Analysis of the Guanine Nucleotide-Bound State of KRAS by Ion-Pair Reversed-Phase High-Performance Liquid Chromatography.

Guanine nucleotides can be quantitatively analyzed by high-performance liquid chromatography (HPLC). Here we describe an ion-pair reversed-phase HPLC (IP-RP-HPLC)-based method, which enables analyzing GDP and GTP bound to small GTPases immunoprecipitated from cells. The activation status of FLAG-KRAS expressed in HEK293T cells can be investigated with the IP-RP-HPLC method. This method also can be adapted to determine the effects of compounds such as the KRAS/G12C inhibitor sotorasib on the activation status of FLAG-KRAS in the cells.

Decreased conformational stability in the oncogenic N92I mutant of Ras-related C3 botulinum toxin substrate 1.

Ras-related C3 botulinum toxin substrate 1 (Rac1) functions as a molecular switch by cycling between an inactive guanosine diphosphate (GDP)-bound state and an active guanosine triphosphate (GTP)-bound state. An oncogenic mutant of Rac1, an N92I mutant, strongly promotes cell proliferation and subsequent oncogenic activities by facilitating the intrinsic GDP dissociation in the inactive GDP-bound state. Here, we used solution nuclear magnetic resonance spectroscopy to investigate the activation mechanism of the N92I mutant. We found that the static structure of the GDP binding site is not markedly perturbed by the mutation, but the overall conformational stability decreases in the N92I mutant, which then facilitates GDP dissociation by lowering the activation energy for the dissociation reaction. On the basis of these results, we proposed the activation mechanism of the N92I mutant, in which the decreased conformational stability plays important roles in its activation process.

Conformational landscape alternations promote oncogenic activities of Ras-related C3 botulinum toxin substrate 1 as revealed by NMR.

Ras-related C3 botulinum toxin substrate 1 (Rac1) plays critical roles in the maintenance of cell morphology by cycling between inactive guanosine diphosphate (GDP)-bound and active guanosine triphosphate (GTP)-bound states. Rac1 P29S mutant is known to strongly promote oncogenesis by facilitating its intrinsic GDP dissociation and thereby increasing the level of the GTP-bound state. Here, we used solution nuclear magnetic resonance spectroscopy to investigate the activation mechanism of the oncogenic P29S mutant. We demonstrate that the conformational landscape is markedly altered in the mutant, and the preexisting equilibrium is shifted toward the conformation with reduced affinity for Mg2+ , a cofactor that is critical for maintaining stable GDP binding. Our results suggest that the alternation of the preexisting conformational equilibrium of proteins is one of the fundamental mechanisms underlying their oncogenic activities.

ADP-ribosylation factor-like 8b is required for the development of mouse models of systemic lupus erythematosus.

Toll-like receptor 7 (TLR7) and type I interferons (IFN-1) are essential for the development of systemic lupus erythematosus (SLE) models such as BXSB.Yaa and 2,6,10,14-tetramethyl-pentadecane (TMPD)-induced experimental lupus. However, the mechanism underlying the development of SLE remains undefined. We report a requirement for ADP-ribosylation factor-like 8b (Arl8b) for TLR7-dependent IFN-1 production in plasmacytoid dendritic cells (pDCs). We analyzed whether Arl8b plays a role in two SLE models by comparing wild-type and Arl8b-deficient Arl8b GeneTrap (Arl8bGt/Gt) mice. We found that BXSB.Yaa Arl8bGt/Gt mice showed none of the abnormalities characterized in BXSB.Yaa mice. TMPD treatment of Arl8bGt/Gt mice significantly inhibited the development of SLE. pDCs were required for TMPD-induced peritonitis. Our data demonstrate that Arl8b contributes to disease pathogenesis in two SLE models via IFN-1-dependent and -independent mechanisms and suggest that Arl8b is an attractive new target for therapeutic intervention in SLE.

GEF mechanism revealed by the structure of SmgGDS-558 and farnesylated RhoA complex and its implication for a chaperone mechanism.

