Dynamic localization of Mps1 kinase to kinetochores is essential for accurate spindle microtubule attachment

The spindle assembly checkpoint (SAC) is a conserved signaling pathway that monitors faithful chromosome segregation during mitosis. As a core component of SAC, the evolutionarily conserved kinase monopolar spindle 1 (Mps1) has been implicated in regulating chromosome alignment, but the underlying molecular mechanism remains unclear. Our molecular delineation of Mps1 activity in SAC led to discovery of a previously unidentified structural determinant underlying Mps1 function at the kinetochores. Here, we show that Mps1 contains an internal region for kinetochore localization (IRK) adjacent to the tetratricopeptide repeat domain. Importantly, the IRK region determines the kinetochore localization of inactive Mps1, and an accumulation of inactive Mps1 perturbs accurate chromosome alignment and mitotic progression. Mechanistically, the IRK region binds to the nuclear division cycle 80 complex (Ndc80C), and accumulation of inactive Mps1 at the kinetochores prevents a dynamic interaction between Ndc80C and spindle microtubules (MTs), resulting in an aberrant kinetochore attachment. Thus, our results present a previously undefined mechanism by which Mps1 functions in chromosome alignment by orchestrating Ndc80C–MT interactions and highlight the importance of the precise spatiotemporal regulation of Mps1 kinase activity and kinetochore localization in accurate mitotic progression.Faithful distribution of the duplicated genome into two daughter cells during mitosis depends on proper kinetochore–microtubule (MT) attachments. Defects in kinetochore–MT attachments result in chromosome missegregation, causing aneuploidy, a hallmark of cancer (1, 2). To ensure accurate chromosome segregation, cells use the spindle assembly checkpoint (SAC) to monitor kinetochore biorientation and to control the metaphase-to-anaphase transition. Cells enter anaphase only after the SAC is satisfied, requiring that all kinetochores be attached to MTs and be properly bioriented (3, 4). The core components of SAC signaling include mitotic arrest deficient-like 1 (Mad1), Mad2, Mad3/BubR1 (budding uninhibited by benzimidazole-related 1), Bub1, Bub3, monopolar spindle 1 (Mps1), and aurora B. The full SAC function requires the correct centromere/kinetochore localization of all SAC proteins (5).Among the SAC components, Mps1 was identified originally in budding yeast as a gene required for duplication of the spindle pole body (6). Subsequently, Mps1 orthologs were found in various species, from fungi to mammals. The stringent requirement of Mps1 for SAC activity is conserved in evolution (6–13). Human Mps1 kinase (also known as “TTK”) is expressed in a cell-cycle–dependent manner and has highest expression levels and activity during mitosis. Its localization is also dynamic (8, 14). Although the molecular mechanism remains unclear, Mps1 is required to recruit Mad1 and Mad2 to unattached kinetochores, supporting its essential role in SAC activity (15–18). It also is clear that aurora B kinase activity and the outer-layer kinetochore protein nuclear division cycle 80 (Ndc80)/Hec1 are required for Mps1 localization to kinetochores, as evidenced by recent work, including ours (17, 19–24). How Mps1 activates the SAC is now becoming clear. Mps1 recruits Bub1/Bub3 and BubR1/Bub3 to kinetochores through phosphorylation of KNL1, the kinetochore receptor protein of Bub1 and BubR1 (25–30).Despite much progress in understanding Mps1 functions, it remains unclear how Mps1 is involved in regulating chromosome alignment. In budding yeast mitosis, Mps1 regulates mitotic chromosome alignment by promoting kinetochore biorientation independently of Ipl1 (aurora B in humans) (31), but in budding yeast meiosis Mps1 must collaborate with Ipl1 to mediate meiotic kinetochore biorientation (32). In humans, Mps1 regulates chromosomal alignment by modulating aurora B kinase activity (33), but recent chemical biology studies show that Mps1 kinase activity is important for proper chromosome alignment and segregation, independently of aurora B (22, 34–36). Therefore whether Mps1 regulates chromosome alignment through modulation of aurora B kinase activity is still under debate (37).In this study, we reexamined the function of human Mps1 in chromosome alignment. We found that chromosomal alignment is largely achieved in Mps1 knockdown cells, provided that cells are arrested in metaphase in the presence of MG132, a proteasome inhibitor. However, disrupting Mps1 activity via small molecule inhibitors perturbs chromosomal alignment, even in the presence of MG132. This chromosome misalignment is caused by the abnormal accumulation of inactive Mps1 in the kinetochore and the subsequent failure of correct kinetochore–MT attachments. Further, we demonstrate that inactive Mps1 does not depend on the previously reported tetratricopeptide repeat (TPR) domain for localizing to kinetochores, and we identify a previously unidentified region adjacent to the C terminus of the TPR domain that is responsible for localizing inactive Mps1 to kinetochores. Thus, our work highlights that Mps1 kinase activity is necessary in regulating chromosome alignment and that it must be tightly regulated in space and time to ensure proper localization of Mps1 at kinetochores.     

