Browsing by Subject "Proto-Oncogene Proteins c-vav"
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Item Characterization of Internal Dynamics in Vav1: Method Development, Mutual Coupling and Functional Relevance(2009-09-04) Li, Pilong; Rosen, Michael K.Protein motions are important to activity, but quantitative relationships between internal dynamics and function are not well understood. The Dbl homology (DH) domain of the proto-oncoprotein and guanine nucleotide exchange factor Vav1 is autoinhibited through interactions between its catalytic surface and a helix from an N-terminal acidic region. Phosphorylation of the helix relieves autoinhibition. Here I show by NMR spectroscopy that the autoinhibited DH domain (AD) exists in equilibrium between a ground state, where the active site is blocked by the inhibitory helix, and an excited state, where the helix is dissociated. Across a series of mutants that differentially sample these states, catalytic activity of the autoinhibited protein and its rate of phosphorylation are linearly dependent on the population of the excited state. Thus, internal dynamics are required for and control both basal activity and the rate of full activation of the autoinhibited DH domain. Vav1 belong to a class of multi-domain signaling proteins exhibit complex behaviors due to cooperative interactions between domains. In many such proteins there is a core regulatory interaction, involving binding of an inhibitory element to the active site of a functional domain like the inhibitory helix to DH in Vav1. The core interaction is cooperatively enhanced by additional intramolecular domain-domain contacts. The physical basis of this cooperativity, and thus the energetic construction of multi-domain systems, is not well understood. Dynamics analysis of AD reveals that the closed and open populations are about 10:1 for the core interaction in isolation. In the full five-domain regulatory fragment of Vav1, interactions between domains outside of the core further bias this inhibitory equilibrium ~10-fold toward the closed state, further suppressing activity. Thus, Vav1 is controlled by two, weakly biasing, but thermodynamically coupled equilibria--an energetic construction that is probably general among multi-domain proteins. The dynamic landscape of AD is composed of two ?s-ms time scale motions: one is the inhibitory helix binding to and dissociating from the DH domain and another is intrinsic to the DH domain. Interestingly relative populations and exchange rates of the second process are altered upon perturbations to the inhibitory helix, suggesting that the two dynamic processes are energetically and kinetically coupled. A strategy has been established to quantify the thermodynamic and kinetic coupling strengths between the two processes via direction parameterization of four-state equilibria using NMR Carr-Purcell-Meiboom-Gill measurement. The coupling strengths between the two dynamic processes in AD are 1.0~1.5 kcal M-1 comparable to the coupling strength between the modulatory interaction and the helix-DH interactions in the full five-domain regulatory fragment of Vav1. The coupling strength is relatively weak consistent with the coupling strengths reported for many other signaling proteins such as Src tyrosine kinase. These findings suggest that weakly coupling may be a common theme in regulatory molecules.Item Identification and Characterization of Novel Mechanisms of Functional and Structural Synapse Remodeling: Focus on Vav Guanine Nucleotide Exchange Factors and MEF2 Transcription Factors(2014-07-23) Hale, Carly Fenwick; Huber, Kimberly M.; Cowan, Christopher W.; Green, Carla B.; Kim, Tae-KyungProper development of synaptic connectivity is a dynamic process requiring formation, elimination, maintenance, and plasticity of synapses. During early postnatal development, excess synapses are formed in most neural circuits, which are subsequently pruned during adolescence in a sensory- and activity-dependent mechanism. The brain also exhibits experience-dependent synaptic modifications that may enhance or weaken functional synapse strength. Investigation of numerous neurodevelopmental and psychiatric disorders reveals dysfunctions in synapse formation and function; however, underlying molecular mechanisms remain poorly understood. In Part One of this study, I identify a novel role for Vav guanine nucleotide exchange factors (GEFs) in brain-derived neurotrophic factor (BDNF)-dependent synapse plasticity. BNDF and its receptor, TrkB, are well-established positive modulators of hippocampal long-term potentiation (LTP), and increasing