Binding between the Junctional Proteins Afadin and PLEKHA7 and Implication in the Formation of Adherens Junction in Epithelial Cells

Adherens junction (AJ) is a specialized cell-cell junction structure that plays a role in mechanically connecting adjacent cells to resist strong contractile forces and to maintain tissue structure, particularly in the epithelium. AJ is mainly comprised of cell adhesion molecules cadherin and nectin and their associating cytoplasmic proteins including β-catenin, α-catenin, p120^ctn, and afadin. Our series of studies have revealed that nectin first forms cell-cell adhesion and then recruits cadherin to form AJ. The recruitment of cadherin by nectin is mediated by the binding of α-catenin and p120^ctn to afadin. Recent studies showed that PLEKHA7 binds to p120^ctn, which is associated with E-cadherin, and maintains the integrity of AJ in epithelial cells. In this study, we showed that PLEKHA7 bound to afadin in addition to p120^ctn and was recruited to the nectin-3α-based cell-cell adhesion site in a manner dependent on afadin, but not on p120^ctn. The binding of PLEKHA7 to afadin was required for the proper formation of AJ, but not for the formation of tight junction, in EpH4 mouse mammary gland epithelial cells. These results indicate that PLEKHA7 plays a cooperative role with nectin and afadin in the proper formation of AJ in epithelial cells.     

Allosteric Regulation of E-Cadherin Adhesion

Cadherins are transmembrane adhesion proteins that maintain intercellular cohesion in all tissues, and their rapid regulation is essential for organized tissue remodeling. Despite some evidence that cadherin adhesion might be allosterically regulated, testing of this has been hindered by the difficulty of quantifying altered E-cadherin binding affinity caused by perturbations outside the ectodomain binding site. Here, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that treatment with activating, anti-E-cadherin antibodies or the dephosphorylation of a cytoplasmic binding partner, p120^ctn, increased the homophilic binding affinity of E-cadherin. Results obtained with Colo 205 cells, which express inactive E-cadherin and do not aggregate, demonstrated that four treatments, which induced Colo 205 aggregation and p120^ctn dephosphorylation, triggered quantitatively similar increases in E-cadherin affinity. Several processes can alter cell aggregation, but these results directly demonstrated the allosteric regulation of cell surface E-cadherin by p120^ctn dephosphorylation.     

The Juxtamembrane Region of the Cadherin Cytoplasmic Tail Supports Lateral Clustering, Adhesive Strengthening, and Interaction with p120ctn

Cadherin cell–cell adhesion molecules form membrane-spanning molecular complexes that couple homophilic binding by the cadherin ectodomain to the actin cytoskeleton. A fundamental issue in cadherin biology is how this complex converts the weak intrinsic binding activity of the ectodomain into strong adhesion. Recently we demonstrated that cellular cadherins cluster in a ligand-dependent fashion when cells attached to substrata coated with the adhesive ectodomain of Xenopus C-cadherin (CEC1-5). Moreover, forced clustering of the ectodomain alone significantly strengthened adhesiveness (Yap, A.S., W.M. Brieher, M. Pruschy, and B.M. Gumbiner. Curr. Biol. 7:308–315). In this study we sought to identify the determinants of the cadherin cytoplasmic tail responsible for clustering activity. A deletion mutant of C-cadherin (CT669) that retained the juxtamembrane 94–amino acid region of the cytoplasmic tail, but not the β-catenin–binding domain, clustered upon attachment to substrata coated with CEC1-5. Like wild-type C-cadherin, this clustering was ligand dependent. In contrast, mutant molecules lacking either the complete cytoplasmic tail or just the juxtamembrane region did not cluster. The juxtamembrane region was itself sufficient to induce clustering when fused to a heterologous membrane-anchored protein, albeit in a ligand-independent fashion. The CT669 cadherin mutant also displayed significant adhesive activity when tested in laminar flow detachment assays and aggregation assays. Purification of proteins binding to the juxtamembrane region revealed that the major associated protein is p120^ctn. These findings identify the juxtamembrane region of the cadherin cytoplasmic tail as a functionally active region supporting cadherin clustering and adhesive strength and raise the possibility that p120^ctn is involved in clustering and cell adhesion.     

