p116Rip is a novel filamentous actin-binding protein.
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.
Division of Cellular Biochemistry and Centre for Biomedical Genetics, The Netherlands Cancer Institute, Amsterdam.
Jacqueline
J
Mulder
Mieke
M
Poland
Martijn F B G
MF
Gebbink
Jero
J
Calafat
Wouter H
WH
Moolenaar
Onno
O
Kranenburg
D016475:3T3 Cells; D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D066298:In Vitro Techniques; D007700:Kinetics; D008246:Lysophospholipids / Q000494:pharmacology; D051379:Mice; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008854:Microscopy, Electron; D024462:Myosin Type II / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D000199:Actins; D008246:Lysophospholipids; D008840:Microfilament Proteins; D011994:Recombinant Proteins; D024462:Myosin Type II; D020742:rhoA GTP-Binding Protein