evidence suggests that BDNF/TrkB facilitates LTP in part through the stimulation of Rho GTPases and subsequent F-actin remodeling and dendritic spine structural dynamics. I report that Vav-family GEFs are activated by BDNF/TrkB signaling, and are required for BDNF-induced Rac-GTP formation. Vav GEFs, which are enriched at hippocampal glutamatergic synapses, are necessary for rapid BDNF-induced dendritic spine growth and CA3-CA1 LTP. Furthermore, Vav2/3-deficient mice have impaired contextual fear conditioning, as well as reduced anxiety. Together, findings support a role for Vav-dependent F-actin dynamics in BDNF-stimulated dendritic spine head enlargement and LTP, and normal hippocampal-dependent learning and memory and anxiety in mice. Part Two of this study reports the identification of common MEF2 and FMRP mRNA targets that are required for MEF2-induced synapse elimination. The activity-dependent transcription factor myocyte enhancer factor 2 (MEF2) is a key negative regulator of excitatory synapse number, promoting synapse removal in neurons through a complex program of gene expression. The RNA binding protein and translational regulator fragile X mental retardation protein (FMRP) was recently identified as an essential downstream component of MEF2-induced synapse elimination, suggesting that these autism-linked proteins coordinate transcriptional and translational control of common transcripts to mediate proper synaptic connectivity. Using high throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) of FMRP, I find a large overlap of MEF2-induced transcripts and FMRP-associated mRNAs, consistent with their shared roles in synapse elimination. More specifically, protocadherin 17 (Pcdh17) mRNA is induced by MEF2 and exhibits differential binding to FMRP following MEF2 activation. Reducing Pcdh17 alone does not alter basal synapse number, but reducing Pcdh17 levels blocks MEF2-induced dendritic spine elimination of hippocampal neurons. These data suggest that MEF2-induced synapse elimination requires Pcdh17 – a MEF2 target gene and FMRP-associated transcript.Item Structural and Mechanistic Studies of Two Regulatory Factors in Actin Cytoskeletal Signaling: Vav and VopL(2011-12-15) Yu, Bingke; Rosen, Michael K.Proper control of actin cytoskeletal dynamics is essential for cell survival. The goals of my thesis work have been to characterize the structural and biophysical properties of two regulatory proteins in actin cytoskeletal rearrangement pathway: Vav and Vibrio outer protein L (VopL). Vav proteins are guanine nucleotide exchange factors for Rho family GTPases. They play key roles in actin regulatory pathways and control diverse cellular processes like T cell maturation and activation, cell migration and phagocytosis. They belong to a group of multi-domain signaling proteins which display complex behaviors because of the collective regulation from multiple domains. Previous work has shown that Vav is autoinhibited in the resting state through the cooperative suppression of N-terminal Calponin domain and Acidic region, with the physical mechanism yet to be determined. Here through structural, energetic and biochemical studies, I demonstrate that the Calponin homology domain of Vav binds to the Pleckstrin homology domain, restrains the inhibitory helix in the Acidic region, and shifts the Dbl homology domain - inhibitory helix equilibrium to a more closed state. This construction enables strong suppression and an efficient activation process. The energetic basis of Vav autoinhibition may turn out to be widespread in multi-domain systems. VopL, a pathogenic effector from Vibrio parahaemalyticus, is an actin nucleation factor that induces stress fibers during bacterial infection. It contains three N-terminal Wiskott-Aldrich Homology 2 (WH2) motifs and a unique VopL C-terminal domain (VCD). It potently promotes actin filament nucleation in vitro. However, the physical basis of VopL mediated nucleation has not been understood. Here I performed structural and biochemical studies to investigate the mechanism of actin filament nucleation by VopL. I found that both the WH2 element and VCD are required for VopL activity. The crystal structure of VCD revealed a U-shaped dimer that is stabilized by a terminal coiled-coil. Dimerization of the WH2 motifs as well as contacts between VCD and actin contribute to the nucleation activity of VopL. My studies suggest the formation of a structurally organized actin cluster involving lateral contacts during nucleation. Stabilization of these lateral contacts may be a common feature of actin filament nucleation by WH2-based factors.