AIDA-1/ANKS1B Binds to the SynGAP Family RasGAPs with High Affinity and Specificity.

AIDA-1, encoded by ANKS1B, is an abundant postsynaptic scaffold protein essential for brain development. Mutations of ANKS1B are closely associated with various psychiatric disorders. However, very little is known regarding the molecular mechanisms underlying AIDA-1's involvements under physiological and pathophysiological conditions. Here, we discovered an interaction between AIDA-1 and the SynGAP family Ras-GTPase activating protein (GAP) via affinity purification using AIDA-1d as the bait. Biochemical studies showed that the PTB domain of AIDA-1 binds to an extended NPx[F/Y]-motif of the SynGAP family proteins with high affinities. The high-resolution crystal structure of AIDA-1 PTB domain in complex with the SynGAP NPxF-motif revealed the molecular mechanism governing the specific interaction between AIDA-1 and SynGAP. Our study not only explains why patients with ANKS1B or SYNGAP1 mutations share overlapping clinical phenotypes, but also allows identification of new AIDA-1 binding targets such as Ras and Rab interactors.

Diverse p120RasGAP interactions with doubly phosphorylated partners EphB4, p190RhoGAP, and Dok1.

RasGAP (p120RasGAP), the founding member of the GTPase-activating protein (GAP) family, is one of only nine human proteins to contain two SH2 domains and is essential for proper vascular development. Despite its importance, its interactions with key binding partners remains unclear. In this study we provide a detailed viewpoint of RasGAP recruitment to various binding partners and assess their impact on RasGAP activity. We reveal the RasGAP SH2 domains generate distinct binding interactions with three well-known doubly phosphorylated binding partners: p190RhoGAP, Dok1, and EphB4. Affinity measurements demonstrate a 100-fold weakened affinity for RasGAP-EphB4 binding compared to RasGAP-p190RhoGAP or RasGAP-Dok1 binding, possibly driven by single versus dual SH2 domain engagement with a dominant N-terminal SH2 interaction. Small-angle X-ray scattering reveals conformational differences between RasGAP-EphB4 binding and RasGAP-p190RhoGAP binding. Importantly, these interactions do not impact catalytic activity, implying RasGAP utilizes its SH2 domains to achieve diverse spatial-temporal regulation of Ras signaling in a previously unrecognized fashion.

Elucidating microscopic events driven by GTP hydrolysis reaction in the Ras-GAP system with semi-reactive molecular dynamics simulations: the alternative role of a phosphate binding loop for mechanical energy storage.

ATPase and GTPase have been widely found as chemical energy-mechanical work transducers, whereas the physicochemical mechanisms are not satisfactorily understood. We addressed the problem by examining John Ross' conjecture that repulsive Coulomb interaction between ADP/GDP and inorganic phosphate (Pi) does the mechanical work upon the system. We effectively simulated the consequence of a GTP hydrolysis reaction in a complex system of Rat sarcoma (Ras) and GTPase activation protein (GAP) in the framework of classical molecular dynamics by switching force field parameters between the reactant and product systems. We then observed a ca. 5 kcal mol-1 increase of potential energy about the phosphate-binding loop (P-loop) in the Ras protein, indicating that the mechanical work generated via the GTP hydrolysis is converted into the local interaction energy and stored in the P-loop. Interestingly, this local energy storage in the P-loop depends on neither impulsive nor consecutive collisions of GDP and Pi with the P-loop. Instead, GTP-GDP conversion itself does work on the Ras system, elevating the potential energy. These observations encourage us to challenge a conjecture previously given by Ross. We assert that triphosphate nucleotide hydrolyses do mechanical work by producing emergent steric interaction accompanied by relaxation, namely, a shift of the biomolecular system to the non-equilibrium state on the reshaped potential energy landscape. Recalling the universality of the P-loop motif among GTPases and ATPases, the observations that we obtained through this study would progress the physicochemical understanding of the operating principles of GTP/ATP hydrolysis-driven biological nano-machines.