Phosphoproteomic analysis identifies the tumor suppressor PDCD4 as a RSK substrate negatively regulated by 14-3-3

The Ras/MAPK signaling cascade regulates various biological functions, including cell growth and proliferation. As such, this pathway is frequently deregulated in several types of cancer, including most cases of melanoma. RSK (p90 ribosomal S6 kinase) is a MAPK-activated protein kinase required for melanoma growth and proliferation, but relatively little is known about its exact function and the nature of its substrates. Herein, we used a quantitative phosphoproteomics approach to define the signaling networks regulated by RSK in melanoma. To more accurately predict direct phosphorylation substrates, we defined the RSK consensus phosphorylation motif and found significant overlap with the binding consensus of 14-3-3 proteins. We thus characterized the phospho-dependent 14-3-3 interactome in melanoma cells and found that a large proportion of 14-3-3 binding proteins are also potential RSK substrates. Our results show that RSK phosphorylates the tumor suppressor PDCD4 (programmed cell death protein 4) on two serine residues (Ser76 and Ser457) that regulate its subcellular localization and interaction with 14-3-3 proteins. We found that 14-3-3 binding promotes PDCD4 degradation, suggesting an important role for RSK in the inactivation of PDCD4 in melanoma. In addition to this tumor suppressor, our results suggest the involvement of RSK in a vast array of unexplored biological functions with relevance in oncogenesis.The Ras/MAPK pathway plays a key role in transducing extracellular signals to intracellular targets involved in cell growth and proliferation (reviewed in ref. 1). Inappropriate regulation of this pathway leads to a variety of diseases, including cancer (2). In this pathway, the small GTPase Ras activates the Raf isoforms, which are Ser/Thr kinases frequently mutated in human cancers (3). One prominent example is melanoma, which harbors activating B-Raf mutations (V600E) in a majority of cases (4). In turn, activated Raf phosphorylates and activates MEK1/2, which themselves phosphorylate and activate the MAPKs ERK1/2 (5). Once activated, ERK1/2 phosphorylate many substrates, including members of the p90 ribosomal S6 kinase (RSK) family of proteins (6). Although the requirement of ERK1/2 signaling in melanoma is well established, relatively little is known regarding RSK signaling.The RSK family is composed of four Ser/Thr kinases (RSK1–4) that share 73–80% sequence identity and belong to the AGC family of basophilic protein kinases (6). The RSK isoforms have been shown to regulate a number of substrates involved in cell growth and proliferation, and accordingly, inhibition of their activity reduces the proliferation of several cancer cell lines (7, 8). Consistent with this, RSK1 and RSK2 were shown to be overexpressed in breast and prostate cancers (7, 8) and hyperactivated in melanoma (9). Although RSK plays an important role in melanoma (10), relatively little is known about the substrates it regulates.The 14-3-3 family of pSer/Thr-binding proteins dynamically regulates the activity of various client proteins involved in diverse biological processes (11). In response to growth factors, 14-3-3 proteins orchestrate a complex network of molecular interactions to achieve well-controlled physiological outputs, such as cell growth and proliferation. Many 14-3-3-binding proteins contain sequences that match its general consensus motif, which consists of RSXpS/pTXP (12). Based on the requirement for an Arg residue at the −3 position, 14-3-3 client proteins are often phosphorylated by basophilic protein kinases, such as members of the AGC family.Quantitative phosphoproteomics has emerged as a powerful tool in the elucidation of complex signaling networks. In this study, we used quantitative liquid chromatography mass spectrometry (LC-MS) to define the RSK phosphoproteome in melanoma cells. We characterized the primary sequence motif specificity of RSK and observed significant overlap with the 14-3-3 binding motif. Characterization of the 14-3-3 interactome in melanoma cells resulted in the identification of a large number of potential RSK substrates. We characterized the tumor suppressor programmed cell death protein 4 (PDCD4) and found that RSK promotes its degradation in a 14-3-3–dependent manner. Together, these results cast insights on the diverse biological functions regulated by RSK in cancer cells.     

SAC phosphoinositide phosphatases at the tonoplast mediate vacuolar function in Arabidopsis

