P21-activated Kinase-interacting Exchange Factor Research Articles

The Discovery of GIT1/β-Pix Inhibitors: Virtual Screening and Biological Evaluation of New Small-molecule Compounds with Anti-invasion Effect in Gastrointestinal Neoplasms.

GIT1 (G-protein-coupled receptor kinase interacting protein-1) has been found to be highly related with cancer cell invasion and metastasis in many cancer types. β-Pix (p21-activated kinase-interacting exchange factor) is one of the proteins that interact with GIT1. Targeting GIT1/β-Pix complex might be a potential therapeutic strategy for interfering cancer metastasis. However, at present, no well-recognized small-molecule inhibitor targeting GIT1/β-Pix is available. Thus, we aim to discover novel GIT1/β-Pix inhibitors with simple scaffold, high activity and low toxicity to develop new therapeutic strategies to restrain cancer metastasis. GIT1/β-Pix inhibitors were identified from ChemBridge by virtual screening. Briefly, the modeling of GIT1 was performed and the establishment of GIT1/β-Pix binding pocket enabled the virtual screening to identify the inhibitor. In addition, direct binding of the candidate molecules to GIT1 was detected by biolayer interferometry (BLI) to discover the hit compound. Furthermore, the inhibitory effect on invasion of stomach and colon cancer cells in vitro was carried out by the transwell assay and detection of epithelial-mesenchymal transition (EMT)-related proteins. Finally, the binding mode of hit compound to GIT1 was estimated by molecular dynamics simulation to analyze the key amino residues to guide further optimization. We selected the top 50 compounds from the ChemBridge library by virtual screening. Then, by skeleton similarity analysis nine compounds were selected for further study. Furthermore, the direct interaction of nine compounds to GIT1 was detected by BLI to obtain the best affinitive compound. Finally, 17302836 was successfully identified (KD = 84.1±2.0 μM). In vitro tests on 17302836 showed significant anti-invasion effect on gastric cancer and colorectal cancer. We discovered a new GIT1/β-Pix inhibitor (17302836) against gastrointestinal cancer invasion and metastasis. This study provides a promising candidate for developing new GIT1/β-Pix inhibitors for tumor treatment.

β-PIX plays an important role in regulation of intestinal epithelial restitution by interacting with GIT1 and Rac1 after wounding.

Early gut mucosal restitution is a process by which intestinal epithelial cells (IECs) migrate over the wounded area, and its defective regulation occurs commonly in various critical pathological conditions. This rapid reepithelialization is mediated by different activating small GTP-binding proteins, but the exact mechanism underlying this process remains largely unknown. Recently, it has been reported that interaction between p21-activated kinase-interacting exchange factor (β-PIX) and G protein-coupled receptor kinase-interacting protein 1 (GIT1) activates small GTPases and plays an important role in the regulation of cell motility. Here, we show that induced association of β-PIX with GIT1 is essential for the stimulation of IEC migration after wounding by activating Rac1. Levels of β-PIX and GIT1 proteins and their association in differentiated IECs (line of IEC-Cdx2L1) were much higher than those observed in undifferentiated IECs (line of IEC-6), which was associated with an increase in IEC migration after wounding. Decreased levels of endogenous β-PIX by its gene-silencing destabilized β-PIX/GIT1 complexes, repressed Rac1 activity and inhibited cell migration over the wounded area. In contrast, ectopic overexpression of β-PIX increased the levels of β-PIX/GIT1 complexes, stimulated Rac1 activity, and enhanced intestinal epithelial restitution. Increased levels of cellular polyamines also stimulated β-PIX/GIT1 association, increased Rac1 activity, and promoted the epithelial restitution. Moreover, polyamine depletion decreased cellular abundances of β-PIX/GIT1 complex and repressed IEC migration after wounding, which was rescued by ectopic overexpression of β-PIX or GIT1. These results indicate that β-PIX/GIT1/Rac1 association is necessary for stimulation of IEC migration after wounding and that this signaling pathway is tightly regulated by cellular polyamines. NEW & NOTEWORTHY Our current study demonstrates that induced association of β-PIX with GIT1 is essential for the stimulation of intestinal epithelial restitution by activating Rac1, and this signaling pathway is tightly regulated by cellular polyamines.