IQGAP1 Is a Scaffold of the Core Proteins of the Hippo Pathway and Negatively Regulates the Pro-Apoptotic Signal Mediated by This Pathway.

The Hippo pathway regulates a complex signalling network which mediates several biological functions including cell proliferation, organ size and apoptosis. Several scaffold proteins regulate the crosstalk of the members of the pathway with other signalling pathways and play an important role in the diverse output controlled by this pathway. In this study we have identified the scaffold protein IQGAP1 as a novel interactor of the core kinases of the Hippo pathway, MST2 and LATS1. Our results indicate that IQGAP1 scaffolds MST2 and LATS1 supresses their kinase activity and YAP1-dependent transcription. Additionally, we show that IQGAP1 is a negative regulator of the non-canonical pro-apoptotic pathway and may enable the crosstalk between this pathway and the ERK and AKT signalling modules. Our data also show that bile acids regulate the IQGAP1-MST2-LATS1 signalling module in hepatocellular carcinoma cells, which could be necessary for the inhibition of MST2-dependent apoptosis and hepatocyte transformation.

IQGAP1 causes choroidal neovascularization by sustaining VEGFR2-mediated Rac1 activation.

Loss of visual acuity in neovascular age-related macular degeneration (nAMD) occurs when factors activate choroidal endothelial cells (CECs) to transmigrate the retinal pigment epithelium into the sensory retina and develop into choroidal neovascularization (CNV). Active Rac1 (Rac1GTP) is required for CEC migration and is induced by different AMD-related stresses, including vascular endothelial growth factor (VEGF). Besides its role in pathologic events, Rac1 also plays a role in physiologic functions. Therefore, we were interested in a method to inhibit pathologic activation of Rac1. We addressed the hypothesis that IQGAP1, a scaffold protein with a Rac1 binding domain, regulates pathologic Rac1GTP in CEC migration and CNV. Compared to littermate Iqgap1+/+, Iqgap1-/- mice had reduced volumes of laser-induced CNV and decreased Rac1GTP and phosphorylated VEGFR2 (p-VEGFR2) within lectin-stained CNV. Knockdown of IQGAP1 in CECs significantly reduced VEGF-induced Rac1GTP, mediated through p-VEGFR2, which was necessary for CEC migration. Moreover, sustained activation of Rac1GTP induced by VEGF was eliminated when CECs were transfected with an IQGAP1 construct that is unable to bind Rac1. IQGAP1-mediated Src activation was involved in initiating Rac1 activation, CEC migration, and tube formation. Our findings indicate that CEC IQGAP1 interacts with VEGFR2 to mediate Src activation and subsequent Rac1 activation and CEC migration. In addition, IQGAP1 binding to Rac1GTP results in sustained activation of Rac1, leading to CEC migration toward VEGF. Our study supports a role of IQGAP1 and the interaction between IQGAP1 and Rac1GTP to restore CECs quiescence and, therefore, prevent vision-threatening CNV in nAMD.

Flightless anchors IQGAP1 and R-ras to mediate cell extension formation and matrix remodeling.

Tractional remodeling of collagen fibrils by fibroblasts requires long cell extensions that mediate fibril alignment. The formation of these cell extensions involves flightless I (FliI), an actin-binding protein that contains a leucine-rich-repeat (LRR), which binds R-ras and may regulate cdc42. We considered that FliI interacts with small GTPases and their regulators to mediate assembly of cell extensions. Mass spectrometry analyses of FliI immunoprecipitates showed abundant Ras GTPase-activating-like protein (IQGAP1), which in immunostained samples colocalized with FliI at cell adhesions. Knockdown of IQGAP1 reduced the numbers of cell extensions and the alignment of collagen fibrils. In experiments using dominant negative mutants, cdc42 activity was required for the formation of short extensions while R-ras was required for the formation of long extensions. Immunoprecipitation of wild-type and mutant constructs showed that IQGAP1 associated with cdc42 and R-ras; this association required the GAP-related domain (1004-1237 aa) of IQGAP1. In cells transfected with FliI mutants, the LRR of FliI, but not its gelsolin-like domains, mediated association with cdc42, R-ras, and IQGAP1. We conclude that FliI interacts with IQGAP1 and co-ordinates with cdc42 and R-ras to control the formation of cell extensions that enable collagen tractional remodeling.