Gα12 binds to the N-terminal regulatory domain of p120, and downregulates p120 tyrosine phosphorylation induced by Src family kinases via a RhoA independent mechanism

p120 catenin (p120^ctn) regulates cadherin stability, and thus facilitates strong cell-cell adhesion. Previously, we demonstrated that Gα₁₂ interacts with p120^ctn. In the present study, we have delineated a region of p120^ctn that binds to Gα₁₂. We report that the N-terminal region of p120^ctn (amino acids 1-346) is necessary and sufficient for the interaction. While the coiled-coiled domain and a charged region, comprising a.a 102-120, were found to be dispensable, amino acids 121-323 were required for p120^ctn binding to Gα₁₂. This region harbors the phosphorylation domain of p120^ctn and has been postulated as important for RhoA regulation. Downregulation of Src family kinase-induced tyrosine phosphorylation of p120^ctn was observed in the presence of activated Gα₁₂. This down-regulation was triggered by three different Gα₁₂ mutants uncoupled from RhoA signalling. Furthermore, a dominant active form of RhoA did not reduce Src-induced phosphoryaltion of p120^ctn. In summary, our results suggest that Gα₁₂ binds to p120^ctn and modulates its phosphorylation status through a Rho-independent mechanism. Gα₁₂ emerges as an important regulator of p120^ctn function, and possibly of cadherin-mediated adhesion and/or cell motility.     

Polarized trafficking of E-cadherin is regulated by Rac1 and Cdc42 in Madin-Darby canine kidney cells

E-cadherin is a major cell-cell adhesion protein of epithelia that is trafficked to the basolateral cell surface in a polarized fashion. The exact post-Golgi route and regulation of E-cadherin transport have not been fully described. The Rho GTPases Cdc42 and Rac1 have been implicated in many cell functions, including the exocytic trafficking of other proteins in polarized epithelial cells. These Rho family proteins are also associated with the cadherin-catenin complexes at the cell surface. We have used functional mutants of Rac1 and Cdc42 and inactivating toxins to demonstrate specific roles for both Cdc42 and Rac1 in the post-Golgi transport of E-cadherin. Dominant-negative mutants of Cdc42 and Rac1 accumulate E-cadherin at a distinct post-Golgi step. This accumulation occurs before p120^ctn interacts with E-cadherin, because p120^ctn localization was not affected by the Cdc42 or Rac1 mutants. Moreover, the GTPase mutants had no effect on the trafficking of a targeting mutant of E-cadherin, consistent with the selective involvement of Cdc42 and Rac1 in basolateral trafficking. These results provide a new example of Rho GTPase regulation of basolateral trafficking and demonstrate novel roles for Cdc42 and Rac1 in the post-Golgi transport of E-cadherin. Rho family GTPases; catenin; polarity; sorting; actin     

Unraveling the distinct distributions of VE- and N-cadherins in endothelial cells: A key role for p120-catenin

Endothelial cells express two different classical cadherins, vascular endothelial (VE) cadherin and neural (N) cadherin, having distinct functions in the vascular system. VE-cadherin is specific to endothelial adherens junctions and is strictly necessary for vascular morphogenesis. On the contrary, N-cadherin shows diffuse localization on the cell surface and interacts with mural cells for vessel stabilization. In this study, we sought to clarify the cellular mechanisms leading to the distinct cellular locations and functions of the two cadherins in the endothelium. VE-cadherin has been shown to be responsible for the junctional exclusion of N-cadherin. Using several endothelial models, we demonstrate that this property is dependent on VE-cadherin binding to p120 catenin (p120^ctn). Moreover, although in the absence of VE-cadherin N-cadherin can localize to cell contacts, angiogenesis remains impaired, demonstrating that endothelial junction formation is not sufficient for normal vessel development. Interestingly, we show that VE-cadherin, but not N-cadherin, is partially associated with cholesterol-enriched microdomains. Lipid raft-associated-VE-cadherin is characterized by a very high level of p120^ctn association, and this association is necessary for VE-cadherin recruitment into lipid rafts. Altogether, our results indicate a critical role for p120^ctn in regulating the membrane distribution of endothelial cadherins with functional consequences in terms of cadherin stabilization and intracellular signaling.     