SmgGDS has dual functions in cells and regulates small GTPases as both a guanine nucleotide exchange factor (GEF) for the Rho family and a molecular chaperone for small GTPases possessing a C-terminal polybasic region followed by four C-terminal residues called the CaaX motif, which is posttranslationally prenylated at its cysteine residue. Our recent structural work revealed that SmgGDS folds into tandem copies of armadillo-repeat motifs (ARMs) that are not present in other GEFs. However, the precise mechanism of GEF activity and recognition mechanism for the prenylated CaaX motif remain unknown because SmgGDS does not have a typical GEF catalytic domain and lacks a pocket to accommodate a prenyl group. Here, we aimed to determine the crystal structure of the SmgGDS/farnesylated RhoA complex. We found that SmgGDS induces a significant conformational change in the switch I and II regions that opens up the nucleotide-binding site, with the prenyl group fitting into the cryptic pocket in the N-terminal ARMs. Taken together, our findings could advance the understanding of the role of SmgGDS and enable drug design strategies for targeting SmgGDS and small GTPases.

Regulation of ciliary retrograde protein trafficking by the Joubert syndrome proteins ARL13B and INPP5E.

ARL13B (a small GTPase) and INPP5E (a phosphoinositide 5-phosphatase) are ciliary proteins encoded by causative genes of Joubert syndrome. We here showed, by taking advantage of a visible immunoprecipitation assay, that ARL13B interacts with the IFT46 -: IFT56 (IFT56 is also known as TTC26) dimer of the intraflagellar transport (IFT)-B complex, which mediates anterograde ciliary protein trafficking. However, the ciliary localization of ARL13B was found to be independent of its interaction with IFT-B, but dependent on the ciliary-targeting sequence RVEP in its C-terminal region. ARL13B-knockout cells had shorter cilia than control cells and exhibited aberrant localization of ciliary proteins, including INPP5E. In particular, in ARL13B-knockout cells, the IFT-A and IFT-B complexes accumulated at ciliary tips, and GPR161 (a negative regulator of Hedgehog signaling) could not exit cilia in response to stimulation with Smoothened agonist. This abnormal phenotype was rescued by the exogenous expression of wild-type ARL13B, as well as by its mutant defective in the interaction with IFT-B, but not by its mutants defective in INPP5E binding or in ciliary localization. Thus, ARL13B regulates IFT-A-mediated retrograde protein trafficking within cilia through its interaction with INPP5E.

Variegated RHOA mutations in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATLL) is a distinct form of peripheral T-cell lymphoma with poor prognosis, which is caused by the human T-lymphotropic virus type 1 (HTLV-1). In contrast to the unequivocal importance of HTLV-1 infection in the pathogenesis of ATLL, the role of acquired mutations in HTLV-1 infected T cells has not been fully elucidated, with a handful of genes known to be recurrently mutated. In this study, we identified unique RHOA mutations in ATLL through whole genome sequencing of an index case, followed by deep sequencing of 203 ATLL samples. RHOA mutations showed distinct distribution and function from those found in other cancers. Involving 15% (30/203) of ATLL cases, RHOA mutations were widely distributed across the entire coding sequence but almost invariably located at the guanosine triphosphate (GTP)-binding pocket, with Cys16Arg being most frequently observed. Unexpectedly, depending on mutation types and positions, these RHOA mutants showed different or even opposite functional consequences in terms of GTP/guanosine diphosphate (GDP)-binding kinetics, regulation of actin fibers, and transcriptional activation. The Gly17Val mutant did not bind GTP/GDP and act as a dominant negative molecule, whereas other mutants (Cys16Arg and Ala161Pro) showed fast GTP/GDP cycling with enhanced transcriptional activation. These findings suggest that both loss- and gain-of-RHOA functions could be involved in ATLL leukemogenesis. In summary, our study not only provides a novel insight into the molecular pathogenesis of ATLL but also highlights a unique role of variegation of heterologous RHOA mutations in human cancers.

The C. elegans Hypodermis Couples Progenitor Cell Quiescence to the Dietary State.