Interaction between the scaffold proteins CBP by IQGAP1 provides an interface between gene expression and cytoskeletal activity.

Crosstalk between cellular pathways is often mediated through scaffold proteins that function as platforms for the assembly of signaling complexes. Based on yeast two-hybrid analysis, we report here the interaction between two complex scaffold proteins, CREB-binding protein (CBP) and the Ras GTPase-activating-like protein 1 (IQGAP1). Dissection of the interaction between the two proteins reveals that the central, thus far uncharacterized, region of IQGAP1 interacts with the HAT domain and the C-terminal intrinsically disordered region of CBP (termed ID5). Structural analysis of ID5 by solution NMR spectroscopy and SAXS reveals the presence of two regions with pronounced helical propensity. The ID5 region(s) involved in the interaction of nanomolar affinity were delineated by solution NMR titrations and pull-down assays. Moreover, we found that IQGAP1 acts as an inhibitor of the histone acetyltransferase (HAT) activity of CBP. In in vitro assays, the CBP-binding region of IQGAP1 positively and negatively regulates the function of HAT proteins of different families including CBP, KAT5 and PCAF. As many signaling pathways converge on CBP and IQGAP1, their interaction provides an interface between transcription regulation and the coordination of cytoskeleton. Disruption or alteration of the interaction between these scaffold proteins may lead to cancer development or metastatic processes, highlighting the importance of this interaction.

Ubiquitination of the scaffold protein IQGAP1 diminishes its interaction with and activation of the Rho GTPase CDC42.

IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a scaffold protein that interacts with numerous binding partners and thereby regulates fundamental biological processes. The functions of IQGAP1 are modulated by several mechanisms, including protein binding, self-association, subcellular localization, and phosphorylation. Proteome-wide screens have indicated that IQGAP1 is ubiquitinated, but the possible effects of this post-translational modification on its function are unknown. Here we characterized and evaluated the function of IQGAP1 ubiquitination. Using MS-based analysis in HEK293 cells, we identified six lysine residues (Lys-556, -1155, -1230, -1465, -1475, and -1528) as ubiquitination sites in IQGAP1. To elucidate the biological consequences of IQGAP1 ubiquitination, we converted each of these lysines to arginine and found that replacing two of these residues, Lys-1155 and Lys-1230, in the GAP-related domain of IQGAP1 (termed IQGAP1 GRD-2K) reduces its ubiquitination. Moreover, IQGAP1 GRD-2K bound a significantly greater proportion of the two Rho GTPases cell division cycle 42 (CDC42) and Rac family small GTPase 1 (RAC1) than did WT IQGAP1. Consistent with this observation, reconstitution of IQGAP1-null cells with IQGAP1 GRD-2K significantly increased the amount of active CDC42 and enhanced cell migration significantly more than WT IQGAP1. Our results reveal that ubiquitination of the CDC42 regulator IQGAP1 alters its ability to bind to and activate this GTPase, leading to physiological effects. Collectively, these findings expand our view of the role of ubiquitination in cell signaling and provide additional insight into CDC42 regulation.

Pressure Sensitivity of SynGAP/PSD-95 Condensates as a Model for Postsynaptic Densities and Its Biophysical and Neurological Ramifications.

