IQGAP1 scaffolding links phosphoinositide kinases to cytoskeletal reorganization

IQGAP1 is a multidomain scaffold protein that coordinates the direction and impact of multiple signaling pathways by scaffolding its various binding partners. However, the spatial and temporal resolution of IQGAP1 scaffolding remains unclear. Here, we use fluorescence imaging and correlation methods that allow for real-time live-cell changes in IQGAP1 localization and complex formation during signaling. We find that IQGAP1 and PIPKIγ interact on both the plasma membrane and in cytosol. Epidermal growth factor (EGF) stimulation, which can initiate cytoskeletal changes, drives the movement of the cytosolic pool toward the plasma membrane to promote cytoskeletal changes. We also observe that a significant population of cytosolic IQGAP1-PIPKIγ complexes localize to early endosomes, and in some instances form aggregated clusters which become highly mobile upon EGF stimulation. Our imaging studies show that PIPKIγ and PI3K bind simultaneously to IQGAP1, which may accelerate conversion of PI4P to PI(3,4,5)P₃ that is required for cytoskeletal changes. Additionally, we find that IQGAP1 is responsible for PIPKIγ association with two proteins associated with cytoskeletal changes, talin and Cdc42, during EGF stimulation. These results directly show that IQGAP1 provides a physical link between phosphoinositides (through PIPKIγ), focal adhesion formation (through talin), and cytoskeletal reorganization (through Cdc42) upon EGF stimulation. Taken together, our results support the importance of IQGAP1 in regulating cell migration by linking phosphoinositide lipid signaling with cytoskeletal reorganization.     

The Pseudomonas aeruginosa N-Acylhomoserine Lactone Quorum Sensing Molecules Target IQGAP1 and Modulate Epithelial Cell Migration

Quorum sensing (QS) signaling allows bacteria to control gene expression once a critical population density is achieved. The Gram-negative human pathogen Pseudomonas aeruginosa uses N-acylhomoserine lactones (AHL) as QS signals, which coordinate the production of virulence factors and biofilms. These bacterial signals can also modulate human cell behavior. Little is known about the mechanisms of the action of AHL on their eukaryotic targets. Here, we found that N-3-oxo-dodecanoyl-L-homoserine lactone 3O-C₁₂-HSL modulates human intestinal epithelial Caco-2 cell migration in a dose- and time-dependent manner. Using new 3O-C₁₂-HSL biotin and fluorescently-tagged probes for LC-MS/MS and confocal imaging, respectively, we demonstrated for the first time that 3O-C₁₂-HSL interacts and co-localizes with the IQ-motif-containing GTPase-activating protein IQGAP1 in Caco-2 cells. The interaction between IQGAP1 and 3O-C₁₂-HSL was further confirmed by pull-down assay using a GST-tagged protein with subsequent Western blot of IQGAP1 and by identifying 3O-C₁₂-HSL with a sensor bioassay. Moreover, 3O-C₁₂-HSL induced changes in the phosphorylation status of Rac1 and Cdc42 and the localization of IQGAP1 as evidenced by confocal and STED microscopy and Western blots. Our findings suggest that the IQGAP1 is a novel partner for P.aeruginosa 3O-C₁₂-HSL and likely the integrator of Rac1 and Cdc42- dependent altered cell migration. We propose that the targeting of IQGAP1 by 3O-C₁₂-HSL can trigger essential changes in the cytoskeleton network and be an essential component in bacterial – human cell communication.     

