Regulation of Rho and Rac Signaling to the Actin Cytoskeleton by Paxillin during Drosophila Development

Abstract

Paxillin is a prominent focal adhesion docking protein that regulates cell adhesion and migration. Although numerous paxillin-binding proteins have been identified and paxillin is required for normal embryogenesis, the precise mechanism by which paxillin functions in vivo has not yet been determined. We identified an ortholog of mammalian paxillin in Drosophila (Dpax) and have undertaken a genetic analysis of paxillin function during development. Overexpression of Dpax disrupted leg and wing development, suggesting a role for paxillin in imaginal disc morphogenesis. These defects may reflect a function for paxillin in regulation of Rho family GTPase signaling as paxillin interacts genetically with Rac and Rho in the developing eye. Moreover, a gain-of-function suppressor screen identified a genetic interaction between Dpax and cdi in wing development. cdi belongs to the cofilin kinase family, which includes the downstream Rho target, LIM kinase (LIMK). Significantly, strong genetic interactions were detected between Dpax and Dlimk, as well as downstream effectors of Dlimk. Supporting these genetic data, biochemical studies indicate that paxillin regulates Rac and Rho activity, positively regulating Rac and negatively regulating Rho. Taken together, these data indicate the importance of paxillin modulation of Rho family GTPases during development and identify the LIMK pathway as a critical target of paxillin-mediated Rho regulation.

Genetic interactions betweenDrosophila melanogasterAtg1 and paxillin reveal a role for paxillin in autophagosome formation

“…1A). 8 To identify additional components of the paxillin-mediated signaling pathway, we conducted a misexpression screen for genes that can suppress the dPax-induced wing defects. In a screen of approximately 1,500 independent EP insertion lines (transposable elements), three lines were identified that significantly suppress the frequency and severity of dPax-induced wing blisters.…”

“…In a screen of approximately 1,500 independent EP insertion lines (transposable elements), three lines were identified that significantly suppress the frequency and severity of dPax-induced wing blisters. 8 One of the lines, EP3348, which strongly suppresses dPax-induced blistered wings, harbors a single transposable element inserted in the 5' untranslated region of the Drosophila dAtg1 gene (Fig. 1A and B).…”

“…8). Briefly, adult male flies were lysed in RIPA buffer, proteins were resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and the blot was probed with dPax antibody (1:5000) and tubulin antibody (1:1000, DSHB), followed by HRP-conjugated secondary antibody, and signal was detected by enhanced chemiluminescence (ECL; Amersham).…”

“…8 DSRF/blistered mutants often exhibit blistered wing phenotypes. 16,17 Thus, like paxillin, DSRF/blistered has been implicated in regulating adhesion between the two wing surfaces.…”

Phosphorylation of Actin-Depolymerizing Factor/Cofilin by LIM-Kinase Mediates Amyloid -Induced Degeneration: A Potential Mechanism of Neuronal Dystrophy in Alzheimer's Disease

“…LIMK1 is a key downstream effector of the Rho family of small GTPases and is activated by phosphorylation on Thr508 by Rho kinase ROCK, or by Rac-Cdc42 p21-activated kinase (PAK) (Edwards et al, 1999). Recent observations showed that a multiprotein complex containing LIMK1 and its kinase activator PAK can be recruited into the focal adhesion complex by p95PKL-mediated binding to Paxillin (Turner, 2000;Chen et al, 2005), raising the possibility that LIMK1 may be required for A␤-induced actin filament remodeling in focal adhesions. To gain additional insight into the molecular mechanism that leads to neuronal dystrophy in AD, we analyzed the involvement of LIMK1, ADF/ cofilin, and actin cytoskeleton in A␤-induced neuritic dystrophy.…”