Biomolecular condensates consisting of proteins and nucleic acids can serve critical biological functions, so that some condensates are referred as membraneless organelles. They can also be disease-causing, if their assembly is misregulated. A major physicochemical basis of the formation of biomolecular condensates is liquid-liquid phase separation (LLPS). In general, LLPS depends on environmental variables, such as temperature and hydrostatic pressure. The effects of pressure on the LLPS of a binary SynGAP/PSD-95 protein system mimicking postsynaptic densities, which are protein assemblies underneath the plasma membrane of excitatory synapses, were investigated. Quite unexpectedly, the model system LLPS is much more sensitive to pressure than the folded states of typical globular proteins. Phase-separated droplets of SynGAP/PSD-95 were found to dissolve into a homogeneous solution already at ten-to-hundred bar levels. The pressure sensitivity of SynGAP/PSD-95 is seen here as a consequence of both pressure-dependent multivalent interaction strength and void volume effects. Considering that organisms in the deep sea are under pressures up to about 1 kbar, this implies that deep-sea organisms have to devise means to counteract this high pressure sensitivity of biomolecular condensates to avoid harm. Intriguingly, these findings may shed light on the biophysical underpinning of pressure-related neurological disorders in terrestrial vertebrates.

Ca2+-Dependent Switch of Calmodulin Interaction Mode with Tandem IQ Motifs in the Scaffolding Protein IQGAP1.

IQ domain GTPase-activating scaffolding protein 1 (IQGAP1) mediates cytoskeleton, cell migration, proliferation, and apoptosis events. Calmodulin (CaM) modulates IQGAP1 functions by binding to its four tandem IQ motifs. Exactly how CaM binds the IQ motifs and which functions of IQGAP1 CaM regulates and how are fundamental mechanistic questions. We combine experimental pull-down assays, mutational data, and molecular dynamics simulations to understand the IQ-CaM complexes with and without Ca2+ at the atomic level. Apo-CaM favors the IQ3 and IQ4 motifs but not the IQ1 and IQ2 motifs that lack two hydrophobic residues for interactions with apo-CaM's hydrophobic pocket. Ca2+-CaM binds all four IQ motifs, with both N- and C-lobes tightly wrapped around each motif. Ca2+ promotes IQ-CaM interactions and increases the amount of IQGAP1-loaded CaM for IQGAP1-mediated signaling. Collectively, we describe IQ-CaM binding in atomistic detail and feature the emergence of Ca2+ as a key modulator of the CaM-IQGAP1 interactions.

The Specificity of EGF-Stimulated IQGAP1 Scaffold Towards the PI3K-Akt Pathway is Defined by the IQ3 motif.

Epidermal growth factor receptor (EGFR) and its downstream phosphoinositide 3-kinase (PI3K) pathway are commonly deregulated in cancer. Recently, we have shown that the IQ motif-containing GTPase-activating protein 1 (IQGAP1) provides a molecular platform to scaffold all the components of the PI3K-Akt pathway and results in the sequential generation of phosphatidylinositol-3,4,5-trisphosphate (PI3,4,5P3). In addition to the PI3K-Akt pathway, IQGAP1 also scaffolds the Ras-ERK pathway. To define the specificity of IQGAP1 for the control of PI3K signaling, we have focused on the IQ3 motif in IQGAP1 as PIPKIα and PI3K enzymes bind this region. An IQ3 deletion mutant loses interactions with the PI3K-Akt components but retains binding to ERK and EGFR. Consistently, blocking the IQ3 motif of IQGAP1 using an IQ3 motif-derived peptide mirrors the effect of IQ3 deletion mutant by reducing Akt activation but has no impact on ERK activation. Also, the peptide disrupts the binding of IQGAP1 with PI3K-Akt pathway components, while IQGAP1 interactions with ERK and EGFR are not affected. Functionally, deleting or blocking the IQ3 motif inhibits cell proliferation, invasion, and migration in a non-additive manner to a PIPKIα inhibitor, establishing the functional specificity of IQ3 motif towards the PI3K-Akt pathway. Taken together, the IQ3 motif is a specific target for suppressing activation of the PI3K-Akt but not the Ras-ERK pathway. Although EGFR stimulates the IQGAP1-PI3K and -ERK pathways, here we show that IQGAP1-PI3K controls migration, invasion, and proliferation independent of ERK. These data illustrate that the IQ3 region of IQGAP1 is a promising therapeutic target for PI3K-driven cancer.

