies to the V3 region were also shown to correlate with protection. The integrin receptor α4β7 was shown to interact with the LDI/V motif on V2 loop but recent studies suggest that additional regions of V2 loop could interact with the α4β7. Thus, there may be several regions on the V2 and possibly V3 loops that may be involved in this binding. Using a cell line, that constitutively expressed α4β7 receptors but lacked CD4, we examined the contribution of V2 and V3 loops and the ability of V2 peptide-, V2 integrin-, V3-specific monoclonal antibodies (mAbs), and purified IgG from RV144 vaccinees to block the V2/V3-α4β7 interaction.

Results

We demonstrate that α4β7 on RPMI8866 cells bound specifically to its natural ligand mucosal addressin cell adhesion molecule-1 (MAdCAM-1) as well as to cyclic-V2 and cyclic-V3 peptides. This binding was inhibited by anti-α4β7-specific monoclonal antibody (mAb) ACT-1, mAbs specific to either V2 or V3 loops, and by purified primary virions or infectious molecular clones expressing envelopes from acute or chronic subtypes A, C, and CRF01_AE viruses. Plasma from HIV-1 infected Thai individuals as well as purified IgG from uninfected RV144 vaccinees inhibited (0–50%) the binding of V2 and V3 peptides to α4β7.

Conclusion

Our results indicate that in addition to the tripeptide LDI/V motif, other regions of the V2 and V3 loops of gp120 were involved in binding to α4β7 receptors and this interaction was blocked by anti-V2 peptide, anti-V2 integrin, and anti-V3 antibodies. The ability of purified IgG from some of the uninfected RV144 vaccinees to inhibit α4β7 raises the hypothesis that anti-V2 and anti-V3 antibodies may play a role in blocking the gp120-α4β7 interaction after vaccination and thus prevent HIV-1 acquisition.     

Developmentally spliced PKCβII provides a possible link between mTORC2 and Akt kinase to regulate 3T3-L1 adipocyte insulin-stimulated glucose transport

Functional adipocyte glucose disposal is a key component of global glucose homeostasis. PKCβII is involved in rat skeletal muscle cell ISGT. Western blot analysis and real-time PCR revealed 3T3-L1 cells developmentally regulated PKCβ splicing such that PKCβI was downregulated and PKCβII was upregulated during the course of differentiation. An initial glucose uptake screen using PKC inhibitor LY379196 pointed to a PKC isozyme other than PKCζ mediating 3T3-L1 adipocyte ISGT. Subsequent use of PKCβII inhibitor CGP53353 pointed to a role for PKCβII in ISGT. Western blot analysis showed that CGP53353 specifically inhibited phosphorylation of PKCβII Serine 660. Subcellular fractionation and immunofluorescence demonstrated that PKCβII regulates GLUT4 translocation. Further Western blot, immunofluorescence and co-immunoprecipitation analysis reveal that PKCβII inhibition does not affect mTORC2 activity yet abrogates phosphorylation of Akt Serine 473. PKCβII regulates GLUT4 translocation by regulating Akt phosphorylation and thus activity.