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Rho GTPase Control of the Actin Cytoskeleton
Summary: Michael Rosen's research is directed toward understanding the structural, biochemical, and cell biological mechanisms of cytoskeletal regulation by the Rho GTPases. His long-term objective is to understand quantitatively how the cytoskeleton integrates biological inputs to create complex but coherent outputs.
Actin dynamics play important roles in eukaryotic cellular processes ranging from motility, division, and polarization to bacterial infection. To achieve complex cellular behaviors, actin rearrangements must be regulated both spatially and temporally and integrated with functions that include gene expression and membrane trafficking. The rapid nucleation of new filaments underlies many actin-based processes. Since nucleation is inherently slow, two ubiquitous machineries, Arp2/3 complex and the formin proteins, have evolved to accelerate it in vivo. The Rho-family GTPases Cdc42, Rac, and Rho are prominent players in the transmission and integration of signals that control the cytoskeleton, and they exert many of their functions by acting on these two nucleators.
My lab takes a multidisciplinary approach to understand mechanisms of Rho GTPase signaling that control Arp2/3 complex and formins. We study the basic structural, thermodynamic, and kinetic properties of these proteins and how interactions between them drive conformational changes that alter their activities. We also use novel engineered reagents that report on and control rapid signaling events in vivo to study how these physical properties guide information transfer in signaling pathways in the cell.
Signaling to Arp2/3 Complex
Cdc42 communicates with the actin cytoskeleton in large part through its interactions with the Wiskott-Aldrich syndrome protein (WASP), the archetypal member of a large group of proteins that integrate GTPase and other signals to control Arp2/3 complex. Members of the family, including neuronal-WASP (N-WASP) and the WAVE proteins, are characterized by a conserved C-terminal VCA domain that stimulates the actin-nucleating activity of Arp2/3 complex. In WASP this domain is inhibited by intramolecular binding of an N-terminal regulatory element, the GTPase-binding domain (GBD). Interaction of the GBD with Cdc42 displaces the VCA, leading to activation toward Arp2/3 complex and establishing a signaling pathway from the GTPase to the cytoskeleton.
Our early work focused on the activation of WASP by Cdc42. Using nuclear magnetic resonance (NMR) spectroscopy, we solved the solution structures of autoinhibited WASP and of the WASP GBD in complex with Cdc42. The former structure, coupled with NMR studies of the intact 240-kDa Arp2/3 complex and its interactions with VCA peptides, revealed that the GBD sequesters residues of the VCA needed for Arp2/3 activation, thus explaining the structural basis of autoinhibition in WASP. Comparison of the two structures indicated that Cdc42 activates WASP by destabilizing its folded autoinhibited domain. We later demonstrated that because of this mechanism, efficient phosphorylation and dephosphorylation of WASP in the GBD are both contingent on binding to activated Cdc42. The requirement for contingency in both phosphorylation and dephosphorylation enables long-term storage of information by WASP following decay of GTPase signals.
Recently, we developed a quantitative thermodynamic model for WASP that predicts its hydrogen exchange behavior, Cdc42-binding affinity, and activity toward Arp2/3 complex. Unexpectedly, the model also revealed that the nucleotide switch in small GTPases can control not only affinity for effectors (potency of GTPase activation, in the parlance of pharmacology) but also the efficiency by which the GTPase-effector complex is driven toward activation (efficacy of activation), a mechanism that increases fidelity of signaling.
Future work in this area will focus on understanding how the Rac GTPase communicates to Arp2/3 complex through the WASP family member WAVE. In contrast to WASP, WAVE is regulated intermolecularly through formation of a large assembly with four other proteins. The assembly is linked to GTPase pathways through binding of one subunit to Rac. It is also linked to RNA metabolism through interaction with the fragile X mental retardation protein (FMRP), an RNA-binding protein involved in translational regulation. Our goal is to reconstitute the WAVE regulatory assembly and use a combination of structural and biochemical analyses to understand how Rac and other molecules use it to control cytoskeletal dynamics and integrate them with RNA metabolism.
Actin Regulation by Formins
The formin proteins, the second major actin nucleation machinery, control production of unbranched filament arrays. Many formins, including yeast Bni1p and the mDia proteins, are downstream targets of Rho GTPases and mediate GTPase effects on actin dynamics in structures such as the cytokinetic ring and stress fibers. The conserved formin homology 2 (FH2) domain nucleates new filaments, binds tightly to filament barbed ends, and inhibits capping proteins, but paradoxically permits rapid addition and loss of actin monomers, leading to its description as a leaky or processive cap.
