Research in the Rosen lab is directed toward understanding the structural, biochemical, and cell biological mechanisms of cytoskeletal regulation by the Rho GTPases. Our 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. Actin rearrangements must be regulated both spatially and temporally and integrated with functions including 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.
We take 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 actin 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, contain 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 activation of WASP by Cdc42. Using NMR spectroscopy, we solved the structures of autoinhibited WASP and of the WASP GBD in complex with Cdc42. The former structure revealed that the GBD sequesters residues of the VCA needed for Arp2/3 activation, explaining the structural basis of autoinhibition. Comparison of the two structures showed that Cdc42 activates WASP by destabilizing its folded autoinhibited domain. We also developed a quantitative thermodynamic model based on this structural mechanism that predicts WASP?s hydrogen exchange behavior, Cdc42-binding affinity, and activity toward Arp2/3 complex. We have also demonstrated that 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.
Future work in this area will focus on understanding how the Rac GTPase communicates to Arp2/3 complex through the WASP family member WAVE. Unlike WASP, WAVE is regulated intermolecularly through formation of a 400 kDa 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 it biochemically to discover the repertoire of ligands that control WAVE activity in vivo. We will also use a combination of structural and biochemical analyses to understand the molecular mechanisms by which these molecules control cytoskeletal dynamics through WAVE 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 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 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. 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 a blocked configuration and an open configuration that differ 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.
Our current work on formins is in 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 formin mDia1 to understand how its FH2 domain is inhibited by intramolecular binding to its N-terminal regulatory domain. Finally, we are planning single-molecule fluorescence studies of formins bound to the ends of actin filaments to test key dynamic features of our model for processive capping.
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. In full-length Vav, autoinhibition is enhanced through interactions of CH, PH 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.
RESEARCH INTERESTS
structural biology
biochemistry
NMR spectroscopy
protein allostery and engineering
cell biology of the actin cytoskeleton
RECENT PUBLICATIONS
Kreishman-Deitrick, M., Goley, E., Denison, C. Burdine, L., C., Egile, C., Li, R., Murali, N., Kodadek, T. J., Welch, M. D., Rosen, M. K., "NMR Analyses of the Activation of Arp2/3 Complex by Neuronal Wiskott-Aldrich Syndrome Protein." Biochemistry, 44:15247-15256, 2005
Torres, E. and Rosen, M. K., "Protein Tyrosine Kinase and GTPase Signals Cooperate to Phosphorylate and Activate WASP/N-WASP" J. Biol. Chem, 281:3513-3520, 2006
Seth, A. and Rosen, M. K., "Autoinhibition Regulates the Actin Assembly Activity and Cellular Localization of the Diaphanous-Related Formins FRLa and mDia1" Cell, submitted 2006
Li, P., Martins, I. R. S., Amarasinghe, G. K. and Rosen, M. K., "Dynamic Origins of Interdomain Cooperativity in the Vav Proto-oncoprotein" Nature, submitted 2007
SIGNIFICANT PUBLICATIONS
Kim, A. S., Kakalis, L. T., Abdul-Manan, N., Liu, G. A., Rosen, M. K., "Autoinhibition and activation mechanisms of the Wiskott-Aldrich syndrome protein" Nature, 404:151-158, 2000
Torres, E. and Rosen, M. K., "Contingent Phosphorylation/Dephosphorylation Provides Mechanisms of Signal Integration and Molecular Memory in WASP" Mol. Cell, 11:1215-1227, 2003
Peterson, J. R., Bickford, L. C., Morgan, D., Kim, A. S., Kirschner, M. W., Rosen, M. K., "Chemical Inhibition of N-WASP by Stabilization of a Native Autoinhibited Conformation" Nature Struct. Mol. Biol., 11:747-755, 2004
Otomo, T., Tomchick, R. R., Otomo, C., Panchal, S. C., Machius, M., Rosen, M. K., "Structural Basis of Actin Filament Nucleation and Processive Capping by a Formin Homology 2 Domain" Nature, 433:488-494, 2005
Otomo, T., Otomo, C., Tomchick, D. R., Machius, M., Rosen, M. K., "Structural Basis of Rho GTPase-Mediated Activation of the Formin mDia1" Mol. Cell, 18:273-281, 2005
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