Diversity of mechanisms in Ras-GAP catalysis of guanosine triphosphate hydrolysis revealed by molecular modeling.

The mechanism of the deceptively simple reaction of guanosine triphosphate (GTP) hydrolysis catalyzed by the cellular protein Ras in complex with the activating protein GAP is an important issue because of the significance of this reaction in cancer research. We show that molecular modeling of GTP hydrolysis in the Ras-GAP active site reveals a diversity of mechanisms of the intrinsic chemical reaction depending on molecular groups at position 61 in Ras occupied by glutamine in the wild-type enzyme. First, a comparison of reaction energy profiles computed at the quantum mechanics/molecular mechanics (QM/MM) level shows that an assignment of the Gln61 side chain in the wild-type Ras either to QM or to MM parts leads to different scenarios corresponding to the glutamine-assisted or the substrate-assisted mechanisms. Second, replacement of Gln61 by the nitro-analog of glutamine (NGln) or by Glu, applied in experimental studies, results in two more scenarios featuring the so-called two-water and the concerted-type mechanisms. The glutamine-assisted mechanism in the wild-type Ras-GAP, in which the conserved Gln61 plays a decisive role, switching between the amide and imide tautomer forms, is consistent with the known experimental results of structural, kinetic and spectroscopy studies. The results emphasize the role of the Ras residue Gln61 in Ras-GAP catalysis and explain the retained catalytic activity of the Ras-GAP complex towards GTP hydrolysis in the Gln61NGln and Gln61Glu mutants of Ras.

Ras-Specific GTPase-Activating Proteins-Structures, Mechanisms, and Interactions.

Ras-specific GTPase-activating proteins (RasGAPs) down-regulate the biological activity of Ras proteins by accelerating their intrinsic rate of GTP hydrolysis, basically by a transition state stabilizing mechanism. Oncogenic Ras is commonly not sensitive to RasGAPs caused by interference of mutants with the electronic or steric requirements of the transition state, resulting in up-regulation of activated Ras in respective cells. RasGAPs are modular proteins containing a helical catalytic RasGAP module surrounded by smaller domains that are frequently involved in the subcellular localization or contributing to regulatory features of their host proteins. In this review, we summarize current knowledge about RasGAP structure, mechanism, regulation, and dual-substrate specificity and discuss in some detail neurofibromin, one of the most important negative Ras regulators in cellular growth control and neuronal function.

Amide-imide tautomerization in the glutamine side chain in enzymatic and photochemical reactions in proteins.

Amide-imide tautomerization presents a pervasive class of chemical transformations in organic chemistry of natural compounds. In this Perspective, we describe two distinctively different protein systems, in which the amide-imide tautomerization in the glutamine side chain takes place in enzymatic or photochemical reactions. First, hydrolysis of guanosine triphosphate (GTP) catalyzed by the Ras-GAP protein complex suggests the occurrence of the imide tautomer of glutamine in reaction intermediates. Second, photoexcitation of flavin-binding protein domains (BLUFs) initiates a chain of reactions in the chromophore-binding pocket, including amide-imide tautomerization of glutamine. Mechanisms of these reactions at the atomic level have been revealed in quantum mechanics/molecular mechanics (QM/MM) simulations. To reinforce conclusions on the critical role of amide-imide tautomerization of glutamine in these reactions we describe results of new quantum chemistry and QM/MM calculations for relevant molecular model systems. We reexamine results of the recent IR spectroscopy studies of BLUF domains, which provide experimental evidences of Gln tautomerization in proteins. We also propose to validate the glutamine-assisted mechanism of enzymatic GTP hydrolysis by using IR spectroscopy in a proper range of wavenumbers.

