Mammalian Actin-binding Protein-1/Hip-55 Interacts with FHL2 and Negatively Regulates Cell Invasion

Lindsy R Boateng,David Bennin,Sofia De Oliveira,Anna Huttenlocher

doi:10.1074/jbc.m116.725739

Lindsy R Boateng, David Bennin + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m116.725739

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Abstract

Mammalian actin-binding protein-1 (mAbp1) is an adaptor protein that binds actin and modulates scission during endocytosis. Recent studies suggest that mAbp1 impairs cell invasion; however, the mechanism for the inhibitory effects of mAbp1 remain unclear. We performed a yeast two-hybrid screen and identified the adaptor protein, FHL2, as a novel binding partner that interacts with the N-terminal actin depolymerizing factor homology domain (ADFH) domain of mAbp1. Here we report that depletion of mAbp1 or ectopic expression of the ADFH domain of mAbp1 increased Rho GTPase signaling and breast cancer cell invasion. Moreover, cell invasion induced by the ADFH domain of mAbp1 required the expression of FHL2. Taken together, our findings show that mAbp1 and FHL2 are novel binding partners that differentially regulate Rho GTPase signaling and MTLn3 breast cancer cell invasion.

Highlights

Invasive migration of cancer cells is dependent on a dynamic actin cytoskeleton [1]
These findings identify an inhibitory role for Mammalian actin-binding protein-1 (mAbp1) during invasive migration of breast cancer cell lines
We report a novel interaction between mAbp1 and the multifunctional adaptor protein fourand-a-half LIM domain protein 2 (FHL2)

Summary

Experimental Procedures

Reagents and Antibodies—Alexa Fluor 405 phalloidin and rhodamine phalloidin were purchased from Invitrogen. Retroviral transduction of MDA-MB-231 cells was performed with the exception that virus from 24, 48, 72, and 96 h was used for infection and virus-containing media remained on cells 24 h instead of 6. For GFP immunoprecipitation experiments, transiently transfected HEK 293 cells were cultured to ϳ80% confluency on 10-cm plates, washed once with PBS, and lysed in lysis buffer. A sample of lysate was removed for Western blot loading control, and the remainder of lysate was placed in a tube containing 60 ␮g of Rhotekin-RBD beads to capture active Rho. Tubes were incubated for 30 min at 4 °C before centrifuging for 1 min at 1000 rpm and washed 3ϫ with wash buffer (25 mM Tris, pH 7.5, 30 mM MgCl2, 40 mM NaCl, and 1% PMSF, leupeptin, and aprotinin). Statistical Analyses—Statistical analyses used were unpaired t test or one-way analysis of variance (ANOVA) with Tukey post tests. p Ͻ 0.05 was considered significant

Results

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