The nutritional status of an organism can greatly impact the function and behavior of stem and progenitor cells [1]. However, the regulatory circuits that inform these cells about the dietary environment remain to be elucidated. Newly hatched C. elegans larvae (L1s) halt development in "L1 arrest" or "L1 diapause" until ample food is encountered and triggers stem and progenitor cells to exit from quiescence [2]. The insulin/insulin-like growth factor signaling (IIS) pathway plays a key role in this reactivation [3, 4], but its site(s) of action have not been elucidated nor have the nutrient molecule(s) that stimulate the pathway been identified. By tissue-specifically modulating the activity of its components, we demonstrate that the IIS pathway acts in the hypodermis to regulate nutrition-responsive reactivation of neural and mesodermal progenitor cells. We identify ethanol, a likely component of the natural Caenorhabditis habitat, and amino acids as nutrients that synergistically reactivate somatic progenitor cells and upregulate expression of insulin-like genes in starved L1 larvae. The hypodermis likely senses the availability of amino acids because forced activation of the amino-acid-responsive Rag-TORC1 (target of rapamycin complex 1) pathway in this tissue can also release somatic progenitor cell quiescence in the presence of ethanol. Finally, there appears to be crosstalk between the IIS and Rag-TORC1 pathways because constitutive activation of the IIS pathway requires Rag to promote reactivation. This work demonstrates that ethanol and amino acids act as dietary cues via the IIS and Rag-TORC1 pathways in the hypodermis to coordinately control progenitor cell behavior.

Concentration of Sec12 at ER exit sites via interaction with cTAGE5 is required for collagen export.

Mechanisms for exporting variably sized cargo from the endoplasmic reticulum (ER) using the same machinery remain poorly understood. COPII-coated vesicles, which transport secretory proteins from the ER to the Golgi apparatus, are typically 60-90 nm in diameter. However, collagen, which forms a trimeric structure that is too large to be accommodated by conventional transport vesicles, is also known to be secreted via a COPII-dependent process. In this paper, we show that Sec12, a guanine-nucleotide exchange factor for Sar1 guanosine triphosphatase, is concentrated at ER exit sites and that this concentration of Sec12 is specifically required for the secretion of collagen VII but not other proteins. Furthermore, Sec12 recruitment to ER exit sites is organized by its direct interaction with cTAGE5, a previously characterized collagen cargo receptor component, which functions together with TANGO1 at ER exit sites. These findings suggest that the export of large cargo requires high levels of guanosine triphosphate-bound Sar1 generated by Sec12 localized at ER exit sites.

Active transport and diffusion barriers restrict Joubert Syndrome-associated ARL13B/ARL-13 to an Inv-like ciliary membrane subdomain.

Cilia are microtubule-based cell appendages, serving motility, chemo-/mechano-/photo- sensation, and developmental signaling functions. Cilia are comprised of distinct structural and functional subregions including the basal body, transition zone (TZ) and inversin (Inv) compartments, and defects in this organelle are associated with an expanding spectrum of inherited disorders including Bardet-Biedl syndrome (BBS), Meckel-Gruber Syndrome (MKS), Joubert Syndrome (JS) and Nephronophthisis (NPHP). Despite major advances in understanding ciliary trafficking pathways such as intraflagellar transport (IFT), how proteins are transported to subciliary membranes remains poorly understood. Using Caenorhabditis elegans and mammalian cells, we investigated the transport mechanisms underlying compartmentalization of JS-associated ARL13B/ARL-13, which we previously found is restricted at proximal ciliary membranes. We now show evolutionary conservation of ARL13B/ARL-13 localisation to an Inv-like subciliary membrane compartment, excluding the TZ, in many C. elegans ciliated neurons and in a subset of mammalian ciliary subtypes. Compartmentalisation of C. elegans ARL-13 requires a C-terminal RVVP motif and membrane anchoring to prevent distal cilium and nuclear targeting, respectively. Quantitative imaging in more than 20 mutants revealed differential contributions for IFT and ciliopathy modules in defining the ARL-13 compartment; IFT-A/B, IFT-dynein and BBS genes prevent ARL-13 accumulation at periciliary membranes, whereas MKS/NPHP modules additionally inhibit ARL-13 association with TZ membranes. Furthermore, in vivo FRAP analyses revealed distinct roles for IFT and MKS/NPHP genes in regulating a TZ barrier to ARL-13 diffusion, and intraciliary ARL-13 diffusion. Finally, C. elegans ARL-13 undergoes IFT-like motility and quantitative protein complex analysis of human ARL13B identified functional associations with IFT-B complexes, mapped to IFT46 and IFT74 interactions. Together, these findings reveal distinct requirements for sequence motifs, IFT and ciliopathy modules in defining an ARL-13 subciliary membrane compartment. We conclude that MKS/NPHP modules comprise a TZ barrier to ARL-13 diffusion, whereas IFT genes predominantly facilitate ARL-13 ciliary entry and/or retention via active transport mechanisms.