Chromosomal Mapping of HumanarmadilloGenes Belonging to the p120/Plakophilin Subfamily

Armadillo-like proteins are characterized by a series of armadillo repeats that are typically 42 to 45 amino acids in length. Three major subfamilies of Armadillo-like proteins can be distinguished on the basis of their number of repeats, their overall sequence similarity, and dispersion of the repeats throughout the protein. One of these is the p120^ctn/plakophilin subfamily, which contains at least six members. We mapped the corresponding human genes by PCR on a monochromosomal cell hybrid mapping panel and by fluorescencein situhybridization. The gene for plakophilin-1 (PKP1) was located at 1q32, the plakophilin-2 gene (PKP2) was located at 12p13, while the gene for p0071 was located at 2q23–q31. We confirmed the chromosomal localization of the p120^ctngene (CTNND1) at 11q11, the ARVCF gene at 22q11, and the δ-catenin/NPRAP gene (CTNND2) at 5p15. Although some of the Armadillo proteins are highly related to one another, the corresponding genes are dispersed throughout the human genome.     

Altered Localization of p120 Catenin in the Cytoplasm Rather than the Membrane Correlates with Poor Prognosis in Esophageal Squamous Cell Carcinoma

Background

P120 catenin (p120ctn), a regulator of cell adhesion, has previously been found in many malignancies, and suggested a role in invasion, metastasis and survival. The aim of this study was to investigate correlations between altered localization of p120ctn and clinical-pathological characteristics in esophageal squamous cell carcinoma (ESCC).

Methods

Immunohistochemical staining for p120^ctn was performed on tissue samples from 118 patients with ESCC. The expression of p120^ctn was scored for intensity and cellular localization by Image-pro Plus 6.0. Correlations between immunohistochemical staining of p120^ctn and pathological characteristics and clinical prognosis were determined using SPSS 18.0 software.

Results

Membrane expression of p120^ctn in ESCCs was lower than that in adjacentnormal esophageal epithelial tissues (P = 0.041), while overall cellular expression of p120^ctn was not different between the two tissue types (P = 0.787). Furthermore, neither overall cellular expression nor localized membrane expression was associated with histological and clinical variables. The high ratio of membrane expression to overall cellular expression (M/C) of p120^ctn was inversely associated with lymph node invasion (P = 0.001), tumor differentiation (P = 0.012) and advanced tumor stage (P = 0.005); however, it was poorly associated with T stage (P = 0.274). The high M/C ratio of p120^ctn was inversely correlated with poor survival; the 5-year OS (overall survival) and the 5-year DFS (disease free survival) for the high M/C ratio group were significantly higher than those of the low M/C ratio group (41.0% vs. 6.7%, P = 0.000; 44.1% vs. 24.9%, P = 0.007). Both the M/C ratio of p120^ctn and N status were independent variables for the prediction of overall survival (P = 0.007 and P = 0.027). The M/C of p120^ctn predicted a 0.49-fold risk of ESCC death (p = 0.007, 95% CI 0.29–0.83).

Conclusions

The reduced M/C ratio of p120^ctn acted as an independent prognostic factor for ESCC patient survival and for the migration and invasive behavior of the disease.     

The Polybasic Insertion in Autotaxin α Confers Specific Binding to Heparin and Cell Surface Heparan Sulfate Proteoglycans

Autotaxin (ATX) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), playing a key role in diverse physiological and pathological processes. ATX exists in distinct splice variants, but isoform-specific functions remain elusive. Here we characterize the ATXα isoform, which differs from the canonical form (ATXβ) in having a 52-residue polybasic insertion of unknown function in the catalytic domain. We find that the ATXα insertion is susceptible to cleavage by extracellular furin-like endoproteases, but cleaved ATXα remains structurally and functionally intact due to strong interactions within the catalytic domain. Through ELISA and surface plasmon resonance assays, we show that ATXα binds specifically to heparin with high affinity (K_d ∼10⁻⁸ m), whereas ATXβ does not; furthermore, heparin moderately enhanced the lysophospholipase D activity of ATXα. We further show that ATXα, but not ATXβ, binds abundantly to SKOV3 carcinoma cells. ATXα binding was abolished after treating the cells with heparinase III, but not after chondroitinase treatment. Thus, the ATXα insertion constitutes a cleavable heparin-binding domain that mediates interaction with heparan sulfate proteoglycans, thereby targeting LPA production to the plasma membrane.     