The cytoskeleton regulates symmetry transitions in moving amoeboid cells.

Symmetry and symmetry breaking are essential in biology. Symmetry comes in different forms: rotational symmetry, mirror symmetry and alternating right-left symmetry (for example, gliding reflection symmetry). Especially the transitions between the different symmetry forms are important because they specify crucial points in cell biology, including gastrulation in development, formation of the cleavage furrow in cell division, or the front in cell polarity. However, the mechanisms of these symmetry transitions are not well understood. Here, we have investigated the fundamental properties of symmetry and symmetry transitions of the cytoskeleton during cell movement. Our data show that the dynamic shape changes of amoeboid cells are far from random, but are the consequence of refined symmetries and symmetry changes that are orchestrated by small G-proteins and the cytoskeleton, with local stimulation by F-actin and Scar, and local inhibition by IQGAP2 and myosin.

Species-conserved SYNGAP1 phenotypes associated with neurodevelopmental disorders.

SYNGAP1 loss-of-function variants are causally associated with intellectual disability, severe epilepsy, autism spectrum disorder and schizophrenia. While there are hundreds of genetic risk factors for neurodevelopmental disorders (NDDs), this gene is somewhat unique because of the frequency and penetrance of loss-of-function variants found in patients combined with the range of brain disorders associated with SYNGAP1 pathogenicity. These clinical findings indicate that SYNGAP1 regulates fundamental neurodevelopmental processes that are necessary for brain development. Here, we describe four phenotypic domains that are controlled by Syngap1 expression across vertebrate species. Two domains, the maturation of cognitive functions and maintenance of excitatory-inhibitory balance, are defined exclusively through a review of the current literature. Two additional domains are defined by integrating the current literature with new data indicating that SYNGAP1/Syngap1 regulates innate survival behaviors and brain structure. These four phenotypic domains are commonly disrupted in NDDs, suggesting that a deeper understanding of developmental Syngap1 functions will be generalizable to other NDDs of known or unknown etiology. Therefore, we discuss the known molecular and cellular functions of Syngap1 and consider how these functions may contribute to the emergence of disease-relevant phenotypes. Finally, we identify major unexplored areas of Syngap1 neurobiology and discuss how a deeper understanding of this gene may uncover general principles of NDD pathobiology.

Unraveling the molecular mechanism of interactions of the Rho GTPases Cdc42 and Rac1 with the scaffolding protein IQGAP2.

IQ motif-containing GTPase-activating proteins (IQGAPs) are scaffolding proteins playing central roles in cell-cell adhesion, polarity, and motility. The Rho GTPases Cdc42 and Rac1, in their GTP-bound active forms, interact with all three human IQGAPs. The IQGAP-Cdc42 interaction promotes metastasis by enhancing actin polymerization. However, despite their high sequence identity, Cdc42 and Rac1 differ in their interactions with IQGAP. Two Cdc42 molecules can bind to the Ex-domain and the RasGAP site of the GTPase-activating protein (GAP)-related domain (GRD) of IQGAP and promote IQGAP dimerization. Only one Rac1 molecule might bind to the RasGAP site of GRD and may not facilitate the dimerization, and the exact mechanism of Cdc42 and Rac1 binding to IQGAP is unclear. Using all-atom molecular dynamics simulations, site-directed mutagenesis, and Western blotting, we unraveled the detailed mechanisms of Cdc42 and Rac1 interactions with IQGAP2. We observed that Cdc42 binding to the Ex-domain of GRD of IQGAP2 (GRD2) releases the Ex-domain at the C-terminal region of GRD2, facilitating IQGAP2 dimerization. Cdc42 binding to the Ex-domain promoted allosteric changes in the RasGAP site, providing a binding site for the second Cdc42 in the RasGAP site. Of note, the Cdc42 "insert loop" was important for the interaction of the first Cdc42 with the Ex-domain. By contrast, differences in Rac1 insert-loop sequence and structure precluded its interaction with the Ex-domain. Rac1 could bind only to the RasGAP site of apo-GRD2 and could not facilitate IQGAP2 dimerization. Our detailed mechanistic insights help decipher how Cdc42 can stimulate actin polymerization in metastasis.