The microRNA miR-235 couples blast-cell quiescence to the nutritional state.

The coordination of stem- and blast-cell behaviours, such as self-renewal, differentiation and quiescence, with physiological changes underlies growth, regeneration and tissue homeostasis. Germline stem and somatic blast cells in newly hatched Caenorhabditis elegans larvae can suspend postembryonic development, which consists of diverse cellular events such as migration, proliferation and differentiation, until the nutritional state becomes favourable (termed L1 diapause). Although previous studies showed that the insulin/insulin-like growth factor (IGF) signalling (IIS) pathway regulates this developmental quiescence, the detailed mechanism by which the IIS pathway enables these multipotent cells to respond to nutrient availability is unknown. Here we show in C. elegans that the microRNA (miRNA) miR-235, a sole orthologue of mammalian miR-92 from the oncogenic miR-17-92 cluster, acts in the hypodermis and glial cells to arrest postembryonic developmental events in both neuroblasts and mesoblasts. Expression of mir-235 persists during L1 diapause, and decreases upon feeding in a manner dependent on the IIS pathway. Upregulation of one of the miR-235 targets, nhr-91, which encodes an orthologue of mammalian germ cell nuclear factor, is responsible for defects caused by loss of the miRNA. Our findings establish a novel role of a miR-92 orthologue in coupling blast-cell behaviours to the nutritional state.

Transforming mutations of RAC guanosine triphosphatases in human cancers.

Members of the RAS superfamily of small guanosine triphosphatases (GTPases) transition between GDP-bound, inactive and GTP-bound, active states and thereby function as binary switches in the regulation of various cellular activities. Whereas HRAS, NRAS, and KRAS frequently acquire transforming missense mutations in human cancer, little is known of the oncogenic roles of other small GTPases, including Ras-related C3 botulinum toxin substrate (RAC) proteins. We show that the human sarcoma cell line HT1080 harbors both NRAS(Q61K) and RAC1(N92I) mutant proteins. Whereas both of these mutants were able to transform fibroblasts, knockdown experiments indicated that RAC1(N92I) may be the essential growth driver for this cell line. Screening for RAC1, RAC2, or RAC3 mutations in cell lines and public databases identified several missense mutations for RAC1 and RAC2, with some of the mutant proteins, including RAC1(P29S), RAC1(C157Y), RAC2(P29L), and RAC2(P29Q), being found to be activated and transforming. P29S, N92I, and C157Y mutants of RAC1 were shown to exist preferentially in the GTP-bound state as a result of a rapid transition from the GDP-bound state, rather than as a result of a reduced intrinsic GTPase activity. Activating mutations of RAC GTPases were thus found in a wide variety of human cancers at a low frequency; however, given their marked transforming ability, the mutant proteins are potential targets for the development of new therapeutic agents.

Critical roles of type III phosphatidylinositol phosphate kinase in murine embryonic visceral endoderm and adult intestine.

The metabolism of membrane phosphoinositides is critical for a variety of cellular processes. Phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P(2)] controls multiple steps of the intracellular membrane trafficking system in both yeast and mammalian cells. However, other than in neuronal tissues, little is known about the physiological functions of PtdIns(3,5)P(2) in mammals. Here, we provide genetic evidence that type III phosphatidylinositol phosphate kinase (PIPKIII), which produces PtdIns(3,5)P(2), is essential for the functions of polarized epithelial cells. PIPKIII-null mouse embryos die by embryonic day 8.5 because of a failure of the visceral endoderm to supply the epiblast with maternal nutrients. Similarly, although intestine-specific PIPKIII-deficient mice are born, they fail to thrive and eventually die of malnutrition. At the mechanistic level, we show that PIPKIII regulates the trafficking of proteins to a cell's apical membrane domain. Importantly, mice with intestine-specific deletion of PIPKIII exhibit diarrhea and bloody stool, and their gut epithelial layers show inflammation and fibrosis, making our mutants an improved model for inflammatory bowel diseases. In summary, our data demonstrate that PIPKIII is required for the structural and functional integrity of two different types of polarized epithelial cells and suggest that PtdIns(3,5)P(2) metabolism is an unexpected and critical link between membrane trafficking in intestinal epithelial cells and the pathogenesis of inflammatory bowel disease.

cTAGE5 mediates collagen secretion through interaction with TANGO1 at endoplasmic reticulum exit sites.