GIT1/βPIX signaling proteins and PAK1 kinase regulate microtubule nucleation

Microtubule nucleation from γ-tubulin complexes, located at the centrosome, is an essential step in the formation of the microtubule cytoskeleton. However, the signaling mechanisms that regulate microtubule nucleation in interphase cells are largely unknown. In this study, we report that γ-tubulin is in complexes containing G protein-coupled receptor kinase-interacting protein 1 (GIT1), p21-activated kinase interacting exchange factor (βPIX), and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1) in various cell lines. Immunofluorescence microscopy revealed association of GIT1, βPIX and activated PAK1 with centrosomes. Microtubule regrowth experiments showed that depletion of βPIX stimulated microtubule nucleation, while depletion of GIT1 or PAK1 resulted in decreased nucleation in the interphase cells. These data were confirmed for GIT1 and βPIX by phenotypic rescue experiments, and counting of new microtubules emanating from centrosomes during the microtubule regrowth. The importance of PAK1 for microtubule nucleation was corroborated by the inhibition of its kinase activity with IPA-3 inhibitor. GIT1 with PAK1 thus represent positive regulators, and βPIX is a negative regulator of microtubule nucleation from the interphase centrosomes. The regulatory roles of GIT1, βPIX and PAK1 in microtubule nucleation correlated with recruitment of γ-tubulin to the centrosome. Furthermore, in vitro kinase assays showed that GIT1 and βPIX, but not γ-tubulin, serve as substrates for PAK1. Finally, direct interaction of γ-tubulin with the C-terminal domain of βPIX and the N-terminal domain of GIT1, which targets this protein to the centrosome, was determined by pull-down experiments. We propose that GIT1/βPIX signaling proteins with PAK1 kinase represent a novel regulatory mechanism of microtubule nucleation in interphase cells.

Affinity Enhancement by Ligand Clustering Effect Inspired by Peptide Dendrimers-Shank PDZ Proteins Interactions.

High-affinity binders are desirable tools to probe the function that specific protein−protein interactions play in cell. In the process of seeking a general strategy to design high-affinity binders, we found a clue from the βPIX (p21-activated kinase interacting exchange factor)−Shank PDZ interaction in synaptic assembly: three PDZ-binding sites are clustered by a parallel coiled-coil trimer but bind to Shank PDZ protein with 1:1 stoichiometry (1 trimer/1 PDZ). Inspired by this architecture, we proposed that peptide dendrimer, mimicking the ligand clustering in βPIX, will also show enhanced binding affinity, yet with 1:1 stoichiometry. This postulation has been proven here, as we synthesized a set of monomeric, dimeric and trimeric peptides and measured their binding affinity and stoichiometry with Shank PDZ domains by isothermal titration calorimetry, native mass spectrometry and surface plasmon resonance. This affinity enhancement, best explained by proximity effect, will be useful to guide the design of high-affinity blockers for protein−protein interactions.

Microtubule nucleation in mouse bone marrow-derived mast cells is regulated by the concerted action of GIT1/βPIX proteins and calcium.

Ag-mediated activation of mast cells initiates signaling events leading to Ca(2+) response, release of allergic mediators from cytoplasmic granules, and synthesis of cytokines and chemokines. Although microtubule rearrangement during activation has been described, the molecular mechanisms that control their remodeling are largely unknown. Microtubule nucleation is mediated by complexes that are formed by γ-tubulin and γ-tubulin complex proteins. In this study, we report that, in bone marrow-derived mast cells (BMMCs), γ-tubulin interacts with p21-activated kinase interacting exchange factor β (βPIX) and G protein-coupled receptor kinase-interacting protein (GIT)1. Microtubule regrowth experiments showed that the depletion of βPIX in BMMCs stimulated microtubule nucleation, whereas depletion of GIT1 led to the inhibition of nucleation compared with control cells. Phenotypic rescue experiments confirmed that βPIX and GIT1 represent negative and positive regulators of microtubule nucleation in BMMCs, respectively. Live-cell imaging disclosed that both proteins are associated with centrosomes. Immunoprecipitation and pull-down experiments revealed that an enhanced level of free cytosolic Ca(2+) affects γ-tubulin properties and stimulates the association of GIT1 and γ-tubulin complex proteins with γ-tubulin. Microtubule nucleation also was affected by Ca(2+) level. Moreover, in activated BMMCs, γ-tubulin formed complexes with tyrosine-phosphorylated GIT1. Further experiments showed that GIT1 and βPIX are involved in the regulation of such important physiological processes as Ag-induced chemotaxis and degranulation. Our study provides for the first time, to our knowledge, a possible mechanism for the concerted action of tyrosine kinases, GIT1/βPIX proteins, and Ca(2+) in the propagation of signals leading to the regulation of microtubule nucleation in activated mast cells.