Phosphatidylinositol (PtdIns) is a structural phospholipid that can be phosphorylated into various lipid signaling molecules, designated polyphosphoinositides (PPIs). The reversible phosphorylation of PPIs on the 3, 4, or 5 position of inositol is performed by a set of organelle-specific kinases and phosphatases, and the characteristic head groups make these molecules ideal for regulating biological processes in time and space. In yeast and mammals, PtdIns3P and PtdIns(3,5)P₂ play crucial roles in trafficking toward the lytic compartments, whereas the role in plants is not yet fully understood. Here we identified the role of a land plant-specific subgroup of PPI phosphatases, the suppressor of actin 2 (SAC2) to SAC5, during vacuolar trafficking and morphogenesis in Arabidopsis thaliana. SAC2–SAC5 localize to the tonoplast along with PtdIns3P, the presumable product of their activity. In SAC gain- and loss-of-function mutants, the levels of PtdIns monophosphates and bisphosphates were changed, with opposite effects on the morphology of storage and lytic vacuoles, and the trafficking toward the vacuoles was defective. Moreover, multiple sac knockout mutants had an increased number of smaller storage and lytic vacuoles, whereas extralarge vacuoles were observed in the overexpression lines, correlating with various growth and developmental defects. The fragmented vacuolar phenotype of sac mutants could be mimicked by treating wild-type seedlings with PtdIns(3,5)P₂, corroborating that this PPI is important for vacuole morphology. Taken together, these results provide evidence that PPIs, together with their metabolic enzymes SAC2–SAC5, are crucial for vacuolar trafficking and for vacuolar morphology and function in plants.Polyphosphoinositides (PPIs) are a class of signaling membrane lipids, comprising the phosphorylated products of phosphatidylinositol (PtdIns). PPIs perform a dual function as scaffolding signals and precursors for other molecular messengers, which, together with their specific distribution at different intracellular membranes, makes PPIs important mediators of a wide variety of cellular processes, such as membrane trafficking and homeostasis, cytoskeleton organization, nuclear signaling, and stress responses (1–5). The metabolism of PPIs is regulated by specific kinases, phosphatases, and phospholipases to tightly control the concentration and intracellular localization of different lipid pools (2, 6, 7).In yeast, two phosphoinositide (PI) types, PtdIns3P and PtdIns(3,5)P₂, and their interconversion have been shown to play crucial roles in trafficking toward the vacuole, regulation of vacuolar pH, and vacuolar membrane fusion and fission (8–11). In yeast and mammals, production and degradation of PtdIns(3,5)P₂ involve the PtdIns3P 5-kinase Fab1p/PIKfyve and the antagonistic phosphatase factor-induced gene/suppressor of actin 3 (Fig4/Sac3), respectively (8, 12–14). Impairment of genes implicated in PtdIns(3,5)P₂ metabolism has deleterious consequences in yeast, plants, and mammals (8, 15–19), demonstrating an essential function of this minor phospholipid. Recent observations also hint at a role for PPIs in plant vacuoles (18–20), but the data are scarce and remain inconclusive.Advances in deciphering various cellular roles of PIs include insights into the phosphatases responsible for hydrolyzing PPIs. A group of phosphatases, designated suppressor of actin (SAC) domain phosphatases, has been identified in lower eukaryotes, mammals, and plants (21). Whereas yeast and humans have only five genes harboring the SAC domain, the genome of the model plant Arabidopsis thaliana contains nine genes, of which some have been functionally characterized and demonstrated to be involved in the regulation of stress responses (22–24), polarized root hair expansion (3), or cell wall formation (25).Here we show that the functionally uncharacterized group of Arabidopsis SAC2–SAC5 proteins that is orthologous to the yeast Fig4p is involved in PPI metabolism. SAC2–SAC5 localize along with PtdIns3P to the tonoplast and maintain the morphology of storage and lytic vacuoles. Our results demonstrate the crucial role of PPIs and SAC domain phosphatases in the function and morphology of vacuoles in plants.     

From the Cover: PNAS Plus: Cell-cycle regulation of formin-mediated actin cable assembly