Arachidonic Acid Induces Direct Interaction of the p67phox-Rac Complex with the Phagocyte Oxidase Nox2, Leading to Superoxide Production

The phagocyte NADPH oxidase Nox2, heterodimerized with p22^phox in the membrane, is dormant in resting cells but becomes activated upon cell stimulation to produce superoxide, a precursor of microbicidal oxidants. Nox2 activation requires two switches to be turned on simultaneously: a conformational change of the cytosolic protein p47^phox and GDP/GTP exchange on the small GTPase Rac. These proteins, in an active form, bind to their respective targets, p22^phox and p67^phox, leading to productive oxidase assembly at the membrane. Although arachidonic acid (AA) efficiently activates Nox2 both in vivo and in vitro, the mechanism has not been fully understood, except that AA induces p47^phox conformational change. Here we show that AA elicits GDP-to-GTP exchange on Rac at the cellular level, consistent with its role as a potent Nox2 activator. However, even when constitutively active forms of p47^phox and Rac1 are both expressed in HeLa cells, superoxide production by Nox2 is scarcely induced in the absence of AA. These active proteins also fail to effectively activate Nox2 in a cell-free reconstituted system without AA. Without affecting Rac-GTP binding to p67^phox, AA induces the direct interaction of Rac-GTP-bound p67^phox with the C-terminal cytosolic region of Nox2. p67^phox-Rac-Nox2 assembly and superoxide production are both abrogated by alanine substitution for Tyr-198, Leu-199, and Val-204 in the p67^phox activation domain that localizes the C-terminal to the Rac-binding domain. Thus the “third” switch (AA-inducible interaction of p67^phox·Rac-GTP with Nox2) is required to be turned on at the same time for Nox2 activation.     

VE-cadherin: adhesion at arm's length

VE-cadherin was first identified in the early 1990s and quickly emerged as an important endothelial cell adhesion molecule. The past decade of research has revealed key roles for VE-cadherin in vascular permeability and in the morphogenic events associated with vascular remodeling. The details of how VE-cadherin functions in adhesion became apparent with structure-function analysis of the cadherin extracellular domain and with the identification of the catenins, a series of cytoplasmic proteins that bind to the cadherin tail and mediate interactions between cadherins and the cytoskeleton. Whereas early work focused on the armadillo family proteins -catenin and plakoglobin, more recent investigations have identified p120-catenin (p120^ctn) and a related group of armadillo family members as key binding partners for the cadherin tail. Furthermore, a series of new studies indicate a key role for p120^ctn in regulating cadherin membrane trafficking in mammalian cells. These recent studies place p120^ctn at the hub of a cadherin-catenin regulatory mechanism that controls cadherin plasma membrane levels in cells of both epithelial and endothelial origin. endothelial cell; cytoskeleton; -catenin; p120^ctn; cell adhesion; vascular endothelial cadherin     

Compensatory and long-range changes in picosecond-nanosecond main-chain dynamics upon complex formation: 15N relaxation analysis of the free and bound states of the ubiquitin-like domain of human plexin-B1 and the small GTPase Rac1

The formation of a complex between Rac1 and the cytoplasmic domain of plexin-B1 is one of the first documented cases of a direct interaction between a small guanosine 5′-triphosphatase (GTPase) and a transmembrane receptor. Structural studies have begun to elucidate the role of this interaction for the signal transduction mechanism of plexins. Mapping of the Rac1 GTPase surface that contacts the Rho GTPase binding domain of plexin-B1 by solution NMR spectroscopy confirms the plexin domain as a GTPase effector protein. Regions neighboring the GTPase switch I and II regions are also involved in the interaction and there is considerable interest to examine the changes in protein dynamics that take place upon complex formation. Here we present main-chain nitrogen-15 relaxation measurements for the unbound proteins as well as for the Rho GTPase binding domain and Rac1 proteins in their complexed state. Derived order parameters, S², show that considerable motions are maintained in the bound state of plexin. In fact, some of the changes in S² on binding appear compensatory, exhibiting decreased as well as increased dynamics. Fluctuations in Rac1, already a largely rigid protein on the picosecond-nanosecond timescale, are overall diminished, but isomerization dynamics in the switch I and II regions of the GTPase are retained in the complex and appear to be propagated to the bound plexin domain. Remarkably, fluctuations in the GTPase are attenuated at sites, including helices α6 (the Rho-specific insert helix), α7 and α8, that are spatially distant from the interaction region with plexin. This effect of binding on long-range dynamics appears to be communicated by hinge sites and by subtle conformational changes in the protein. Similar to recent studies on other systems, we suggest that dynamical protein features are affected by allosteric mechanisms. Altered protein fluctuations are likely to prime the Rho GTPase-plexin complex for interactions with additional binding partners.     