Function of Ahnak protein in aortic smooth muscle cell migration through Rac activation

Ahnak protein acts as a scaffold protein networking phospholipase C-γ and protein kinase C-α, which subsequently stimulate an extracellular signal-regulated kinase (Erk) pathway. In mouse aortic smooth muscle cells (ASMCs), the activation of the signalling cascade ultimately promotes the cell migration through an unknown mechanism. We aimed to dissect the Ahnak-mediated cell signalling network involved in the migration of ASMCs. Migration of ASMCs from wild-type mice was significantly increased by platelet-derived growth factor (PDGF) stimulation in transwell chamber and wound-healing assays, whereas migration of ASMCs from Ahnak knockout mice was reduced. Consistently, stimulation of wild-type ASMCs with PDGF resulted in Rac activation-mediated lamellipodial protrusion in migrating cells. In contrast, Ahnak knockout ASMCs displayed lower activation of Rac in response to PDGF and slow lamellipodial protrusion rate and cell migration. Ahnak signalling complex was analysed by immunoprecipitation with antibody to p21-activated protein kinase (PAK). Ahnak protein was shown to function as the signalling scaffold interacting with the multiple protein complex of Erk, PAK, and p21-activated kinase-interacting exchange factor β. The proposed role of Ahnak in cell migration was examined using a restenosis model in which the carotid arteries of mice were subjected to post-ligation injury. We show neointimal formation and SMC migration after ligation injury in Ahnak knockout mice were significantly retarded compared with wild-type mice. Ahnak protein plays an important scaffolding function connecting Erk and Rac activation in PDGF-dependent migration of ASMC.

Structural Basis for Asymmetric Association of the βPIX Coiled Coil and Shank PDZ

βPIX (p21-activated kinase interacting exchange factor) and Shank/ProSAP protein form a complex acting as a protein scaffold that integrates signaling pathways and regulates postsynaptic structure. Complex formation is mediated by the C-terminal PDZ binding motif of βPIX and the Shank PDZ domain. The coiled-coil (CC) domain upstream of the PDZ binding motif allows multimerization of βPIX, which is important for its physiological functions. We have solved the crystal structure of the βPIX CC–Shank PDZ complex and determined the stoichiometry of complex formation. The βPIX CC forms a 76-Å-long parallel CC trimer. Despite the fact that the βPIX CC exposes three PDZ binding motifs in the C-termini, the βPIX trimer associates with a single Shank PDZ. One of the C-terminal ends of the CC forms an extensive β-sheet interaction with the Shank PDZ, while the other two ends are not involved in ligand binding and form random coils. The two C-terminal ends of βPIX have significantly lower affinity than the first PDZ binding motif due to the steric hindrance in the C-terminal tails, which results in binding of a single PDZ domain to the βPIX trimer. The structure shows canonical class I PDZ binding with a β-sheet interaction extending to position −6 of βPIX. The βB–βC loop of Shank PDZ undergoes a conformational change upon ligand binding to form the β-sheet interaction and to accommodate the bulky side chain of Trp −5. This structural study provides a clear picture of the molecular recognition of the PDZ ligand and the asymmetric association of βPIX CC and Shank PDZ.