Assembly of appropriately oriented actin cables nucleated by formin proteins is necessary for many biological processes in diverse eukaryotes. However, compared with knowledge of how nucleation of dendritic actin filament arrays by the actin-related protein-2/3 complex is regulated, the in vivo regulatory mechanisms for actin cable formation are less clear. To gain insights into mechanisms for regulating actin cable assembly, we reconstituted the assembly process in vitro by introducing microspheres functionalized with the C terminus of the budding yeast formin Bni1 into extracts prepared from yeast cells at different cell-cycle stages. EM studies showed that unbranched actin filament bundles were reconstituted successfully in the yeast extracts. Only extracts enriched in the mitotic cyclin Clb2 were competent for actin cable assembly, and cyclin-dependent kinase 1 activity was indispensible. Cyclin-dependent kinase 1 activity also was found to regulate cable assembly in vivo. Here we present evidence that formin cell-cycle regulation is conserved in vertebrates. The use of the cable-reconstitution system to test roles for the key actin-binding proteins tropomyosin, capping protein, and cofilin provided important insights into assembly regulation. Furthermore, using mass spectrometry, we identified components of the actin cables formed in yeast extracts, providing the basis for comprehensive understanding of cable assembly and regulation.Eukaryotic cells contain populations of actin structures with distinct architectures and protein compositions, which mediate varied cellular processes (1). Understanding how F-actin polymerization is regulated in time and space is critical to understanding how actin structures provide mechanical forces for corresponding biological processes. Branched actin filament arrays, which concentrate at sites of clathrin-mediated endocytosis (2, 3) and at the leading edge of motile cells (4), are nucleated by the actin-related protein-2/3 (Arp2/3) complex. In contrast, bundles of unbranched actin filaments, which sometimes mediate vesicle trafficking or form myosin-containing contractile bundles, often are nucleated by formin proteins (5–14).Much has been learned about how branched actin filaments are polymerized by the Arp2/3 complex and how these filaments function in processes such as endocytosis (2, 15). In contrast, relatively little is known about how actin cables are assembled under physiological conditions. In previous studies, branched actin filaments derived from the Arp2/3 complex have been reconstituted using purified proteins (16–19) or cellular extracts (20–25). When microbeads were coated with nucleation-promoting factors for the Arp2/3 complex and then were incubated in cell extracts, actin comet tails were formed by sequential actin nucleation, symmetry breaking, and tail elongation. Importantly, the motility behavior of F-actin assembled by the Arp2/3 complex using defined, purified proteins differs from that of F-actin assembled by the Arp2/3 complex in the full complexity of cytoplasmic extracts (19, 26–28).Formin-based actin filament assembly using purified proteins also has been reported (29, 30). However, reconstitution of formin-derived actin cables under the more physiological conditions represented by cell extracts has not yet been reported.The actin nucleation activity of formin proteins is regulated by an inhibitory interaction between the N- and C-terminal domains, which can be released when GTP-bound Rho protein binds to the formin N-terminal domain, allowing access of the C terminus (FH1-COOH) to actin filament barbed ends (31–40). In yeast, the formin Bni1 N terminus also has an inhibitory effect on actin nucleation through binding to the C terminus (41).Interestingly, several recent reports provided evidence for cell-cycle regulation of F-actin dynamics in oocytes and early embryos (42–45). However, which specific types of actin structures are regulated by the cell cycle and what kind of nucleation factors and actin interacting-proteins are involved remain to be determined.Here, we report a reconstitution of actin cables in yeast extracts from microbeads derivatized with Bni1 FH1-COOH, identifying the proteins involved, increasing the inventory of the proteins that regulate actin cable dynamics and establishing that the actin cable reconstitution in cytoplasmic extracts is cell-cycle regulated.     