Allosteric Regulation of Focal Adhesion Kinase by PIP2 and ATP

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that regulates cell signaling, proliferation, migration, and development. A major mechanism of regulation of FAK activity is an intramolecular autoinhibitory interaction between two of its domains—the catalytic and FERM domains. Upon cell adhesion to the extracellular matrix, FAK is being translocated toward focal adhesion sites and activated. Interactions of FAK with phosphoinositide phosphatidylinsositol-4,5-bis-phosphate (PIP₂) are required to activate FAK. However, the molecular mechanism of the activation remains poorly understood. Recent fluorescence resonance energy transfer experiments revealed a closure of the FERM-kinase interface upon ATP binding, which is reversed upon additional binding of PIP₂. Here, we addressed the allosteric regulation of FAK by performing all-atom molecular-dynamics simulations of a FAK fragment containing the catalytic and FERM domains, and comparing the dynamics in the absence or presence of ATP and PIP₂. As a major conformational change, we observe a closing and opening motion upon ATP and additional PIP₂ binding, respectively, in good agreement with the fluorescence resonance energy transfer experiments. To reveal how the binding of the regulatory PIP₂ to the FERM F2 lobe is transduced to the very distant F1/N-lobe interface, we employed force distribution analysis. We identified a network of mainly charged residue-residue interactions spanning from the PIP₂ binding site to the distant interface between the kinase and FERM domains, comprising candidate residues for mutagenesis to validate the predicted mechanism of FAK activation.     

A temporal model of cofilin regulation and the early peak of actin barbed ends in invasive tumor cells

Cofilin is an important regulator of actin polymerization, cell migration, and chemotaxis. Recent experimental data on mammary carcinoma cells reveal that stimulation by epidermal growth factor (EGF) generates a pool of active cofilin that results in a peak of actin filament barbed ends on the timescale of 1 min. Here, we present results of a mathematical model for the dynamics of cofilin and its transition between several pools in response to EGF stimulation. We describe the interactions of phospholipase C, membrane lipids (PIP₂), and cofilin bound to PIP₂ and to F-actin, as well as diffusible cofilin in active G-actin-monomer-bound or phosphorylated states. We consider a simplified representation in which the thin cell edge (lamellipod) and the cell interior are represented by two compartments that are linked by diffusion. We demonstrate that a high basal level of active cofilin stored by binding to PIP₂, as well as the highly enriched local milieu of F-actin at the cell edge, is essential to capture the EGF-induced barbed-end amplification observed experimentally.     

Involvement of IQGAP3, a regulator of Ras/ERK‐related cascade,in hepatocyte proliferation in mouse liver regeneration and development

The spatio‐temporal regulation of hepatocyte proliferation is a critical issue in liver regeneration. Here, in normal and regenerating liver as well as in developing liver, we examined its expression/localization of IQGAP3, which was most recently reported as a Ras/Rac/Cdc42‐binding proliferation factor associated with cell–cell contacts in epithelial‐type cells. In parallel, the expression/localization of Rac/Cdc42‐binding IQGAP1/2 was examined. IQGAP3 showed a specific expression in proliferating hepatocytes positive for the proliferating marker Ki‐67, the levels of expressions of mRNAs and proteins were significantly increased in hepatocytes in liver regeneration and development. In immunofluorescence, IQGAP3 was highly enriched at cell–cell contacts of hepatocytes. IQGAP1 and IQGAP2 were exclusively expressed in Kupffer and sinusoidal endothelial cells, respectively, in normal, regenerating, and developing liver. The expression of IQGAP1, but not of IQGAP2, was increased in CCl₄‐induced (but not in partial hepatectomy‐induced) liver regeneration. Exclusive expression/localization of IQGAP3 to hepatocytes in the liver likely reflects the specific involvement of the IQGAP3/Ras/ERK signaling cascade in hepatocyte proliferation in addition to the previously identified signaling pathways, possibly by integrating cell–cell contact‐related proliferating signaling events. On the other hand, the Rac/Cdc42‐binding properties of IQGAP1/2/3 may be related to the distinct modes of remodeling due to the different strategies which induced proliferation of liver cells; partial hepatectomy, CCl₄ injury, or embryonic development. Thus, the functional orchestration of Ras and the Ras homologous (Rho) family proteins Rac/Cdc42 likely plays a critical role in liver regeneration and development. J. Cell. Physiol. 220: 621–631, 2009. © 2009 Wiley‐Liss, Inc.     

