Masaki Fukata

Rac1 and Cdc42 Capture Microtubules through IQGAP1 and CLIP-170

Linkage of microtubules to special cortical regions is essential for cell polarization. CLIP-170 binds to the growing ends of microtubules and plays pivotal roles in orientation. We have found that IQGAP1, an effector of Rac1 and Cdc42, interacts with CLIP-170. In Vero fibroblasts, IQGAP1 localizes at the polarized leading edge. Expression of carboxy-terminal fragment of IQGAP1, which includes the CLIP-170 binding region, delocalizes GFP-CLIP-170 from the tips of microtubules and alters the microtubule array. Activated Rac1/Cdc42, IQGAP1, and CLIP-170 form a tripartite complex. Furthermore, expression of an IQGAP1 mutant defective in Rac1/Cdc42 binding induces multiple leading edges. These results indicate that Rac1/Cdc42 marks special cortical spots where the IQGAP1 and CLIP-170 complex is targeted, leading to a polarized microtubule array and cell polarization.

Roles of Rho-family GTPases in cell polarisation and directional migration.

Polarised cell migration is a tightly regulated process that occurs in tissue development, chemotaxis and wound healing. Rho-family GTPases, including Cdc42, Rac1 and RhoA, play a central role in establishing cell polarisation, which requires asymmetric and ordered distribution of the signalling molecules and the cytoskeleton. Recent advances reveal that Rho GTPases, together with phosphatidylinositol 3-kinase, contribute to asymmetric phosphatidylinositol 3,4,5-trisphosphate distribution via a positive-feedback loop. Phosphatidylinositol 3,4,5-trisphosphate thereby activates the signalling cascades to the cytoskeleton as a second messenger. Rho GTPases also capture and stabilise microtubules through their effectors (e.g. IQGAP1, mDia and Par6) near the cell cortex, leading to polarised cell morphology and directional cell migration. Thus, elucidation of the signal transduction cascades from receptors to Rho GTPases and, subsequently, from Rho GTPases to microtubules has begun.

Rho-family GTPases in cadherin-mediated cell — cell adhesion

Cell?cell adhesions are rearranged dynamically during tissue development and tumour metastasis. Recently, Rho-family GTPases, including RhoA, Rac1 and Cdc42, have emerged as key regulators of cadherin-mediated cell?cell adhesion. Following the identification and characterization of regulators and effectors of Rho GTPases, signal transduction pathways from cadherin to Rho GTPases and, in turn, from Rho GTPases to cadherin, are beginning to be clarified.

Identification of PSD-95 Palmitoylating Enzymes

Palmitoylation is a lipid modification that plays a critical role in protein trafficking and function throughout the nervous system. Palmitoylation of PSD-95 is essential for its regulation of AMPA receptors and synaptic plasticity. The enzymes that mediate palmitoyl acyl transfer to PSD-95 have not yet been identified; however, proteins containing a DHHC cysteine-rich domain mediate palmitoyl acyl transferase activity in yeast. Here, we isolated 23 mammalian DHHC proteins and found that a subset specifically palmitoylated PSD-95 in vitro and in vivo. These PSD-95 palmitoyl transferases (P-PATs) showed substrate specificity, as they did not all enhance palmitoylation of Lck, SNAP-25b, Galpha(s), or H-Ras in cultured cells. Inhibition of P-PAT activity in neurons reduced palmitoylation and synaptic clustering of PSD-95 and diminished AMPA receptor-mediated neurotransmission. This study suggests that P-PATs regulate synaptic function through PSD-95 palmitoylation.

Protein palmitoylation in neuronal development and synaptic plasticity

Protein palmitoylation, a classical and common lipid modification, regulates diverse aspects of neuronal protein trafficking and function. The reversible nature of palmitoylation provides a potential general mechanism for protein shuttling between intracellular compartments. The recent discovery of palmitoylating enzymes — a large DHHC (Asp-His-His-Cys) protein family — and the development of new proteomic and imaging methods have accelerated palmitoylation analysis. It is becoming clear that individual DHHC enzymes generate and maintain the specialized compartmentalization of substrates in polarized neurons. Here, we discuss the regulatory mechanisms for dynamic protein palmitoylation and the emerging roles of protein palmitoylation in various aspects of pathophysiology, including neuronal development and synaptic plasticity.

Epilepsy-Related Ligand/Receptor Complex LGI1 and ADAM22 Regulate Synaptic Transmission

Abnormally synchronized synaptic transmission in the brain causes epilepsy. Most inherited forms of epilepsy result from mutations in ion channels. However, one form of epilepsy, autosomal dominant partial epilepsy with auditory features (ADPEAF), is characterized by mutations in a secreted neuronal protein, LGI1. We show that ADAM22, a transmembrane protein that when mutated itself causes seizure, serves as a receptor for LGI1. LGI1 enhances AMPA receptor-mediated synaptic transmission in hippocampal slices. The mutated form of LGI1 fails to bind to ADAM22. ADAM22 is anchored to the postsynaptic density by cytoskeletal scaffolds containing stargazin. These studies in rat brain indicate possible avenues for understanding human epilepsy.

