Treponema denticola Msp-deduced peptide conjugate, P34BSA, promotes RhoA-dependent actin stress fiber formation independent of its internalization by fibroblasts

• P34BSA, a BSA conjugate of a synthetic 10-mer peptide deduced from Treponema denticola major outer sheath protein (Msp), stabilizes actin filaments in fibroblasts and retards cell motility. We reported previously that it is internalized by cells, binds and bundles actin filaments in vitro, and activates RhoA, yet, its site and mechanism of action were not defined. We have assessed P34BSA's modes of interaction with and signaling to fibroblasts. At 4°C, P34BSA was not internalized, but it bound to the plasma membrane and promoted actin stress fiber formation at ~80% capacity compared with 37°C controls, casting doubt that cellular uptake is a critical step for its cytoskeleton-stabilizing property. In Rho G-LISA™ and co-immunoprecipitation assays, P34BSA was found to activate RhoA, even at 4°C, to promote its interaction with guanosine nucleotide exchange factor p114RhoGEF. It also caused phosphorylation of cofilin. Upon RhoA inhibition, either by C3 transferase RhoA inhibitor or by transfection with a dominant negative RhoA construct, P34BSA did not achieve the stress fiber formation seen with P34BSA alone. By inhibiting phosphatidylinositol-3 kinase (PI 3-K) with LY294002, the P34BSA effects were completely blocked. Depletion of cholesterol with methyl-ß-cyclodextrin (MßCD) partially inhibited P34BSA signaling via the plasma membrane to the cytoskeleton. This suggests that multivalent P34BSA activation of lipid raft components requires active PI 3-K, and initiates the pathway through a RhoGEF and RhoA, which mediates stress fiber formation in fibroblasts. Hence, P34BSA may represent a novel tool to investigate RhoA-dependent processes, such as remodeling filamentous actin in eukaryotic cells. © 2008 Wiley-Liss, Inc.

Nyckelord

Actin stress fibers

Major outer sheath protein

RhoA

RhoGEF

Treponema denticola

MEDICINE

MEDICIN

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