Phosphoinositide signaling regulates the exocyst complex and polarized integrin trafficking in directionally migrating cells

Polarized delivery of signaling and adhesion molecules to the leading edge is required for directional migration of cells. Here, we describe a role for the PIP(2)-synthesizing enzyme, PIPKIγi2, in regulation of exocyst complex control of cell polarity and polarized integrin trafficking during migration. Loss of PIPKIγi2 impaired directional migration, formation of cell polarity, and integrin trafficking to the leading edge. Upon initiation of directional migration, PIPKIγi2 via PIP(2) generation controls the integration of the exocyst complex into an integrin-containing trafficking compartment that requires the talin-binding ability of PIPKIγi2, and talin for integrin recruitment to the leading edge. A PIP(2) requirement is further emphasized by inhibition of PIPKIγi2-regulated directional migration by an Exo70 mutant deficient in PIP(2) binding. These results reveal how phosphoinositide generation orchestrates polarized trafficking of integrin in coordination with talin that links integrins to the actin cytoskeleton, processes that are required for directional migration.     

m-calpain Activation Is Regulated by Its Membrane Localization and by Its Binding to Phosphatidylinositol 4,5-Bisphosphate

m-calpain plays a critical role in cell migration enabling rear de-adhesion of adherent cells by cleaving structural components of the adhesion plaques. Growth factors and chemokines regulate keratinocyte, fibroblast, and endothelial cell migration by modulating m-calpain activity. Growth factor receptors activate m-calpain secondary to phosphorylation on serine 50 by ERK. Concurrently, activated m-calpain is localized to its inner membrane milieu by binding to phosphatidylinositol 4,5-bisphosphate (PIP₂). Opposing this, CXCR3 ligands inhibit cell migration by blocking m-calpain activity secondary to a PKA-mediated phosphorylation in the C2-like domain. The failure of m-calpain activation in the absence of PIP₂ points to a key regulatory role, although whether this PIP₂-mediated membrane localization is regulatory for m-calpain activity or merely serves as a docking site for ERK phosphorylation is uncertain. Herein, we report the effects of two CXCR3 ligands, CXCL11/IP-9/I-TAC and CXCL10/IP-10, on the EGF- and VEGF-induced redistribution of m-calpain in human fibroblasts and endothelial cells. The two chemokines block the tail retraction and, thus, the migration within minutes, preventing and reverting growth factor-induced relocalization of m-calpain to the plasma membrane of the cells. PKA phosphorylation of m-calpain blocks the binding of the protease to PIP₂. Unexpectedly, we found that this was due to membrane anchorage itself and not merely serine 50 phosphorylation, as the farnesylation-induced anchorage of m-calpain triggers a strong activation of this protease, leading notably to an increased cell death. Moreover, the ERK and PKA phosphorylations have no effect on this membrane-anchored m-calpain. However, the presence of PIP₂ is still required for the activation of the anchored m-calpain. In conclusion, we describe a novel mechanism of m-calpain activation by interaction with the plasma membrane and PIP₂ specifically, this phosphoinositide acting as a cofactor for the enzyme. The phosphorylation of m-calpain by ERK and PKA by growth factors and chemokines, respectively, act in cells to regulate the enzyme only indirectly by controlling its redistribution.     

Synergistic Activation of p21-activated Kinase 1 by Phosphatidylinositol 4,5-Bisphosphate and Rho GTPases

Autoinhibited p21-activated kinase 1 (Pak1) can be activated in vitro by the plasma membrane-bound Rho GTPases Rac1 and Cdc42 as well as by the lipid phosphatidylinositol (4,5)-bisphosphate (PIP₂). Activator binding is mediated by a GTPase-binding motif and an adjacent phosphoinositide-binding motif. Whether these two classes of activators play alternative, additive, or synergistic roles in Pak1 activation is unknown, as is their contributions to Pak1 activation in vivo. To address these questions, we developed a system to mimic the membrane anchoring of Rho GTPases by creating liposomes containing both PIP₂ and a Ni²⁺-NTA modified lipid capable of binding hexahistidine-tagged Cdc42. We find that among all biologically relevant phosphoinositides, only PIP₂ is able to synergistically activate Pak1 in concert with Cdc42. Membrane binding of the kinase was highly sensitive to the spatial density of PIP₂ and Pak1 demonstrated dramatically enhanced affinity for Cdc42 anchored in a PIP₂ environment. To validate these findings in vivo, we utilized an inducible recruitment system to drive the ectopic synthesis of PIP₂ on Golgi membranes, which normally have active Cdc42 but lack significant concentrations of PIP₂. Pak1 was recruited to PIP₂-containing membranes in a manner dependent on the ability of Pak1 to bind to both PIP₂ and Cdc42. These findings provide a mechanistic explanation for the essential role of both phosphoinositides and GTPases in Pak1 recruitment and activation. In contrast, Ack, another Cdc42 effector kinase that lacks an analogous phosphoinositide-binding motif, fails to show the same enhancement of membrane binding and activation by PIP₂, thus indicating that regulation by PIP₂ and Cdc42 could provide a combinatorial code for activation of different GTPase effectors in different subcellular locations.     