A New PIXel in the Puzzle

Hypertension is a clinical situation associated with endothelial dysfunction and increased vascular reactive oxygen species (ROS) formation. The observations that antioxidants, such as Tempol or apocynin, lower the blood pressure in spontaneously hypertensive rats gave rise to the concept that ROS essentially contribute to hypertension. Recent work, however, has sharpened this view, and it is now clear that the underlying mechanism of ROS-induced hypertension may not be endothelial dysfunction but rather an ROS-dependent modulation of the sympathetic drive,1 the kidney, and potentially alterations in the immune system.2 Despite these findings, hypertension has been consistently linked to increased ROS formation in the vascular system. It is, therefore, plausible that ROS are not necessarily the cause but potentially the consequence of hypertension. This notion certainly does not exclude that ROS are involved in the pathogenesis of specific aspects of hypertension, such as the development of fibrosis. In keeping with the concept of hypertension-induced ROS formation, it was noted previously that distension of a vessel with an oversized balloon acutely increases ROS formation.3 Moreover, acute elevation of pressure from 80 to 160 mm Hg enhances the ROS formation and induces an ROS-dependent attenuation of the endothelium-dependent relaxation in isolated rat femoral arteries.4 Moreover, even in chronic models of isolated hypertension, such as aortic banding, the ROS formation was consistently higher in the hypertensive rather than in the normotensive part. There is, however, uncertainty regarding the enzymatic sources of ROS, and a contribution of mitochondria, endothelial NO …

Structures of Dimeric GIT1 and Trimeric β-PIX and Implications for GIT–PIX Complex Assembly

GIT (G protein-coupled receptor kinase-interacting protein) and PIX (p21-activated kinase-interacting exchange factor) family proteins integrate signaling pathways involving Arf and Rho family GTPases. GIT1 and beta-PIX form a constitutively associated complex that acts as a scaffold to allow the formation of large multiprotein assemblies that regulate synaptogenesis, cell polarity and cell migration among other physiological processes. Complex formation is mediated by the GIT binding domain (GBD) in beta-PIX, which recognizes the Spa homology domain of GIT1. Both binding domains are adjacent to predicted coiled-coil segments that allow homo-oligomerization of GIT1 and beta-PIX, respectively. Oligomerization of GIT and PIX proteins is important for their physiological functions, and deletion of the coiled-coil domains interferes with correct subcellular localization and the GEF (guanine nucleotide exchange factor) activity of PIX. We have solved the crystal structures of the CC domains of GIT1 and beta-PIX and determined the stoichiometry of complex formation between the two proteins in order to understand the molecular architecture of the GIT1-beta-PIX complex. The crystal structure of the CC domain of GIT1 solved at 1.4 A resolution shows a dimeric, parallel CC that spans 67 A in length. Unexpectedly, and in contrast to prevalent dimeric models, the structure of the CC region of beta-PIX determined at 2.8 A resolution, combined with hydrodynamic studies, reveals that this protein forms a parallel trimer. Furthermore, we demonstrate that dimeric GIT and trimeric PIX form an unusual high-affinity heteropentameric complex in which each Spa homology domain of the GIT1 dimer recognizes one GBD of the beta-PIX trimer, leaving one GBD unoccupied. These results can serve as a basis to better understand oligomerization-dependent GIT1-beta-PIX-regulated signaling events and provide an insight into the architecture of large signaling complexes involving GIT1 and beta-PIX.

A RAC/CDC-42–Independent GIT/PIX/PAK Signaling Pathway Mediates Cell Migration in C. elegans