Parkinson-related LRRK2 mutation R1441C/G/H impairs PKA phosphorylation of LRRK2 and disrupts its interaction with 14-3-3

Leucine-rich repeat kinase 2 (LRRK2) is a multidomain protein implicated in Parkinson disease (PD); however, the molecular mechanism and mode of action of this protein remain elusive. cAMP-dependent protein kinase (PKA), along with other kinases, has been suggested to be an upstream kinase regulating LRRK2 function. Using MS, we detected several sites phosphorylated by PKA, including phosphorylation sites within the Ras of complex proteins (ROC) GTPase domain as well as some previously described sites (S910 and S935). We systematically mapped those sites within LRRK2 and investigated their functional consequences. S1444 in the ROC domain was confirmed as a target for PKA phosphorylation using ROC single-domain constructs and through site-directed mutagenesis. Phosphorylation at S1444 is strikingly reduced in the major PD-related LRRK2 mutations R1441C/G/H, which are part of a consensus PKA recognition site (¹⁴⁴¹RASpS¹⁴⁴⁴). Furthermore, our work establishes S1444 as a PKA-regulated 14-3-3 docking site. Experiments of direct binding to the three 14-3-3 isotypes gamma, theta, and zeta with phosphopeptides encompassing pS910, pS935, or pS1444 demonstrated the highest affinities to phospho-S1444. Strikingly, 14-3-3 binding to phospho-S1444 decreased LRRK2 kinase activity in vitro. Moreover, substitution of S1444 by alanine or by introducing the mutations R1441C/G/H, abrogating PKA phosphorylation and 14-3-3 binding, resulted in increased LRRK2 kinase activity. In conclusion, these data clearly demonstrate that LRRK2 kinase activity is modulated by PKA-mediated binding of 14-3-3 to S1444 and suggest that 14-3-3 interaction with LRRK2 is hampered in R1441C/G/H-mediated PD pathogenesis.Parkinson disease (PD), one of the most prevalent neurodegenerative afflictions, is characterized pathologically by the selective loss of dopaminergic neurons in the midbrain and by the presence of intracellular inclusions in the remaining cells, termed Lewy bodies (1). However, the molecular mechanisms underlying the complex pathological process are poorly understood. Many genetic and environmental factors contribute to the disease, and mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of familial PD. LRRK2 is a large protein of 285 kDa and encodes several structural motifs, such as armadillo, ankyrin, and the namesake leucine-rich repeats, a Ras of complex proteins (ROC) GTPase, a C-terminal of ROC (COR), a kinase domain [with sequence homology to MAP kinase kinase kinase (MAPKKKs)], and a C-terminal WD40 domain (2). Notably, mutations known to cause PD are located within the catalytically active GTPase (ROC) and kinase domains of LRRK2 (see Fig. 3A) (3). Particularly for a single residue located within the ROC domain, three independent PD-associated mutations (R1441C, R1441G, and R1441H) have been found (4), whereas the kinase domain may harbor the most frequent pathogenic mutation, G2019S. Mutations at both these sites have been associated with enhanced kinase activity compared with wild type (5, 6), suggesting that dysregulation of these enzymatic activities may contribute to PD pathogenesis.Open in a separate windowFig. 3.S1444 on LRRK2 is a PKA-induced 14-3-3 binding site. (A) Multidomain structure of LRRK2. ANK, ankyrin repeat region; LRR, leucine-rich repeat domain; ROC, Ras of complex (GTPase); COR, C-terminal of ROC. The potential 14-3-3 interaction motif in LRRK2 is shown and aligned to a mode I 14-3-3 binding consensus sequence. Known pathogenic mutations R1441C/G/H and G2019S are indicated. (B) LRRK2 pull-down with recombinant GST–14-3-3 gamma. LRRK2 WT and LRRK2 WT ∆967 were expressed in Sf9 cells. Lysates were incubated with GST–14-3-3 gamma for 4 h. GST–14-3-3 gamma pulled down both LRRK2 WT full length and LRRK2 WT ∆967. Samples were separated on a 4–12% gradient SDS gel, and the membranes were probed with Strep-Tactin HRP or GST antibody. (C) LRRK2 WT ∆967 was precipitated with GST–14-3-3-agarose in the absence or in the presence of increasing concentrations of chemically synthesized LRRK2 peptides (LFNIKARASSSPVILVGT) phosphorylated or nonphosphorylated at S1444. Sample separation and Western blotting were carried out as described under B. (D) Two micrograms of His-ROC WT, S1443A, S1444A, or S1443A-S1444A mutant proteins was incubated in the presence or absence of PKA for 1 h at 30 °C. Far western blotting was performed using GST–14-3-3 protein as a probe. PKA-induced ROC–GST–14-3-3 interaction could be observed only when S1444 was present. Equal loading was demonstrated using anti-His antibody.LRRK2 is a cytosolic phosphoprotein (7) phosphorylated in vitro by a variety of serine/threonine kinases, including PKC zeta (8), serine protein kinase ataxia telangiectasia mutated (9), the IκB kinase family (10), and cAMP-dependent protein kinase (PKA) (11, 12). PKA is a key regulator of a vast number of signaling molecules and is critical for neuronal functions such as synaptic plasticity, protein trafficking, protein degradation, neuronal excitability, and regulation of dopamine physiology (13–17). In LRKK2, the conserved residue (S935) was shown to be phosphorylated by PKA (12), and recently this site was proposed as a biomarker for LRRK2 activity (10, 18, 19). Phosphorylation of S910 and S935 within LRRK2 promotes binding of 14-3-3 proteins (19), a family of small 29–30 kDa acidic regulatory proteins, highly conserved and ubiquitously expressed in various tissues. The binding of 14-3-3 proteins results in various downstream effects, such as changes in structural conformations, kinase activity, and subcellular localization of the target proteins (20, 21). Nichols et al. (19) proposed a role for 14-3-3 proteins in regulating the cytoplasmic localization of LRRK2, whereas Li et al. (12) observed protection from dephosphorylation of S935 after 14-3-3 binding. Furthermore, recent data from Fraser et al. (22) suggest a regulatory function of 14-3-3 binding in controlling extracellular release of LRRK2. Dysregulation of 14-3-3/client protein interaction has been shown to facilitate the development of several human disorders (23, 24), and an influence of the pathogenic mutation R1441G on LRRK2/14-3-3 interaction has been demonstrated (12, 19). Although these data support an involvement of PKA and 14-3-3 proteins in regulating LRRK2 function, LRRK2 phosphorylation by PKA, as well as 14-3-3 binding and possible (patho)physiological consequences of this interplay, have not yet been addressed in detail.Here, we systematically mapped PKA phosphorylation sites in LRRK2 and further investigated the impact of this phosphorylation in terms of 14-3-3 binding and LRRK2 function. Our results predict an essential function for PKA phosphorylation and subsequent 14-3-3 interaction in the negative regulation of LRRK2 kinase activity.     