RET PLCγ phosphotyrosine binding domain regulates Ca2+ signaling and neocortical neuronal migration

The receptor tyrosine kinase RET plays an essential role during embryogenesis in regulating cell proliferation, differentiation, and migration. Upon glial cell line-derived neurotrophic factor (GDNF) stimulation, RET can trigger multiple intracellular signaling pathways that in concert activate various downstream effectors. Here we report that the RET receptor induces calcium (Ca(2+)) signaling and regulates neocortical neuronal progenitor migration through the Phospholipase-C gamma (PLCγ) binding domain Tyr1015. This signaling cascade releases Ca(2+) from the endoplasmic reticulum through the inositol 1,4,5-trisphosphate receptor and stimulates phosphorylation of ERK1/2 and CaMKII. A point mutation at Tyr1015 on RET or small interfering RNA gene silencing of PLCγ block the GDNF-induced signaling cascade. Delivery of the RET mutation to neuronal progenitors in the embryonic ventricular zone using in utero electroporation reveal that Tyr1015 is necessary for GDNF-stimulated migration of neurons to the cortical plate. These findings demonstrate a novel RET mediated signaling pathway that elevates cytosolic Ca(2+) and modulates neuronal migration in the developing neocortex through the PLCγ binding domain Tyr1015.     

The Phosphatidylinositol (3,4,5)-Trisphosphate-dependent Rac Exchanger 1·Ras-related C3 Botulinum Toxin Substrate 1 (P-Rex1·Rac1) Complex Reveals the Basis of Rac1 Activation in Breast Cancer Cells

The P-Rex (phosphatidylinositol (3,4,5)-trisphosphate (PIP₃)-dependent Rac exchanger) family (P-Rex1 and P-Rex2) of the Rho guanine nucleotide exchange factors (Rho GEFs) activate Rac GTPases to regulate cell migration, invasion, and metastasis in several human cancers. The family is unique among Rho GEFs, as their activity is regulated by the synergistic binding of PIP₃ and Gβγ at the plasma membrane. However, the molecular mechanism of this family of multi-domain proteins remains unclear. We report the 1.95 Å crystal structure of the catalytic P-Rex1 DH-PH tandem domain in complex with its cognate GTPase, Rac1 (Ras-related C3 botulinum toxin substrate-1). Mutations in the P-Rex1·Rac1 interface revealed a critical role for this complex in signaling downstream of receptor tyrosine kinases and G protein-coupled receptors. The structural data indicated that the PIP₃/Gβγ binding sites are on the opposite surface and markedly removed from the Rac1 interface, supporting a model whereby P-Rex1 binding to PIP₃ and/or Gβγ releases inhibitory C-terminal domains to expose the Rac1 binding site.     

CAPS and Munc13 utilize distinct PIP2-linked mechanisms to promote vesicle exocytosis

Phosphoinositides provide compartment-specific signals for membrane trafficking. Plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP₂) is required for Ca²⁺-triggered vesicle exocytosis, but whether vesicles fuse into PIP₂-rich membrane domains in live cells and whether PIP₂ is metabolized during Ca²⁺-triggered fusion were unknown. Ca²⁺-dependent activator protein in secretion 1 (CAPS-1; CADPS/UNC31) and ubMunc13-2 (UNC13B) are PIP₂-binding proteins required for Ca²⁺-triggered vesicle exocytosis in neuroendocrine PC12 cells. These proteins are likely effectors for PIP₂, but their localization during exocytosis had not been determined. Using total internal reflection fluorescence microscopy in live cells, we identify PIP₂-rich membrane domains at sites of vesicle fusion. CAPS is found to reside on vesicles but depends on plasma membrane PIP₂ for its activity. Munc13 is cytoplasmic, but Ca²⁺-dependent translocation to PIP₂-rich plasma membrane domains is required for its activity. The results reveal that vesicle fusion into PIP₂-rich membrane domains is facilitated by sequential PIP₂-dependent activation of CAPS and PIP₂-dependent recruitment of Munc13. PIP₂ hydrolysis only occurs under strong Ca²⁺ influx conditions sufficient to activate phospholipase Cη2 (PLCη2). Such conditions reduce CAPS activity and enhance Munc13 activity, establishing PLCη2 as a Ca²⁺-dependent modulator of exocytosis. These studies provide a direct view of the spatial distribution of PIP₂ linked to vesicle exocytosis via regulation of lipid-dependent protein effectors CAPS and Munc13.     