Cutaneous T-cell lymphoma-associated antigen 5 (cTAGE5), an originally identified tumor antigen, is overexpressed in various cancer cell lines. The cDNA encodes an integral membrane protein containing two coiled-coil motifs and a proline-rich domain. We show that cTAGE5 specifically localizes to the endoplasmic reticulum (ER) exit sites. In addition, cTAGE5 forms a complex with TANGO1 (MIA3), a previously characterized cargo receptor for collagen VII, by the interaction of their coiled-coil motifs. Of interest, cTAGE5, as well as TANGO1, is capable of interacting with the inner-layer coatomer of COPII Sec23/24 complex through their C-terminal proline-rich domains and required for collagen VII secretion. We propose that cTAGE5 acts as a coreceptor of TANGO1 for collagen VII export from the ER.

Repression of cell-cell fusion by components of the C. elegans vacuolar ATPase complex.

Cell-cell fusion initiates fertilization, sculpts tissues during animal development, reprograms stem cells to new differentiated states, and may be a key step in cancer progression. While cell fusion is tightly regulated, the mechanisms that limit fusion to appropriate partners are unknown. Here, we report that the fus-1 gene is essential to repress fusion of epidermal cells in C. elegans: in severe fus-1 mutants, all epidermal cells, except the lateral seam cells, inappropriately fuse into a single large syncytium. This hyperfusion requires EFF-1, an integral membrane protein essential for fusion of epidermal cells into discrete syncytia. FUS-1 is localized to the apical plasma membrane in all epidermal cells potentiated to undergo fusion, whereas it is virtually undetectable in nonfusing seam cells. fus-1 encodes the e subunit of the vacuolar H(+)-ATPase (V-ATPase), and loss of other V-ATPase subunits also causes widespread hyperfusion. These findings raise the possibility of manipulating cell fusion by altering V-ATPase activity.

Cell fusion: EFF is enough.

Developmentally programmed cell-cell fusion in Caenorhabditis elegans requires the EFF-1 protein, which is sufficient to cause normally non-fusing cells to fuse. EFF-1 localizes to fusion-fated membranes, implicating it as a direct fusogen.

Novel role of the small GTPase Rheb: its implication in endocytic pathway independent of the activation of mammalian target of rapamycin.

The Ras-homologous GTPase Rheb that is conserved from yeast to human appears to be involved not only in cell growth but also in nutrient uptake. Recent biochemical analysis revealed that tuberous sclerosis complex (TSC), a GTPase-activating protein (GAP), deactivates Rheb and that phosphatidylinositol 3'-kinase (PI3k)-Akt/PKB kinase pathway activates Rheb through inhibition of the GAP-mediated deactivation. Although mammalian target of rapamycin (mTOR) kinase is implicated in the downstream target of Rheb, the direct effector(s) and exact functions of Rheb have not been fully elucidated. Here we identified that Rheb expression in cultured cells induces the formation of large cytoplasmic vacuoles, which are characterized as late endocytic (late endosome- and lysosome-like) components. The vacuole formation required the GTP form of Rheb, but not the activation of the downstream mTOR kinase. These results suggest that Rheb regulates endocytic trafficking pathway independent of the previously identified mTOR pathway. The physiological roles of the two Rheb-dependent signaling pathways are discussed in terms of nutrient uptake and cell growth or cell cycle progression.

Dual phosphorylation of phosphoinositide 3-kinase adaptor Grb2-associated binder 2 is responsible for superoxide formation synergistically stimulated by Fc gamma and formyl-methionyl-leucyl-phenylalanine receptors in differentiated THP-1 cells.