Syntaxin binding mechanism and disease-causing mutations in Munc18-2

Mutations in either syntaxin 11 (Stx11) or Munc18-2 abolish cytotoxic T lymphocytes (CTL) and natural killer cell (NK) cytotoxicity, and give rise to familial hemophagocytic lymphohistiocytosis (FHL4 or FHL5, respectively). Although Munc18-2 is known to interact with Stx11, little is known about the molecular mechanisms governing the specificity of this interaction or how in vitro IL-2 activation leads to compensation of CTL and NK cytotoxicity. To understand how mutations in Munc18-2 give rise to disease, we have solved the structure of human Munc18-2 at 2.6 Å resolution and mapped 18 point mutations. The four surface mutations identified (R39P, L130S, E132A, P334L) map exclusively to the predicted syntaxin and soluble N-ethylmaleimide–sensitive factor accessory protein receptor binding sites of Munc18-2. We find that Munc18-2 binds the N-terminal peptide of Stx11 with a ∼20-fold higher affinity than Stx3, suggesting a potential role in selective binding. Upon IL-2 activation, levels of Stx3 are increased, favoring Munc18-2 binding when Stx11 is absent. Similarly, Munc18-1, expressed in IL-2–activated CTL, is capable of binding Stx11. These findings provide potential explanations for restoration of Munc18-Stx function and cytotoxicity in IL-2–activated cells.Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells perform a crucial role in host defense, destroying virally infected and tumourigenic cells. CTL or NK recognition of a target cell triggers polarization of secretory lysosomes, containing the cytotoxic proteins perforin and granzymes, toward the immunological synapse formed between the two cells. Fusion of secretory lysosomes with the CTL plasma membrane releases the cytotoxic proteins and destroys the targeted cell.Granule secretion is critical for CTL and NK cytotoxicity. Mutations that disrupt this step give rise to profound immunodeficiencies, including familial hemophagocytic lymphohistiocytosis (FHL), which is characterized by fever, cytopenia, and hepatosplenomegaly, symptoms caused by hyperactive CTL and NK (1, 2). To date, four genetic loci have been associated with FHL. Disease-causing mutations have been mapped onto the genes encoding the pore-forming protein perforin (FHL type 2), the secretory regulator Munc13-4 (FHL type 3), the soluble N-ethylmaleimide–sensitive factor accessory protein receptor (SNARE) protein syntaxin 11 (Stx11, FHL type 4), and Munc18-2 (FHL type 5) (3–7).The molecular mechanisms underlying the killing defect seen in CTL and NK from FHL2 and FHL3 patients have been relatively straightforward to understand, as loss of perforin (FHL2) or inhibition of granule secretion (FHL3) prevents target cell lysis (3, 4). However, understanding the molecular basis of FHL4 or FHL5 mutations has been complicated by the finding that cytotoxicity is often restored by in vitro activation of NK or CTL with IL-2, required for culture of these cells (5–8), limiting investigations and raising the question as to how a genetic deficiency can be overcome by in vitro culture.Munc18-2 belongs to the Sec1/Munc18-like (SM) protein family, whose members are all ∼600 residues long and are involved in regulation of SNARE-mediated membrane fusion events (9, 10). The two closest homologs of Munc18-2 are Munc18-1, which is crucial for neurotransmitter secretion in neurons (11, 12), and Munc18-3, which is more widely expressed and is involved in Glut4 translocation (13). Stx11 is a member of the syntaxin-family of SNARE proteins, comprised of an N-terminal peptide (N peptide) followed by an autonomously folded, three-helical bundle (H_ABC domain) and a single helical SNARE motif. However, although most syntaxins associate with membranes through a C-terminal transmembrane domain, Stx11 is unusual in that it contains a cysteine-rich region at its C terminus, allowing for putative palmitoylation and membrane association (14).Munc18 family proteins regulate SNARE-mediated membrane fusion by binding syntaxins and SNARE complexes, and this is reflected in their structures. All SM proteins analyzed to date adopt an arch-shaped structure formed by three distinct domains (15–19). The cavity within the arch forms the major interface for syntaxin and most likely SNARE complex binding (15, 17, 20). However, the N-terminal peptide of syntaxins extends from this cavity and binds at a spatially distinct site on Munc18 proteins, formed by an acidic groove, a basic region and a hydrophobic pocket (16, 21, 22).Munc18 family proteins have been found to regulate SNARE-mediated membrane fusion both positively and negatively. The cocrystal structure of Stx1A with rat Munc18-1 revealed a “closed” Stx1A conformation, with the SNARE helix bound back on the H_ABC domain, clasped inside the central cavity of Munc18-1 (17, 20). This structural model explains how overexpression of Munc18 proteins might impair secretion by locking syntaxins in an inactive state. Conversely, the ability of the same region in Munc18-1 to bind to fully assembled SNARE complexes explains how Munc18 proteins might also facilitate vesicle docking and catalyze membrane fusion (23–26). Indeed, the yeast homolog Sec1p only binds SNARE complexes and not monomeric syntaxins (27, 28).The functional importance of the N peptide binding site on the surface of Munc18 proteins is not entirely clear. For syntaxin binding, the N peptide is crucial for the interaction between Munc18-3 and Stx4 (29), and between Munc18-2 and Stx3 (30) but not for the interaction between Stx1A and Munc18-1 (22). It has been proposed that the N peptide initiates contact between syntaxins and Munc18 proteins and in this way may lead to high-affinity binding of the full-length syntaxin molecule (31). In polarized epithelial cells the N peptide has also been found to determine which Munc18 isoform is bound and where the syntaxin localizes (32). However, nothing is known about the role of the N peptide in the selection of syntaxin binding when two different syntaxins are both able to bind the same Munc18 protein.An essential role for the N peptide in supporting fusion in vitro has been shown in several studies (24, 33, 34). One recent study has proposed that the role of the N peptide is to facilitate the transition of Munc18-bound syntaxins from a “closed” fusion-incompetent to an “open” conformation that allows SNARE complex formation (31, 35), although a subsequent study provides an alternative hypothesis (36). Although gene complementation in Caenorhabditis elegans in vitro studies (37, 38) and studies in cultured neurons (36) support a critical functional role for the N peptide interaction with Munc18 proteins, conflicting reports exist (39, 40).Although the exact roles of Munc18-2 and Stx11 in CTL and NK are not known, it seems likely that these proteins function together because Munc18-2–deficient patients (FHL5) show decreased levels of Stx11 and the two proteins can be coprecipitated from cell lysates (5, 7, 41).Studies on FHL4 and FHL5 have been hampered by the fact that CTL and NK, which need to be cultured in IL-2, often show restored cytotoxicity (5, 8, 42), suggesting that IL-2 activation can restore Munc18-Stx function. The molecular basis for this is completely unexplored.In this study we ask how Munc18-2 and Stx11 function is linked in CTL and NK, and whether the Stx11 N peptide plays a functional role in Munc18-2 binding. We have solved the crystal structure of human Munc18-2 to 2.6 Å resolution and mapped point mutations that lead to FHL5. Our study identifies four disease-causing surface mutations in Munc18-2, all of which map to either the syntaxin or SNARE binding domains. Using biophysical techniques we reveal that the syntaxin N peptide interaction is likely to be important for the selection of Stx11 over Stx3 by Munc18-2. Furthermore, we analyzed changes in syntaxin protein levels that occur upon activation of resting NK with IL-2, and propose a molecular mechanism for the restoration of cytotoxicity in FHL4 and FHL5 upon IL-2 activation.     