β-Arrestin2 Regulates Lysophosphatidic Acid-Induced Human Breast Tumor Cell Migration and Invasion via Rap1 and IQGAP1

β-arrestins play critical roles in chemotaxis and cytoskeletal reorganization downstream of several receptor types, including G protein-coupled receptors (GPCRs), which are targets for greater than 50% of all pharmaceuticals. Among them, receptors for lysophosphatidic acid (LPA), namely LPA₁ are overexpressed in breast cancer and promote metastatic spread. We have recently reported that β-arrestin2 regulates LPA₁-mediated breast cancer cell migration and invasion, although the underlying molecular mechanisms are not clearly understood. We show here that LPA induces activity of the small G protein, Rap1 in breast cancer cells in a β-arrestin2-dependent manner, but fails to activate Rap1 in non-malignant mammary epithelial cells. We found that Rap1A mRNA levels are higher in human breast tumors compared to healthy patient samples and Rap1A is robustly expressed in human ductal carcinoma in situ and invasive tumors, in contrast to the normal mammary ducts. Rap1A protein expression is also higher in aggressive breast cancer cells (MDA-MB-231 and Hs578t) relative to the weakly invasive MCF-7 cells or non-malignant MCF10A mammary cells. Depletion of Rap1A expression significantly impaired LPA-stimulated migration of breast cancer cells and invasiveness in three-dimensional Matrigel cultures. Furthermore, we found that β-arrestin2 associates with the actin binding protein IQGAP1 in breast cancer cells, and is necessary for the recruitment of IQGAP1 to the leading edge of migratory cells. Depletion of IQGAP1 blocked LPA-stimulated breast cancer cell invasion. Finally, we have identified that LPA enhances the binding of endogenous Rap1A to β-arrestin2, and also stimulates Rap1A and IQGAP1 to associate with LPA₁. Thus our data establish novel roles for Rap1A and IQGAP1 as critical regulators of LPA-induced breast cancer cell migration and invasion.     

Homer3 regulates the establishment of neutrophil polarity

Most chemoattractants rely on activation of the heterotrimeric G-protein Gαi to regulate directional cell migration, but few links from Gαi to chemotactic effectors are known. Through affinity chromatography using primary neutrophil lysate, we identify Homer3 as a novel Gαi2-binding protein. RNA interference–mediated knockdown of Homer3 in neutrophil-like HL-60 cells impairs chemotaxis and the establishment of polarity of phosphatidylinositol 3,4,5-triphosphate (PIP₃) and the actin cytoskeleton, as well as the persistence of the WAVE2 complex. Most previously characterized proteins that are required for cell polarity are needed for actin assembly or activation of core chemotactic effectors such as the Rac GTPase. In contrast, Homer3-knockdown cells show normal magnitude and kinetics of chemoattractant-induced activation of phosphoinositide 3-kinase and Rac effectors. Chemoattractant-stimulated Homer3-knockdown cells also exhibit a normal initial magnitude of actin polymerization but fail to polarize actin assembly and intracellular PIP₃ and are defective in the initiation of cell polarity and motility. Our data suggest that Homer3 acts as a scaffold that spatially organizes actin assembly to support neutrophil polarity and motility downstream of GPCR activation.     