The class Ia phosphoinositide (PI) 3-kinase consisting of p110 catalytic and p85 regulatory subunits is activated by Tyr kinase-linked membrane receptors such as FcgammaRII through the association of p85 with the phosphorylated receptors or adaptors. The heterodimeric PI 3-kinase is also activated by G protein-coupled chemotactic fMLP receptors, and activation of the lipid kinase plays an important role in various immune responses, including superoxide formation in neutrophils. Although fMLP-induced superoxide formation is markedly enhanced in FcgammaRII-primed neutrophils, the molecular mechanisms remain poorly characterized. In this study, we identified two Tyr-phosphorylated proteins, c-Cbl (Casitas B-lineage lymphoma) and Grb2-associated binder 2 (Gab2), as PI 3-kinase adaptors that are Tyr phosphorylated upon the stimulation of FcgammaRII in differentiated neutrophil-like THP-1 cells. Interestingly, Gab2 was, but c-Cbl was not, further Ser/Thr phosphorylated by fMLP. Thus, the adaptor Gab2 appeared to be dually phosphorylated at the Ser/Thr and Tyr residues through the two different types of membrane receptors. The Ser/Thr phosphorylation of Gab2 required the activation of extracellular signal-regulated kinase, and fMLP receptor stimulation indeed activated extracellular signal-regulated kinase in the cells. Enhanced superoxide formation in response to Fcgamma and fMLP was markedly attenuated when the Gab2 Ser/Thr phosphorylation was inhibited. These results show the importance of the dual phosphorylation of PI 3-kinase adaptor Gab2 for the enhanced superoxide formation in neutrophil-type cells.

Di-Ras, a distinct subgroup of ras family GTPases with unique biochemical properties.

The small GTPase Ras family regulates a variety of cell functions including proliferation and differentiation. Here we have identified novel Ras members, human Di-Ras1 and Di-Ras2, belonging to a distinct branch of the GTPase family. Di-Ras1 and Di-Ras2 specifically expressed in heart and brain share 30-40% overall identity with other members of Ras family, however, they have the following characteristic substitutions at highly conserved regions among the Ras family. 1) Thr-63 and Ser-65 in Di-Ras are substituted for Ala-59 and Gln-61 positions in Ha-Ras, respectively, that are known to be critical for GTP hydrolysis. 2) Within the effector domains, Di-Ras has Ile at a position corresponding to Asp-33 in Ha-Ras, which is important for its interaction with the downstream effector Raf. As predicted by these substitutions, Di-Ras has only a quite low level of GTPase activity and exists predominantly as a GTP-bound form upon its expression in living cells. Moreover, Di-Ras fails to interact with the Ras-binding domain of Raf, resulting in no stimulation of mitogen-activated protein kinase. Interestingly, introduction of Di-Ras into HEK293T cells induces large cellular vacuolation. These findings raise the possibility that Di-Ras might regulate cell morphogenesis in a manner distinct from other members of Ras family.

Activation mechanism of Gi and Go by reactive oxygen species.

Reactive oxygen species are proposed to work as intracellular mediators. One of their target proteins is the alpha subunit of heterotrimeric GTP-binding proteins (Galpha(i) and Galpha(o)), leading to activation. H(2)O(2) is one of the reactive oxygen species and activates purified Galpha(i2). However, the activation requires the presence of Fe(2+), suggesting that H(2)O(2) is converted to more reactive species such as c*OH. The analysis with mass spectrometry shows that seven cysteine residues (Cys(66), Cys(112), Cys(140), Cys(255), Cys(287), Cys(326), and Cys(352)) of Galpha(i2) are modified by the treatment with *OH. Among these cysteine residues, Cys(66), Cys(112), Cys(140), Cys(255), and Cys(352) are not involved in *OH-induced activation of Galpha(i2). Although the modification of Cys(287) but not Cys(326) is required for subunit dissociation, the modification of both Cys(287) and Cys(326) is necessary for the activation of Galpha(i2) as determined by pertussis toxin-catalyzed ADP-ribosylation, conformation-dependent change of trypsin digestion pattern or guanosine 5'-3-O-(thio)triphosphate binding. Wild type Galpha(i2) but not Cys(287)- or Cys(326)-substituted mutants are activated by UV light, singlet oxygen, superoxide anion, and nitric oxide, indicating that these oxidative stresses activate Galpha(i2) by the mechanism similar to *OH-induced activation. Because Cys(287) exists only in G(i) family, this study explains the selective activation of G(i)/G(o) by oxidative stresses.