Relaxin-3/RXFP3 system regulates alcohol-seeking

Relapse and hazardous drinking represent the most difficult clinical problems in treating patients with alcohol use disorders. Using a rat model of alcohol use and alcohol-seeking, we demonstrated that central administration of peptide antagonists for relaxin family peptide 3 receptor (RXFP3), the cognate receptor for the highly conserved neuropeptide, relaxin-3, decreased self-administration of alcohol in a dose-related manner and attenuated cue- and stress-induced reinstatement following extinction. By comparison, RXFP3 antagonist treatment did not significantly attenuate self-administration or reinstatement of sucrose-seeking, suggesting a selective effect for alcohol. RXFP3 is densely expressed in the stress-responsive bed nucleus of the stria terminalis, and bilateral injections of RXFP3 antagonist into the bed nucleus of the stria terminalis significantly decreased self-administration and stress-induced reinstatement of alcohol, suggesting that this brain region may, at least in part, mediate the effects of RXFP3 antagonism. RXFP3 antagonist treatment had no effect on general ingestive behavior, activity, or procedural memory for lever pressing in the paradigms assessed. These data suggest that relaxin-3/RXFP3 signaling regulates alcohol intake and relapse-like behavior, adding to current knowledge of the brain chemistry of reward-seeking.Alcohol abuse is a major cause of morbidity and mortality worldwide, accounting for an estimated 3.8% of all global deaths and 4.6% of the global burden of disease and injury (1). Excessive alcohol use may also lead to alcohol dependence (also termed “alcohol addiction”) (2, 3), which has a lifetime prevalence of ∼12.5% (4). Economic costs due to alcohol abuse were in the order of $235 billion in the United States in 2007, or ∼2.7% of GDP (1, 5). Despite the huge impact of alcohol use disorders on society, current first-line therapeutic agents, such as naltrexone and acamprosate, are far from adequate, with high relapse rates during treatment and problems with compliance (6–8). New therapeutic agents are clearly required, particularly for the reduction of hazardous drinking and prevention of relapse (9). To this end, a major goal in addiction neuroscience is to understand the neurobiology and neurocircuitry affected by alcohol use disorders and to identify factors implicated in these conditions, which may lead to improved and more targeted therapies (7–10). Here we investigate the neuropeptide relaxin-3 for its involvement in rodent models of alcohol-seeking and consumption.Relaxin-3 is the highly conserved, ancestral neuropeptide of the relaxin/insulin superfamily, and its cognate G-protein–coupled receptor is relaxin family peptide 3 receptor (RXFP3) (11–16). Relaxin-3 is predominantly expressed in gamma-aminobutyric acid (GABA) neurons in the hindbrain nucleus incertus, which projects widely to forebrain areas, including the amygdala, bed nucleus of the stria terminalis (BNST), hippocampus, and lateral hypothalamus, which also express high levels of RXFP3 (11, 15, 17–22). This pattern of innervation, along with findings that relaxin-3 can modulate (i) food intake (23–25), (ii) responses to stress (20, 26, 27), (iii) arousal (28, 29), and (iv) interactions with the corticotropin-releasing factor (CRF) systems (20, 26), led us to hypothesize that relaxin-3 may modulate aspects of behavior related to substance use and abuse. Such a role would parallel that of other neuropeptides, such as orexin/hypocretin (30, 31), galanin (32), and melanin-concentrating hormone (33).The relaxin-3/RXFP3 system was investigated using rat models of alcohol self-administration followed by cue- and stress-induced reinstatement, which are considered robust models for the human experience of relapse (34, 35). Because native relaxin-3 displays some pharmacological cross-reactivity with other relaxin family receptors, peptide ligands selective for RXFP3 have been developed and characterized (36–38). Central injection of a RXFP3-selective agonist increases food intake in rats, which is prevented by prior injection of a RXFP3-selective antagonist (37, 38). Here, we demonstrate that the RXFP3-selective antagonists R3(B1-22)R and R3(BΔ23–27)R/I5 (37, 38) decrease alcohol intake and reinstatement behavior in rats.     

From the Cover: PNAS Plus: Phosphoinositide 5- and 3-phosphatase activities of a voltage-sensing phosphatase in living cells show identical voltage dependence