Open Conformation of Ezrin Bound to Phosphatidylinositol 4,5-Bisphosphate and to F-actin Revealed by Neutron Scattering

Ezrin is a member of the ezrin-radixin-moesin family (ERM) of adapter proteins that are localized at the interface between the cell membrane and the cortical actin cytoskeleton, and they regulate a variety of cellular functions. The structure representing a dormant and closed conformation of an ERM protein has previously been determined by x-ray crystallography. Here, using contrast variation small angle neutron scattering, we reveal the structural changes of the full-length ezrin upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP₂) and to F-actin. Ezrin binding to F-actin requires the simultaneous binding of ezrin to PIP₂. Once bound to F-actin, the opened ezrin forms more extensive contacts with F-actin than generally depicted, suggesting a possible role of ezrin in regulating the interfacial structure and dynamics between the cell membrane and the underlying actin cytoskeleton. In addition, using gel filtration, we find that the conformational opening of ezrin in response to PIP₂ binding is cooperative, but the cooperativity is disrupted by a phospho-mimic mutation S249D in the 4.1-ezrin/radixin/moesin (FERM) domain of ezrin. Using surface plasmon resonance, we show that the S249D mutation weakens the binding affinity and changes the kinetics of 4.1-ERM to PIP₂ binding. The study provides the first structural view of the activated ezrin bound to PIP₂ and to F-actin.     

PI(3,4,5)P3 potentiates phospholipase C-β activity

Phospholipase C-β (PLC-β) isozymes are key effectors in G protein-coupled signaling pathways. Previously, we showed that PLC-β1 and PLC-β3 bound immobilized PIP₃. In this study, PIP₃ was found to potentiate Ca²⁺-stimulated PLC-β activities using an in vitro reconstitution assay. LY294002, a specific PI 3-kinase inhibitor, significantly inhibited 10 min of agonist-stimulated total IP accumulation. Both LY294002 and wortmannin inhibited 90 sec of agonist-stimulated IP₃ accumulation in intact cells. Moreover, transfected p110CAAX, a constitutively activated PI 3-kinase catalytic subunit, increased 90 sec of oxytocin-stimulated IP₃ accumulation. Receptor-ligand binding assays indicated that LY294002 did not affect G protein-coupled receptors directly, suggesting a physiological role for PIP₃ in directly potentiating PLC-β activity. When coexpressed with p110CAAX, fluorescence-tagged PLC-β3 was increasingly localized to the plasma membrane. Additional observations suggest that the PH domain of PLC-β is not important for p110CAAX-induced membrane association.     

NuMA interacts with phosphoinositides and links the mitotic spindle with the plasma membrane

The positioning and the elongation of the mitotic spindle must be carefully regulated. In human cells, the evolutionary conserved proteins LGN/Gα_i1‐3 anchor the coiled‐coil protein NuMA and dynein to the cell cortex during metaphase, thus ensuring proper spindle positioning. The mechanisms governing cortical localization of NuMA and dynein during anaphase remain more elusive. Here, we report that LGN/Gα_i1‐3 are dispensable for NuMA‐dependent cortical dynein enrichment during anaphase. We further establish that NuMA is excluded from the equatorial region of the cell cortex in a manner that depends on the centralspindlin components CYK4 and MKLP1. Importantly, we reveal that NuMA can directly associate with PtdInsP (PIP) and PtdInsP₂ (PIP₂) phosphoinositides in vitro. Furthermore, chemical or enzymatic depletion of PIP/PIP₂ prevents NuMA cortical localization during mitosis, and conversely, increasing PIP₂ levels augments mitotic cortical NuMA. Overall, our study uncovers a novel function for plasma membrane phospholipids in governing cortical NuMA distribution and thus the proper execution of mitosis.     