P21 activated kinase (PAK), PAK interacting exchange factor (PIX), and G protein coupled receptor kinase interactor (GIT) compose a highly conserved signaling module controlling cell migrations, immune system signaling, and the formation of the mammalian nervous system. Traditionally, this signaling module is thought to facilitate the function of RAC and CDC-42 GTPases by allowing for the recruitment of a GTPase effector (PAK), a GTPase activator (PIX), and a scaffolding protein (GIT) as a regulated signaling unit to specific subcellular locations. Instead, we report here that this signaling module functions independently of RAC/CDC-42 GTPases in vivo to control the cell shape and migration of the distal tip cells (DTCs) during morphogenesis of the Caenorhabditis elegans gonad. In addition, this RAC/CDC-42–independent PAK pathway functions in parallel to a classical GTPase/PAK pathway to control the guidance aspect of DTC migration. Among the C. elegans PAKs, only PAK-1 functions in the GIT/PIX/PAK pathway independently of RAC/CDC42 GTPases, while both PAK-1 and MAX-2 are redundantly utilized in the GTPase/PAK pathway. Both RAC/CDC42–dependent and –independent PAK pathways function with the integrin receptors, suggesting that signaling through integrins can control the morphology, movement, and guidance of DTC through discrete pathways. Collectively, our results define a new signaling capacity for the GIT/PIX/PAK module that is likely to be conserved in vertebrates and demonstrate that PAK family members, which are redundantly utilized as GTPase effectors, can act non-redundantly in pathways independent of these GTPases.

Transgenic Mouse Proteomics Identifies New 14-3-3-associated Proteins Involved in Cytoskeletal Rearrangements and Cell Signaling

Identification of protein-protein interactions is crucial for unraveling cellular processes and biochemical mechanisms of signal transduction. Here we describe, for the first time, the application of the tandem affinity purification (TAP) and LC-MS method to the characterization of protein complexes from transgenic mice. The TAP strategy developed in transgenic mice allows the emplacement of complexes in their physiological environment in contact with proteins that might only be specifically expressed in certain tissues while simultaneously ensuring the right stoichiometry of the TAP protein versus their binding partners and represents a novelty in proteomics approaches used so far. Mouse lines expressing TAP-tagged 14-3-3zeta protein were generated, and protein interactions were determined. 14-3-3 proteins are general regulators of cell signaling and represent up to 1% of the total brain protein. This study allowed the identification of almost 40 novel 14-3-3zeta-binding proteins. Biochemical and functional characterization of some of these interactions revealed new mechanisms of action of 14-3-3zeta in several signaling pathways, such as glutamate receptor signaling via binding to homer homolog 3 (Homer 3) and in cytoskeletal rearrangements and spine morphogenesis by binding and regulating the activity of the signaling complex formed by G protein-coupled receptor kinase-interactor 1 (GIT1) and p21-activated kinase-interacting exchange factor beta (betaPIX).

Crystal structure of the N-terminal SH3 domain of mouse βPIX, p21-activated kinase-interacting exchange factor

The mouse βPIX–SH3 domain, residues 8–63 of P21-activated kinase interacting exchange factor, has been characterized by X-ray diffraction. Crystals belonging to space group P3 221 diffracted to 2.0 Å and the structure was phased by the single-wavelength anomalous diffraction method. The domain is a compact β-barrel with an overall conformation similar to the general SH3 structure. The X-ray structure shows mouse βPIX–SH3 domain binding the way in which the βPIX characteristic amino acids do so for an unconventional ligand binding surface. This arrangement provides a rationale for the unusual ligand recognition motif exhibited by mouse βPIX–SH3 domain. Comparison with another SH3/peptide complex shows that the recognition mode of the mouse βPIX–SH3 domain should be very similar to the RXXK ligand binding mode. The unique large and planar hydrophobic pocket may contribute to the promiscuity of βPIX–SH3 domain resulting in its multiple biological functions.

Characterization of the endogenous GIT1– βPIX complex, and identification of its association to membranes

