Abstract
p116Rip is a ubiquitously expressed protein that was originally identified as a putative binding partner of RhoA in a yeast two-hybrid screen. Overexpression of p116Rip in neuroblastoma cells inhibits RhoA-mediated cell contraction induced by lysophosphatidic acid (LPA); so far, however, the function of p116Rip is unknown. Here we report that p116Rip localizes to filamentous actin (F-actin)-rich structures, including stress fibers and cortical microfilaments, in both serum-deprived and LPA-stimulated cells, with the N terminus (residues 1-382) dictating cytoskeletal localization. In addition, p116Rip is found in the nucleus. Direct interaction or colocalization with RhoA was not detected. We find that p116Rip binds tightly to F-actin (Kd approximately 0.5 microm) via its N-terminal region, while immunoprecipitation assays show that p116Rip is complexed to both F-actin and myosin-II. Purified p116Rip and the F-actin-binding region can bundle F-actin in vitro, as shown by electron microscopy. When overexpressed in NIH3T3 cells, p116Rip disrupts stress fibers and promotes formation of dendrite-like extensions through its N-terminal actin-binding domain; furthermore, overexpressed p116Rip inhibits growth factor-induced lamellipodia formation. Our results indicate that p116Rip is an F-actin-binding protein with in vitro bundling activity and in vivo capability of disassembling the actomyosin-based cytoskeleton.
Highlights
In our ongoing studies to delineate Rho signaling by the lipid growth factor lysophosphatidic acid (LPA) [11, 12], we previously identified a ubiquitously expressed protein of 116 kDa, provisionally named p116Rip, which binds relatively weakly to activated RhoA in a yeast two-hybrid assay [13]
We find that p116Rip, rather than directly binding to RhoA, interacts with filamentous actin (F-actin) via its N-terminal region and colocalizes with dynamic F-actin structures such as stress fibers, cortical microfilaments, filopodia, and lamellipodial ruffles
We show here that p116Rip is an F-actinbinding protein that has bundling activity in vitro, with the actin-binding domain residing in the N-terminal region
Summary
F-actin, filamentous actin; LPA, lysophosphatidic acid; PH, pleckstrin homology; aa, amino acids; HA, hemagglutinin; FL, full-length; GST, glutathione S-transferase; GFP, green fluorescent protein; NT, N terminus; CT, C terminus; BSA, bovine serum albumin; RBD, RhoA-binding domain; PBS, phosphatebuffered saline; PDGF, platelet-derived growth factor
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