Delineation of the Cdc42/Rac-Binding Domain of p21-Activated Kinase

Thompson, Gladstone; Owen, Darerca; Chalk, Peter A.; Lowe, Peter N.

doi:10.1021/bi980140+

Cited by 138 publications

(166 citation statements)

References 28 publications

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Section: Discussionmentioning

confidence: 99%

“…This domain is necessary but not sufficient for a high affinity binding (47,48). The smallest fragment of PAK that binds small G proteins with high affinity consists of residues Ile 70 -Lys 173 (47). Some residues, (PAK1 Met 99 , homologous to the PAK3 Ile 94 , and also the conserved residues Trp 98 , Leu 101 , and Leu 102 ), located in the proximity of the insertion, are involved in interaction with the GTPase (41).…”

Section: Discussionmentioning

confidence: 99%

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A New Constitutively Active Brain PAK3 Isoform Displays Modified Specificities toward Rac and Cdc42 GTPases

Rousseau¹,

Goupille²,

Morin³

et al. 2003

Journal of Biological Chemistry

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p21-activated kinases (PAK) are involved in the control of cytoskeleton dynamics and cell cycle progression. Here we report the characterization of a new mammalian PAK3 mRNA that contains a 45-bp alternatively spliced exon. This exon encodes for 15 amino acids that are inserted in the regulatory domain, inside the autoinhibitory domain but outside the Cdc42 and Rac interactive binding domain. The transcript of the 68-kDa new isoform named PAK3b is expressed in various areas of the adult mouse brain. In contrast to PAK3 without the exon b (PAK3a), whose basal kinase activity is weak in resting cells, PAK3b displays a high kinase activity in starved cells that is not further stimulated by active GTPases. Indeed, we demonstrate that the autoinhibitory domain of PAK3b no longer inhibits the kinase activity of PAK3. Moreover, we show that the 15-amino acid insertion within the autoinhibitory domain impedes the ability of PAK3b to bind to the GTPases Rac and Cdc42 and changes its specificity toward the GTPases. Altogether, our results show that the new PAK3b isoform has unique properties and would signal differently from PAK3a in neurons.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A New Constitutively Active Brain PAK3 Isoform Displays Modified Specificities toward Rac and Cdc42 GTPases

Rousseau¹,

Goupille²,

Morin³

et al. 2003

Journal of Biological Chemistry

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“…2B) indicated a slow interaction process, with heat liberated after each injection taking Ͼ30 min to reach baseline. This indicates either slow interconversion of Y516F to a conformation competent to bind GTP, or a rapid, low-affinity interaction with GTP followed by a slow conformational change to high-affinity binding (20).…”

Section: Gtp Stabilizes a Compact Conformation Of Tg2mentioning

confidence: 99%

Mechanism of allosteric regulation of transglutaminase 2 by GTP

Begg

Carrington

Stokes

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

138

145

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Allosteric regulation is a fundamental mechanism of biological control. Here, we investigated the allosteric mechanism by which GTP inhibits cross-linking activity of transglutaminase 2 (TG2), a multifunctional protein, with postulated roles in receptor signaling, extracellular matrix assembly, and apoptosis. Our findings indicate that at least two components are involved in functionally coupling the allosteric site and active center of TG2, namely (i) GTP binding to mask a conformationally destabilizing switch residue, Arg-579, and to facilitate interdomain interactions that promote adoption of a compact, catalytically inactive conformation and (ii) stabilization of the inactive conformation by an uncommon H bond between a cysteine (Cys-277, an active center residue) and a tyrosine (Tyr-516, a residue located on a loop of the ␤-barrel 1 domain that harbors the GTP-binding site). Although not essential for GTP-mediated inhibition of cross-linking, this H bond enhances the rate of formation of the inactive conformer.protein conformation ͉ GTP inhibition ͉ transamidase activity A llosteric regulation of enzymes by conformational change is an important means of biological control, involving residues that functionally couple ligand binding at the allosteric site to modification of the catalytic site. Transglutaminase type 2 (TG2), also known as tissue TG or G h (high molecular weight GTP-binding protein), is a multifunctional protein that is allosterically regulated by calcium and GTP (1). TG2 catalyzes calcium-dependent transamidation reactions, resulting in posttranslational amine modification of proteins or cross-linking of interchain glutamine and lysine residues to form N (␥-glutamyl)lysine isopeptide bonds, which confer rigidity and protease resistance on protein complexes (2). TG2 is also a GTPase (3) and mediates intracellular signaling by various G protein-coupled receptors (4-6).GDP-bound human TG2 (7) is comprised of four domains: an N-terminal ␤-sandwich, a core domain in which the transamidase active site catalytic triad (Cys-277, His-335, and Asp-358) and transition-state stabilizing residue (Trp-241) (8) are buried and inaccessible to substrate, and two ␤-barrels. Nucleotide binds mainly to residues from the first and last strands (amino acids 476-482 and 580-583) of ␤-barrel 1 and to two core domain residues (Lys-173 and Phe-174) that protrude on a loop to meet ␤-barrel 1 (7, 9, 10). This is postulated to stabilize two ␤-barrel 1 loops that block access to the catalytic site (7). One of these loops protrudes into the core domain localizing Tyr-516 within hydrogen-bonding distance of Cys-277 (7). This is postulated to prevent Cys-277 interaction with the substrate (7,11,12). Calcium-activated TG2 has unique conformational epitopes (13) and is less compact (14-16) and less resistant to protease digestion (1, 10, 14) than GTP-bound TG2. Allosteric mechanisms governing the conformational switch between transamidase and GTPase functions have yet to be elucidated.Mutation of Arg-579 in rat TG2 (Arg-580 in ...