Voltage-sensing phosphatases (VSPs) are homologs of phosphatase and tensin homolog (PTEN), a phosphatidylinositol 3,4-bisphosphate [PI(3,4)P₂] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃] 3-phosphatase. However, VSPs have a wider range of substrates, cleaving 3-phosphate from PI(3,4)P₂ and probably PI(3,4,5)P₃ as well as 5-phosphate from phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] and PI(3,4,5)P₃ in response to membrane depolarization. Recent proposals say these reactions have differing voltage dependence. Using Förster resonance energy transfer probes specific for different PIs in living cells with zebrafish VSP, we quantitate both voltage-dependent 5- and 3-phosphatase subreactions against endogenous substrates. These activities become apparent with different voltage thresholds, voltage sensitivities, and catalytic rates. As an analytical tool, we refine a kinetic model that includes the endogenous pools of phosphoinositides, endogenous phosphatase and kinase reactions connecting them, and four exogenous voltage-dependent 5- and 3-phosphatase subreactions of VSP. We show that apparent voltage threshold differences for seeing effects of the 5- and 3-phosphatase activities in cells are not due to different intrinsic voltage dependence of these reactions. Rather, the reactions have a common voltage dependence, and apparent differences arise only because each VSP subreaction has a different absolute catalytic rate that begins to surpass the respective endogenous enzyme activities at different voltages. For zebrafish VSP, our modeling revealed that 3-phosphatase activity against PI(3,4,5)P₃ is 55-fold slower than 5-phosphatase activity against PI(4,5)P₂; thus, PI(4,5)P₂ generated more slowly from dephosphorylating PI(3,4,5)P₃ might never accumulate. When 5-phosphatase activity was counteracted by coexpression of a phosphatidylinositol 4-phosphate 5-kinase, there was accumulation of PI(4,5)P₂ in parallel to PI(3,4,5)P₃ dephosphorylation, emphasizing that VSPs can cleave the 3-phosphate of PI(3,4,5)P₃.This paper concerns the substrate specificity and voltage dependence of a unique voltage-sensitive phosphoinositide (PI) phosphatase in intact live cells. Bioelectricity, caused by ion channels and differences in ion concentrations between the inside and outside of a cell, regulates essential biological activities like generation, propagation, and processing of neuronal signals; muscle contraction; and secretion of hormones. Voltage-gated ion channels were the first protein family identified that possessed bioelectric voltage-sensing domains (VSDs) and participated in these signaling activities. Recently, a quite unanticipated voltage-sensing enzyme with a VSD was cloned from the sea squirt Ciona intestinalis (1). Biochemical and electrophysiological examination revealed a voltage-dependent phosphatase activity toward polyphospho-PIs that was given the name C. intestinalis voltage-sensing phosphatase (Ci-VSP). Since then, homologs have been discovered in other vertebrates, for example, Danio rerio (zebrafish; Dr-VSP), African frog (Xi-VSP and Xt-VSP), chicken (Gg-VSP), and salamander (Hn-VSP and Cp-VSP) (2–5). In addition, two mammalian homologs have been discovered in human Hs-transmembrane phosphatase with tensin homology (TPTE) and mouse (Mm-VSP) tissues (6, 7). Although knowledge about the physiological function of these two proteins is still lacking, a recent study showed that mouse VSP seems to be localized to intracellular membranes of neuronal cells, suggesting a different role for mammalian VSPs than for plasma membrane-localized Ci-VSP or Dr-VSP (7). As in voltage-gated ion channels, the VSD of VSPs consists of four transmembrane segments, S1–S4, with the charged voltage-sensing S4 segment being moved by the intense electric fields across the plasma membrane upon depolarization (8). However, VSPs are monomeric and have a cytosolic catalytic domain instead of the pore-forming domain (S5–S6) of ion channels. This enzyme domain is homologous to tumor suppressor phosphatase and tensin homolog (PTEN), a phosphoinositide 3-phosphatase that dephosphorylates both phosphatidylinositol 3,4-bisphosphate [PI(3,4)P₂] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃] (1). Three distinctive regions of PTEN, an N-terminal phospholipid-binding motif that anchors the protein at the plasma membrane, a phosphatase domain that has the enzymatic site, and a C-terminal lipid-interacting C2 domain (9, 10), are well conserved in sequence and structure in the VSPs (11–14).Propagation of the depolarization-induced conformational changes of the VSD to the cytosolic catalytic domain activates the unique voltage-activated phosphoinositide phosphatase activity (12, 14, 15). Unlike its analog PTEN, which has only 3-phosphatase activity (9, 16), Ci-VSP was seen initially to cleave the 5-phosphate from phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] and PI(3,4,5)P₃ in response to membrane depolarization, generating phosphatidylinositol 4-phosphate [PI(4)P] and PI(3,4)P₂, respectively (1, 17–19). Subsequently, VSPs were reported to cleave 3-phosphate from PI(3,4)P₂, generating PI(4)P upon larger depolarization (20), and, very recently, Ci-VSP was indicated to cleave 3-phosphate from PI(3,4,5)P₃, generating PI(4,5)P₂ (21, 22). Because different substrate reactions are best seen at different voltages, several authors have suggested that changing electric fields can drive VSPs successively through several catalytically active states that favor one set of substrates or reactions over another (20–22). The active site of VSPs is well conserved among species (12) and shows only a single amino acid difference from the active site of PTEN (1). When this residue in Ci-VSP was mutated to the corresponding amino acid of the PTEN active site, the mutated Ci-VSP still showed both 5- and 3-phosphatase activities toward polyphosphoinositides (14). Therefore, the observed substrate specificity and the voltage-dependent dual phosphatase activity of VSPs might be determined by the environment surrounding the active site rather than only by the active site itself (14). When the entire enzymatic domain of Ci-VSP was replaced by PTEN, the resulting chimera, called VSPTEN, had the enzymatic properties of a voltage-dependent pure 3-phosphatase (23).VSPs are found in remarkably diverse tissues, including the testis and brain of mice (7, 24, 25); testis, brain, and stomach of humans (6); testis and neuronal complex of the ascidian (1); and testis, ovary, kidney, and liver of the African frog (26). Surprisingly, the physiological functions of this widespread enzyme remain a puzzle. Recent work suggests that VSPs might have roles in egg fertilization (26) and in neuronal signaling in the brain (7). To understand its physiological enzymology more completely, we screened Dr-VSP–induced phosphoinositide changes in living cells by engineered Förster resonance energy transfer (FRET) probes that specifically report the cellular dynamics of PI(4)P, PI(3,4)P₂, PI(4,5)P₂, and PI(3,4,5)P₃. Our results show that when exogenous expression of PI(4)P 5-kinase type Iγ (PIPKIγ) was used selectively to counteract the 5-phosphatase activity of Dr-VSP, PI(4,5)P₂ accumulated, revealing an intrinsic 3-phosphatase activity of VSP toward PI(3,4,5)P₃. The voltage dependence and substrate specificity of 3- and 5-phosphatase subreactions of Dr-VSP were then extracted quantitatively by a comprehensive kinetic systems analysis. Together, our data demonstrate that Dr-VSP possesses both 3- and 5-phosphatase activities toward PI(3,4,5)P₃, with the same voltage dependence but with quite different absolute catalytic rates. These results should help clarify the roles of VSPs in fertilization, neural computations, and other signaling events that involve voltage changes.