Characterization of novel calmodulin binding domains within IQ motifs of IQGAP1

IQ motif-containing GTPase-activating protein 1 (IQGAP1), which is a well-known calmodulin (CaM) binding protein, is involved in a wide range of cellular processes including cell proliferation, tumorigenesis, adhesion, and migration. Interaction of IQGAP1 with CaM is important for its cellular functions. Although each IQ domain of IQGAP1 for CaM binding has been characterized in a Ca²⁺-dependent or -independent manner, it was not clear which IQ motifs are physiologically relevant for CaM binding in the cells. In this study, we performed immunoprecipitation using 3xFLAGhCaM in mammalian cell lines to characterize the domains of IQGAP1 that are key for CaM binding under physiological conditions. Interestingly, using this method, we identified two novel domains, IQ(2.7–3) and IQ(3.5–4.4), within IQGAP1 that were involved in Ca²⁺-independent or -dependent CaM binding, respectively. Mutant analysis clearly showed that the hydrophobic regions within IQ(2.7–3) were mainly involved in apoCaM binding, while the basic amino acids and hydrophobic region of IQ(3.5–4.4) were required for Ca²⁺/CaM binding. Finally, we showed that IQ(2.7–3) was the main apoCaM binding domain and both IQ(2.7–3) and IQ(3.5–4.4) were required for Ca²⁺/CaM binding within IQ(1-2-3-4). Thus, we identified and characterized novel direct CaM binding motifs essential for IQGAP1. This finding indicates that IQGAP1 plays a dynamic role via direct interactions with CaM in a Ca²⁺-dependent or -independent manner.     

Identification of ribosomal protein S3a as a candidate for a novel PI 3-kinase target in the nucleus

Phosphatidylinositol 3,4,5-trisphosphate (PIP₃) is an important lipid second messenger that mediates various cell responses. We have searched for the nuclear PIP₃ binding proteins using PIP₃ analogue beads. A 33 kD protein was detected in this method, which was identified as ribosomal protein S3a by the mass spectrometric analysis. The recombinant S3a protein bound specifically to PIP₃. S3a localized not only in the cytosol but also in the nucleus. Interestingly, not cytosolic but nuclear S3a bound to PIP₃, suggesting different roles of S3a in the cytosol and the nucleus. This revised version was published online in August 2006 with corrections to the Cover Date.     

Honokiol inhibits HepG2 migration via down‐regulation of IQGAP1 expression discovered by a quantitative pharmaceutical proteomic analysis

Honokiol (HNK), a natural small molecular product, inhibited proliferation of HepG2 cells and exhibited anti‐tumor activity in nude mice. In this article, we applied a novel sensitive stable isotope labeling with amino acids in cell culture‐based quantitative proteomic method and a model of nude mice to investigate the correlation between HNK and the hotspot migration molecule Ras GTPase‐activating‐like protein (IQGAP1). The quantitative proteomic analysis showed that IQGAP1 was 0.53‐fold down‐regulated under 10 μg/mL HNK exposure for 24 h on HepG2 cells. Migration ability of HepG2 cells under HNK treatment was correlated with its expression level of IQGAP1. In addition, the biochemical validation on HepG2 cells and the tumor xenograft model further demonstrated that HNK decreased the expression level of IQGAP1 and its upstream proteins Cdc42/Rac1. These data supported that HNK can modulate cell adhesion and cell migration by acting on Cdc42/Rac1 signaling via IQGAP1 interactions with its upstream Cdc42/Rac1 proteins, which is a new molecular mechanism of HNK to exert its anti‐tumor activity.     

Flexible pivoting of dynamin pleckstrin homology domain catalyzes fission: insights into molecular degrees of freedom

The neuronal dynamin1 functions in the release of synaptic vesicles by orchestrating the process of GTPase-dependent membrane fission. Dynamin1 associates with the plasma membrane–localized phosphatidylinositol-4,5-bisphosphate (PIP₂) through the centrally located pleckstrin homology domain (PHD). The PHD is dispensable as fission (in model membranes) can be managed, even when the PHD-PIP₂ interaction is replaced by a generic polyhistidine- or polylysine-lipid interaction. However, the absence of the PHD renders a dramatic dampening of the rate of fission. These observations suggest that the PHD-PIP₂–containing membrane interaction could have evolved to expedite fission to fulfill the requirement of rapid kinetics of synaptic vesicle recycling. Here, we use a suite of multiscale modeling approaches to explore PHD–membrane interactions. Our results reveal that 1) the binding of PHD to PIP₂-containing membranes modulates the lipids toward fission-favoring conformations and softens the membrane, and 2) PHD associates with membrane in multiple orientations using variable loops as pivots. We identify a new loop (VL4), which acts as an auxiliary pivot and modulates the orientation flexibility of PHD on the membrane—a mechanism that we believe may be important for high-fidelity dynamin collar assembly. Together, these insights provide a molecular-level understanding of the catalytic role of PHD in dynamin-mediated membrane fission.