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“…Hall (Memorial Sloan-Kettering Cancer Center). The Pak CRIB-encompassing domain (amino acids 70 -118) fused to glutathione S-transferase (GST) was from Dr. Lowe (GlaxoSmithKline, United Kingdom) (Thompson et al, 1998). GFP-tagged and GST-tagged ELMO plasmids were provided by Dr. K. Ravichandran (University of Virginia, Charlottesville, VA).…”

Section: Plasmidsmentioning

confidence: 99%

Interaction of Ezrin with the Novel Guanine Nucleotide Exchange Factor PLEKHG6 Promotes RhoG-dependent Apical Cytoskeleton Rearrangements in Epithelial Cells

D'Angelo

Aresta

Blangy

et al. 2007

MBoC

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The mechanisms underlying functional interactions between ERM (ezrin, radixin, moesin) proteins and Rho GTPases are not well understood. Here we characterized the interaction between ezrin and a novel Rho guanine nucleotide exchange factor, PLEKHG6. We show that ezrin recruits PLEKHG6 to the apical pole of epithelial cells where PLEKHG6 induces the formation of microvilli and membrane ruffles. These morphological changes are inhibited by dominant negative forms of RhoG. Indeed, we found that PLEKHG6 activates RhoG and to a much lesser extent Rac1. In addition we show that ezrin forms a complex with PLEKHG6 and RhoG. Furthermore, we detected a ternary complex between ezrin, PLEKHG6, and the RhoG effector ELMO. We demonstrate that PLEKHG6 and ezrin are both required in macropinocytosis. After down-regulation of either PLEKHG6 or ezrin expression, we observed an inhibition of dextran uptake in EGF-stimulated A431 cells. Altogether, our data indicate that ezrin allows the local activation of RhoG at the apical pole of epithelial cells by recruiting upstream and downstream regulators of RhoG and that both PLEKHG6 and ezrin are required for efficient macropinocytosis. INTRODUCTIONThe membrane-cytoskeleton linker ezrin is mainly expressed in epithelial cells where it associates to the apical actin-rich structures such as microvilli (Berryman et al., 1993(Berryman et al., , 1995. Recent genetic analyses revealed that ezrin is essential for the morphogenesis of epithelial cells (Fiévet et al., 2007). In ezrin Ϫ/Ϫ mice, morphological defects in the apical domain of intestinal and retinal pigment epithelial cells have been observed (Saotome et al., 2004;Bonilha et al., 2006). In parietal cells, ezrin knockdown impairs the formation of canalicular apical membrane, resulting in severe achlorhydria (Tamura et al., 2005).How ezrin participates in the assembly of the apical actinrich structures such as microvilli is still poorly understood. The association of ezrin and the highly related proteins radixin and moesin with the cortical actin cytoskeleton is strictly regulated. ERM (ezrin, radixin, moesin) proteins contain a conserved globular N-terminal domain, called the FERM domain (Four point one ezrin, radixin, moesin), involved in the binding to both phosphatidylinositol 4,5 bisphosphate (PIP 2 ; Barret et al., 2000) and plasma membrane proteins and a C-terminal F-actin-binding domain that resides in the last 34 amino acids (Turunen et al., 1994;Bretscher et al., 2002). In the cytoplasm, ERM proteins exist in a closed conformation because of an intramolecular interaction between the N-terminal domain and the last 100 amino acids called N-and C-ERMAD (ERM association domain), respectively . This intramolecular association masks the binding sites for plasma membrane proteins and F-actin. An activation step is required to disrupt this association that occurs through conformational changes induced by sequential binding to PIP 2 and phosphorylation of a conserved C-terminal threonine residue (T567 in ezrin; Matsui et al., 1998;Fié...

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Delineation of the Cdc42/Rac-Binding Domain of p21-Activated Kinase

Cited by 138 publications

References 28 publications

A New Constitutively Active Brain PAK3 Isoform Displays Modified Specificities toward Rac and Cdc42 GTPases

A New Constitutively Active Brain PAK3 Isoform Displays Modified Specificities toward Rac and Cdc42 GTPases

Mechanism of allosteric regulation of transglutaminase 2 by GTP

Interaction of Ezrin with the Novel Guanine Nucleotide Exchange Factor PLEKHG6 Promotes RhoG-dependent Apical Cytoskeleton Rearrangements in Epithelial Cells

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