Abstract
The two p190RhoGAP proteins, p190RhoGAP-A and -B, are key regulators of Rho GTPase signaling and are essential for actin cytoskeletal structure and contractility. Here we report the discovery of two evolutionarily conserved GTPase-like domains located in the ‘middle domain’, previously thought to be unstructured. Deletion of these domains reduces RhoGAP activity. Crystal structures, MANT-GTPγS binding, thermal denaturation, biochemical assays and sequence homology analysis all strongly support defects in nucleotide-binding activity. Analysis of p190RhoGAP proteins therefore indicates the presence of two previously unidentified domains which represent an emerging group of pseudoenzymes, the pseudoGTPases.
Highlights
The two p190RhoGAP proteins, p190RhoGAP-A and -B, are key regulators of Rho GTPase signaling and are essential for actin cytoskeletal structure and contractility
Two GTPase-like folds are predicted in p190RhoGAP
Our initial analyses indicate that the middle domainsof p190RhoGAP proteins contain unstructured regions, secondary structure prediction algorithms unexpectedly indicate extensive order for the amino-terminal half of the middle domain, corresponding to residues 550–960 of p190RhoGAP-A (Fig. 1a)
Summary
The two p190RhoGAP proteins, p190RhoGAP-A and -B, are key regulators of Rho GTPase signaling and are essential for actin cytoskeletal structure and contractility. We report the discovery of two evolutionarily conserved GTPase-like domains located in the ‘middle domain’, previously thought to be unstructured. GTPases bind to guanosine triphosphate (GTP), hydrolyze γ-phosphate, release guanosine diphosphate (GDP) and re-bind GTP, a process termed ‘GTPase cycling’ This cycling, and consequent signal transduction, is regulated by GTPase activating proteins (GAP) (for GTP hydrolysis) and guanine nucleotide exchange factors (GEF) (for GDP release). The p190RhoGAP proteins, p190RhoGAP-A (ARHGAP35) and p190RhoGAP-B (ARHGAP5)[4,5,6], are key regulators of Rho GTP hydrolysis and are highly important for maintenance of proper Rho signaling They share over 50% sequence identity and a domain organization containing a GTP-binding GTPase domain, four FF domains and a C-terminal GAP domain.
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