We recently solved the crystal structure of the Bni1p FH2 domain in complex with actin. The structure reveals that the FH2 domain binds actin monomers in an orientation closely resembling a short-pitch actin filament, suggesting nucleation occurs through a templating mechanism. FH2 binding leaves the actin pointed end free to bind the bulk filament but blocks the barbed end, explaining how formins attach to filaments and inhibit capping proteins. Functional studies have led us to a model for processive capping in which the FH2 domain exists in a dynamic equilibrium at the barbed end between the blocked configuration and an open configuration that differs in the relative orientation of the two halves of the FH2 dimer. Interconversion between these two states is required for barbed-end elongation and shrinkage in the presence of bound FH2 domain.
We also solved the crystal structure of the N-terminal regulatory domain of mDia1, which binds the C terminus and inhibits the activity of the FH2 domain. We used NMR and mutagenesis to show that Rho activates mDia1 by competing with the C terminus for a common binding site in the N terminus.
Our current work on formins is focused on several areas. First, we have initiated single-particle electron microscopy investigations, in collaboration with Masahide Kikkawa (University of Texas Southwestern Medical Center at Dallas), to image directly the FH2 domain attached to the filament. Second, we are pursuing structural and biochemical analyses of the N+C interactions in mDia1 to understand how these block activity of the FH2 domain.
Finally, a major goal is to understand how formin and Arp2/3 complex pathways are coordinated at structural, biochemical, and cell biological levels. Both actin-nucleating systems are used during processes including motility, cytokinesis, and, as we have recently discovered, phagocytosis. Yet, their respective roles in these processes, and potential interactions between them, are not understood. The PCH proteins have recently emerged as potential physical links between formins and Arp2/3 complex, with the discovery that the Schizosaccharomyces pombe formin, Cdc12, interacts with the PCH protein Cdc15, which in turn assembles the Arp2/3 activator Myo1p. Communication between the pathways could have significant effects on actin filament dynamics, since the linear filaments rapidly generated by formin should activate Arp2/3 complex, thus creating a closed topology of interactions. The response properties of this circuit could also be significantly modified by the physical architecture of the assembly and resultant filaments. In collaboration with Kathleen Gould (HHMI, Vanderbilt University), we are combining computational modeling, biochemical reconstitution, and in vivo analysis of the yeast Cdc12, Cdc15, Myo1p, and Arp2/3 complex to examine these issues.
Along similar lines, we recently found that the formin FRL functions along with Arp2/3 complex during phagocytosis in macrophages. We will continue to use live-cell imaging and coordinate biochemical analyses of macrophages to understand how the different Arp2/3 complex and formin pathways localize and evolve to produce the structures needed for phagocytosis.
Cooperativity in Multidomain Signaling Proteins: The Guanine Nucleotide Exchange Factor Vav
Autoinhibition in multidomain systems is typically achieved through a core active-site repression mechanism whose energetics are modulated by combinations of additional contacts involving other domains. These cooperative interactions suppress activity in the basal state and provide mechanisms of integrating multiple inputs to achieve signaling specificity in vivo. The structural organization of autoinhibited systems suggests that activators function by recognizing poorly populated excited states. Thus, the internal dynamics of such systems and the domain-domain contacts that modulate autoinhibitory energetics likely play key roles in the regulatory process.
We are studying these issues in the protein Vav, a multidomain guanine nucleotide exchange factor (GEF) for Cdc42 and Rac. Our early work revealed that autoinhibition in Vav is mediated by an N-terminal helical extension from the catalytic Dbl homology (DH) domain that folds back into the active site. A key regulatory tyrosine in this helix is buried in the active site, and we showed by NMR that its phosphorylation by upstream kinases leads to release of the helix from the DH domain.
Activation of Vav must occur through an excited state since the regulatory tyrosine on its inhibitory helix is buried in the DH active site in the ground state structure. In full-length Vav, autoinhibition appears to be enhanced through interactions of calponin homology, pleckstrin homology, and zinc finger domains that flank the helix-DH regulatory core. In the future, we will use NMR to examine the role of dynamics in Vav regulation and learn how dynamics and energetics of autoinhibition are altered by domain-domain interactions. These thermodynamic analyses will be married with structure determination of the multidomain systems. Our work will help establish general structural and energetic principles by which autoinhibited systems can be generated, function, and evolve.
This research was also supported by grants from the National Institutes of Health and the Welch Foundation.
Last updated: January 13, 2006
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