Dendrite-like Process Formation and Cytoskeletal Remodeling Regulated by δ-Catenin Expression

Actin- and microtubule-mediated changes in cell shape are essential for many cellular activities. However, the molecular mechanisms underlying the interplay between the two are complex and remain obscure. Here we show that the expression of δ-catenin (or NPRAP/Neurojungin), a member of p120^ctn subfamily of armadillo proteins can induce the branching of dendrite-like processes in 3T3 cells and enhance dendritic morphogenesis in primary hippocampal neurons. This induction of branching phenotype involves initially the disruption of filamentous actin, and requires the growth of microtubules. The carboxyl-terminal truncation mutant of δ-catenin can cluster and redistribute the full-length protein, and dominantly inhibit its branching effect. δ-Catenin forms protein complexes and can bind directly to actin in vitro. The carboxyl-terminal truncation of δ-catenin does not interfere with its actin-binding capability; therefore the actin interaction alone is not sufficient for the induction of dendrite-like processes. When δ-catenin-transformed cells establish elaborate dendrite-like branches, the main cellular processes become stabilized and resist the disruption of both actin filaments and microtubules, as determined by fluorescent light microscopy and time-lapse recording analyses. We suggest that δ-catenin can effect a biphasic cytoskeletal remodeling event which differentially regulates actin and microtubules and promotes cellular morphogenesis.     

Phosphorylation of Rac1 T108 by Extracellular Signal-Regulated Kinase in Response to Epidermal Growth Factor: a Novel Mechanism To Regulate Rac1 Function

Accumulating evidence has implicated Rho GTPases, including Rac1, in many aspects of cancer development. Recent findings suggest that phosphorylation might further contribute to the tight regulation of Rho GTPases. Interestingly, sequence analysis of Rac1 shows that Rac1 T108 within the ¹⁰⁶PNTP¹⁰⁹ motif is likely an extracellular signal-regulated kinase (ERK) phosphorylation site and that Rac1 also has an ERK docking site, ¹⁸³KKRKRKCLLL¹⁹² (D site), at the C terminus. Indeed, we show here that both transfected and endogenous Rac1 interacts with ERK and that this interaction is mediated by its D site. Green fluorescent protein (GFP)-Rac1 is threonine (T) phosphorylated in response to epidermal growth factor (EGF), and EGF-induced Rac1 threonine phosphorylation is dependent on the activation of ERK. Moreover, mutant Rac1 with the mutation of T108 to alanine (A) is not threonine phosphorylated in response to EGF. In vitro ERK kinase assay further shows that pure active ERK phosphorylates purified Rac1 but not mutant Rac1 T108A. We also show that Rac1 T108 phosphorylation decreases Rac1 activity, partially due to inhibiting its interaction with phospholipase C-γ1 (PLC-γ1). T108 phosphorylation targets Rac1 to the nucleus, which isolates Rac1 from other guanine nucleotide exchange factors (GEFs) and hinders Rac1''s role in cell migration. We conclude that Rac1 T108 is phosphorylated by ERK in response to EGF, which plays an important role in regulating Rac1.     

A Conserved Region between the TPR and Activation Domains of p67phox Participates in Activation of the Phagocyte NADPH Oxidase

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The membrane-integrated protein gp91^phox serves as the catalytic core, because it contains a complete electron-transporting apparatus from NADPH to molecular oxygen for superoxide production. Activation of gp91^phox requires the cytosolic proteins p67^phox, p47^phox, and Rac (a small GTPase). p67^phox, comprising 526 amino acids, moves upon cell stimulation to the membrane together with p47^phox and there interacts with Rac; these processes are prerequisite for gp91^phox activation. Here we show that a region of p67^phox (amino acids 190–200) C-terminal to the Rac-binding domain is evolutionarily well conserved and participates in oxidase activation at a later stage in conjunction with an activation domain. Alanine substitution for Tyr-198, Leu-199, or Val-204 abrogates the ability of p67^phox to support superoxide production by gp91^phox-based oxidase as well as its related oxidases Nox1 and Nox3; the activation also involves other invariant residues such as Leu-193, Asp-197, and Gly-200. Intriguingly, replacement of Gln-192 by alanine or that of Tyr-198 by phenylalanine or tryptophan rather enhances superoxide production by gp91^phox-based oxidase, suggesting a tuning role for these residues. Furthermore, the Y198A/V204A or L199A/V204A substitution leads to not only a complete loss of the activity of the reconstituted oxidase system but also a significant decrease in p67^phox interaction with the gp91^phox NADPH-binding domain, although these mutations affect neither the protein integrity nor the Rac binding activity. Thus the extended activation domain of p67^phox (amino acids 190–210) containing the D(Y/F)LGK motif plays an essential role in oxidase activation probably by interacting with gp91^phox.     