Valosin-containing protein (VCP) is a novel IQ motif-containing GTPase activating protein 1 (IQGAP1)-interacting protein.

Scaffold proteins play a pivotal role in making protein complexes, and organize binding partners into a functional unit to enhance specific signaling pathways. IQ motif-containing GTPase activating protein 1 (IQGAP1) is an essential protein for spine formation due to its role in scaffolding multiple signal complexes. However, it remains unclear how IQGAP1 interacts within the brain. In the present study, we screened novel IQGAP1-interacting proteins by a proteomic approach. As a novel IQGAP1-interacting protein, we identified valosin-containing protein (VCP) which is a causative gene in patients with inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD). The physiological interaction of IQGAP1 with VCP was confirmed by an immunoprecipitation assay. Both the N-terminal (N-half) and C-terminal (C-half) fragments of IQGAP1 interacted with the N-terminal region of VCP. Co-localization of IQGAP1 and VCP was observed in the growth corn, axonal shaft, cell body, and dendrites in cultured hippocampal neurons at 4 days in vitro (DIV4). In cultured neurons at DIV14, IQGAP1 co-localized with VCP in dendrites. When HEK293T cells were co-transfected with IQGAP1 and VCP, an immunoprecipitation assay revealed that binding of IQGAP1 with disease-related mutant (R155H or A232E) VCP was markedly reduced compared to wild-type (WT) VCP. These results suggest that reduction of IQGAP1 and VCP interaction may be associated with the pathophysiology of IBMPFD.

Unveiling a VEGF-mimetic peptide sequence in the IQGAP1 protein.

The ability to modulate angiogenesis by chemical tools has several important applications in different scientific fields. With the perspective of finding novel proangiogenic molecules, we searched peptide sequences with a chemical profile similar to that of the QK peptide, a well described VEGF mimetic peptide. We found that residues 1617-1627 of the IQGAP1 protein show molecular features similar to those of the QK peptide sequence. The IQGAP1-derived synthetic peptide was analyzed by NMR spectroscopy and its biological activity was characterized in endothelial cells. These studies showed that this IQGAP1-derived peptide has a biological activity similar to that of VEGF and could be considered as a novel tool for reparative angiogenesis.

The WW domain of the scaffolding protein IQGAP1 is neither necessary nor sufficient for binding to the MAPKs ERK1 and ERK2.

Mitogen-activated protein kinase (MAPK) scaffold proteins, such as IQ motif containing GTPase activating protein 1 (IQGAP1), are promising targets for novel therapies against cancer and other diseases. Such approaches require accurate information about which domains on the scaffold protein bind to the kinases in the MAPK cascade. Results from previous studies have suggested that the WW domain of IQGAP1 binds to the cancer-associated MAPKs ERK1 and ERK2, and that this domain might thus offer a new tool to selectively inhibit MAPK activation in cancer cells. The goal of this work was therefore to critically evaluate which IQGAP1 domains bind to ERK1/2. Here, using quantitative in vitro binding assays, we show that the IQ domain of IQGAP1 is both necessary and sufficient for binding to ERK1 and ERK2, as well as to the MAPK kinases MEK1 and MEK2. Furthermore, we show that the WW domain is not required for ERK-IQGAP1 binding, and contributes little or no binding energy to this interaction, challenging previous models of how WW-based peptides might inhibit tumorigenesis. Finally, we show that the ERK2-IQGAP1 interaction does not require ERK2 phosphorylation or catalytic activity and does not involve known docking recruitment sites on ERK2, and we obtain an estimate of the dissociation constant (Kd ) for this interaction of 8 µm These results prompt a re-evaluation of published findings and a refined model of IQGAP scaffolding.