TITLE: Administrative Supplement for a Synapt G2-Si HDX-MS System ABSTRACT The parent grant (R01-GM102262) supports the study of kinase signaling cascades that impinge on the cytoskeleton. Our focus is to investigate mechanisms underlying specificity and regulation in protein kinase signaling cascades converging on phosphorylation of the cofilin/ADF (actin-depolymerizing factor) group of proteins, key molecules that mediate remodeling of actin filaments. The pathway we are studying is a kinase cascade downstream of RHO family GTPases. This signaling cascade is tightly controlled through multiple mechanisms, including substrate specificity of both the upstream kinases and of LIM kinases themselves, and through autoregulation of the LIM kinases. We have proposed to use Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) to map intramolecular interaction interfaces within LIM kinases that contrain the kinase in an autoinhibited state. This Administrative Supplement for Equipment requests the purchase a Waters Synapt G2-Si Hydrogen-Deuterium Exchange Mass Spectrometry system and will facilitate our study of LIM kinase autoregulation by bringing the technique ?on-site? and by providing improved resolution over currently available instrumentation. Furthermore, on-site availability of the instrument will allow us to investigate how binding of activator proteins to PAK kinases affects dynamics of intramolecular interactions that we have previously observed crystallographically. The equipment request is being shared by ourselves through our MPI grant (TITUS BOGGON and BENJAMIN TURK, R01-GM102262) and with other PIs from Yale who are funded by NIGMS. The cross references for their grants are MARK LEMMON, R35-GM122485; KAREN ANDERSON, R01- GM049551; YA HA, R01-GM112778. Furthermore, the Yale Cancer Biology Institute is committing extensive funds to support the remainder of the purchase price, and the cost of the service contract for 3 years.
Tight control of signaling pathways impinging on the actin cytoskeleton are essential to normal development and homeostasis. The goal of the application is to discover the molecular basis for specificity and regulation in this biologically important pathway focused on the LIM kinase proteins and understanding how the p21-activated kinases are regulated. Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) is a tool to provide a significant improvement in understanding of the molecular interactions of the proteins we are working on. This Administrative Supplement requests partial funding for a Synapt G2-Si HDX-MS system.
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