G protein-coupled receptor kinase interactors (GITs) are adaptor proteins with ADP-ribosylating factor – GTPase-activating protein (ARF–GAP) activity that form complexes with the p21-activated kinase-interacting exchange factor (PIX) guanine nucleotide exchanging factors for Rac and Cdc42. In this study we have characterized the endogenous GIT1/p95-APP1/Cat1 (GIT1)– PIX complexes in neuronal and non-neuronal cells. In COS7 cells, immunocytochemical analysis shows the localization of endogenous GIT1 in the perinuclear region of the cell, as well as at the cell periphery, where GIT1 co-localizes with filamentous actin. The perinuclear localization of endogenous GIT1 was confirmed in avian fibroblasts. In COS7 cells, immunoprecipitation and microsequencing experiments with either anti-GIT1 or anti- βPIX antibodies unequivocally show that βPIX is uniquely associated with GIT1 in lysates from these cells, while GIT2/PKL/p95-APP2/Cat2 (GIT2) is undetectable in the endogenous complexes. Moreover, this analysis demonstrates that βPIX is the limiting factor for the formation of the endogenous complexes, since a small fraction of GIT1 can be co-immunoprecipitated with most βPIX from these cells. Saponin treatment of unfixed cells indicates that βPIX-bound GIT1 is preferentially retained in the saponin-resistant fraction when compared to βPIX-free GIT1. Moreover, analysis by tissue fractionation shows that a significant fraction of the endogenous GIT1– βPIX complex is firmly associated to membranes from brain homogenates. Our findings show the specific localization of the complex at intracellular membranes, and indicate a correlation between the association of GIT1 to βPIX, and the localization of the endogenous complex at membranes.

W14.357 Methylenetetrahydrofolate reductase C677T, moderate hyperhomocysteinemia, and risk of ischemic cardiovascular disease and venous thromboembolism

G protein-coupled receptor kinase interactors (GITs) are adaptor proteins with ADP-ribosylating factor – GTPase-activating protein (ARF–GAP) activity that form complexes with the p21-activated kinase-interacting exchange factor (PIX) guanine nucleotide exchanging factors for Rac and Cdc42. In this study we have characterized the endogenous GIT1/p95-APP1/Cat1 (GIT1)– PIX complexes in neuronal and non-neuronal cells. In COS7 cells, immunocytochemical analysis shows the localization of endogenous GIT1 in the perinuclear region of the cell, as well as at the cell periphery, where GIT1 co-localizes with filamentous actin. The perinuclear localization of endogenous GIT1 was confirmed in avian fibroblasts. In COS7 cells, immunoprecipitation and microsequencing experiments with either anti-GIT1 or anti-βPIX antibodies unequivocally show that βPIX is uniquely associated with GIT1 in lysates from these cells, while GIT2/PKL/p95-APP2/Cat2 (GIT2) is undetectable in the endogenous complexes. Moreover, this analysis demonstrates that βPIX is the limiting factor for the formation of the endogenous complexes, since a small fraction of GIT1 can be co-immunoprecipitated with most βPIX from these cells. Saponin treatment of unfixed cells indicates that βPIX-bound GIT1 is preferentially retained in the saponin-resistant fraction when compared to βPIX-free GIT1. Moreover, analysis by tissue fractionation shows that a significant fraction of the endogenous GIT1–βPIX complex is firmly associated to membranes from brain homogenates. Our findings show the specific localization of the complex at intracellular membranes, and indicate a correlation between the association of GIT1 to βPIX, and the localization of the endogenous complex at membranes.

Leucine Zipper-mediated Homodimerization of the p21-activated Kinase-interacting Factor, βPix: IMPLICATION FOR A ROLE IN CYTOSKELETAL REORGANIZATION

Pix, a p21-activated kinase-interacting exchange factor, is known to be involved in the regulation of Cdc42/Rac GTPases. The 85-kDa betaPix-a protein contains an Src homology 3 domain, the tandem Dbl homology and Pleckstrin homology domains, a proline-rich region, and a GIT1-binding domain. In addition to those domains, betaPix-a also contains a putative leucine zipper domain at the C-terminal end. In this study, we demonstrate that the previously identified putative leucine zipper domain mediates the formation of betaPix-a homodimers. Using in vitro and in vivo methodologies, we show that deletion of the leucine zipper domain is sufficient to abolish betaPix-a homodimerization. In NIH3T3 fibroblast cells, expression of wild type betaPix-a induces the formation of membrane ruffles. However, cells expressing the leucine zipper domain deletion mutant could not form membrane ruffle structures. Moreover, platelet-derived growth factor-mediated cytoskeletal changes were completely blocked by the leucine zipper domain deletion mutant. The results suggest that the leucine zipper domain enables betaPix-a to homodimerize, and homodimerization is essential for betaPix-a signaling functions leading to the cytoskeletal reorganization.