Regulation of calcium signalling by the small GTP-binding proteins Ras and Rac1

In order to investigate a possible interaction of the small GTP-binding proteins Ras and Rac1 with Ca²⁺-mediated signalling cascades the effects of dominant negative mutants of Ras and Rac1 on Ca²⁺ signalling have been studied after stimulation of either the EGFR or the nerve growth factor receptor (TRK). Expression of dominant negative Ras blocks the release of Ca²⁺ from internal stores after activation of EGFR whereas the calcium signal elicited by the activated TRK receptor is unaffected. The sensitivity to dominant negative Ras is determined by the structure of the PLCγ-binding sites of the corresponding receptors. Exchange of the PLCγ-binding domain of the EGFR by the PLCγ-binding site of TRK renders the EGFR-induced calcium signal insensitive to the expression of dominant negative Ras. Substitution of the PLCγ-binding site of TRK by the PLCγ-binding region of EGFR renders TRK sensitive to dominant negative Ras. The inhibition of Ca²⁺ release by dominant negative Ras is accompanied by a reduction in PLCγ binding to the EGFR and a concomitant decrease of EGF-induced inositol-1,3,5-trisphosphate (InsP₃) formation. The depression of PLCγ binding to EGFR is explained by a competition of PLCγ with other SH2-domain containing proteins for the same low affinity binding regions of the EGFR. This conclusion is supported by the observation that microinjection of several SH2-domain containing proteins including Ras-GP, lipase-free fragment of PLCγ or Janus kinase binding protein (JAB), reduces the association of PLCγ to the EGFR, not, however, to TRK. In contrast to dominant negative Ras which does not affect the Ca²⁺ transient induced by the activation of the TRK receptor, a dominant negative mutant of Rac significantly depresses the Ca²⁺ signals induced by EGFR as well as by TRK. The different behavior of Rac and Ras supports the notion that the two small GTP-binding proteins act through separate pathways. It is demonstrated that dominant negative Rac significantly reduces the formation of phosphatidylinositol-4,5-bisphosphate (PIP₂), the substrate of PLCγ. This effect is not observed after expression of dominant negative Ras. In summary, the data provide further evidence for a cross-talk between small GTP-binding proteins and Ca²⁺ signalling in which both G-proteins interfere with the formation of InsP₃ although by different mechanisms.     

Spectrin Tetramer Formation Is Not Required for Viable Development in Drosophila

The dominant paradigm for spectrin function is that (αβ)₂-spectrin tetramers or higher order oligomers form membrane-associated two-dimensional networks in association with F-actin to reinforce the plasma membrane. Tetramerization is an essential event in such structures. We characterize the tetramerization interaction between α-spectrin and β-spectrins in Drosophila. Wild-type α-spectrin binds to both β- and β_H-chains with high affinity, resembling other non-erythroid spectrins. However, α-spec^R22S, a tetramerization site mutant homologous to the pathological α-spec^R28S allele in humans, eliminates detectable binding to β-spectrin and reduces binding to β_H-spectrin ∼1000-fold. Even though spectrins are essential proteins, α-spectrin^R22S rescues α-spectrin mutants to adulthood with only minor phenotypes indicating that tetramerization, and thus conventional network formation, is not the essential function of non-erythroid spectrin. Our data provide the first rigorous test for the general requirement for tetramer-based non-erythroid spectrin networks throughout an organism and find that they have very limited roles, in direct contrast to the current paradigm.     

Alternative splicing of Rac1 generates Rac1b, a self-activating GTPase

Rac1b was recently identified in malignant colorectal tumors as an alternative splice variant of Rac1 containing a 19-amino acid insertion next to the switch II region. The structures of Rac1b in the GDP- and the GppNHp-bound forms, determined at a resolution of 1.75 A, reveal that the insertion induces an open switch I conformation and a highly mobile switch II. As a consequence, Rac1b has an accelerated GEF-independent GDP/GTP exchange and an impaired GTP hydrolysis, which is restored partially by GTPase-activating proteins. Interestingly, Rac1b is able to bind the GTPase-binding domain of PAK but not full-length PAK in a GTP-dependent manner, suggesting that the insertion does not completely abolish effector interaction. The presented study provides insights into the structural and biochemical mechanism of a self-activating GTPase.