Cdc42 and Rac1 Regulate the Interaction of IQGAP1 with β-Catenin

IQGAP1, a target of Cdc42 and Rac1 small GTPases, directly interacts with β-catenin and negatively regulates E-cadherin-mediated cell-cell adhesion by dissociating α-catenin from the cadherin-catenin complex in vivo (Kuroda, S., Fukata, M., Nakagawa, M., Fujii, K., Nakamura, T., Ookubo, T., Izawa, I., Nagase, T., Nomura, N., Tani, H., Shoji, I., Matsuura, Y., Yonehara, S., and Kaibuchi, K. (1998) Science 281, 832–835). Here we investigated how Cdc42 and Rac1 regulate the IQGAP1 function. IQGAP1 interacted with the amino-terminal region (amino acids 1–183) of β-catenin, which contains the α-catenin-binding domain. IQGAP1 dissociated α-catenin from the β-catenin-α-catenin complex in a dose-dependent manner in vitro. Guanosine 5′-(3-O-thio)triphosphate (GTPγS)·glutathioneS-transferase (GST)-Cdc42 and GTPγS·GST-Rac1 inhibited the binding of IQGAP1 to β-catenin in a dose-dependent manner in vitro, whereas neither GDP·GST-Cdc42, GDP·GST-Rac1, nor GTPγS·GST-RhoA did. The coexpression of dominant active Cdc42 with IQGAP1 suppressed the dissociation of α-catenin from the cadherin-catenin complex induced by the overexpression of IQGAP1 in L cells expressing E-cadherin (EL cells). Consistent with this, the overexpression of either dominant negative Cdc42 or Rac1 resulted in the reduction of E-cadherin-mediated cell adhesive activity in EL cells. These results indicate that Cdc42 and Rac1 negatively regulate the IQGAP1 function by inhibiting the interaction of IQGAP1 with β-catenin, leading to stabilization of the cadherin-catenin complex.

p140Sra-1 (Specifically Rac1-associated Protein) Is a Novel Specific Target for Rac1 Small GTPase

Rac1 small GTPase plays pivotal roles in various cell functions such as cell morphology, cell polarity, and cell proliferation. We have previously identified IQGAP1 from bovine brain cytosol as a target for Rac1 by an affinity purification method. By using the same method, we purified a specifically Rac1-associated protein with a molecular mass of about 140 kDa (p140) from bovine brain cytosol. This protein interacted with guanosine 5′-(3-O-thio)triphosphate (GTPγS)·glutathioneS-transferase (GST)-Rac1 but not with the GDP·GST-Rac1, GTPγS·GST-Cdc42, or GTPγS·GST-RhoA. The amino acid sequences of this protein revealed that p140 is identified as a product of KIAA0068 gene. We denoted this protein as Sra-1 (SpecificallyRac1-associated protein). Recombinant Sra-1 interacted with GTPγS·GST-Rac1 and weakly with GDP·Rac1 but not with GST-Cdc42 or GST-RhoA. The N-terminal domain of Sra-1 (1–407 amino acids) was responsible for the interaction with Rac1. Myc-tagged Sra-1 and the deletion mutant capable of interacting with Rac1, but not the mutants unable to bind Rac1, were colocalized with dominant active Rac1Val-12 and cortical actin filament at the Rac1Val-12-induced membrane ruffling area in KB cells. Sra-1 was cosedimented with filamentous actin (F-actin), indicating that Sra-1 directly interacts with F-actin. These results suggest that Sra-1 is a novel and specific target for Rac1.

Regulation of cadherin-mediated cell-cell adhesion by the Rho family GTPases

Reports in the past two years have shown that Cdc42, Rac1, and Rho - belonging to the Rho small GTPase family - participate in the regulation of cadherin-mediated cell-cell adhesion. IQGAP1, an effector of Cdc42 and Rac1, interacts with cadherin and beta-catenin and induces the dissociation of alpha-catenin from the cadherin-catenins complex leading to disruption of cell-cell adhesion: activated Cdc42 and Rac1 counteract the effect of IQGAP1. Thus, Cdc42 and Rac1 appear to regulate cadherin-mediated cell-cell adhesion acting through IQGAP1.

Disruption of LGI1–linked synaptic complex causes abnormal synaptic transmission and epilepsy

Epilepsy is a devastating and poorly understood disease. Mutations in a secreted neuronal protein, leucine-rich glioma inactivated 1 (LGI1), were reported in patients with an inherited form of human epilepsy, autosomal dominant partial epilepsy with auditory features (ADPEAF). Here, we report an essential role of LGI1 as an antiepileptogenic ligand. We find that loss of LGI1 in mice (LGI1−/−) causes lethal epilepsy, which is specifically rescued by the neuronal expression of LGI1 transgene, but not LGI3. Moreover, heterozygous mice for the LGI1 mutation (LGI1+/−) show lowered seizure thresholds. Extracellularly secreted LGI1 links two epilepsy-related receptors, ADAM22 and ADAM23, in the brain and organizes a transsynaptic protein complex that includes presynaptic potassium channels and postsynaptic AMPA receptor scaffolds. A lack of LGI1 disrupts this synaptic protein connection and selectively reduces AMPA receptor–mediated synaptic transmission in the hippocampus. Thus, LGI1 may serve as a major determinant of brain excitation, and the LGI1 gene-targeted mouse provides a good model for human epilepsy.