Activation of a signaling cascade by cytoskeleton stretch.
Cells sense and respond to mechanical force. However, the mechanisms of transduction of extracellular matrix (ECM) forces to biochemical signals are not known. After removing the cell membrane and soluble proteins by Triton X-100 extraction, we found that the remaining complex (Triton cytoskeletons) activated Rap1 upon stretch. Rap1 guanine nucleotide exchange factor, C3G, was required for this activation; C3G as well as the adaptor protein, CrkII, in cell extract bound to Triton cytoskeletons in a stretch-dependent manner. CrkII binding, which was Cas dependent, correlated with stretch-dependent tyrosine phosphorylation of proteins in Triton cytoskeletons including Cas at the contacts with ECM. These in vitro findings were compatible with in vivo observations of stretch-enhanced phosphotyrosine signals, accumulation of CrkII at cell-ECM contacts, and CrkII-Cas colocalization. We suggest that mechanical force on Triton cytoskeletons activates local tyrosine phosphorylation, which provides docking sites for cytosolic proteins, and initiates signaling to activate Rap1.
Tamada
Masako
M
Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Sheetz
Michael P
MP
Sawada
Yasuhiro
Y
D000199:Actins / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D024042:Collagen Type I / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism* / Q000502:physiology; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D003902:Detergents / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D020735:Guanine Nucleotide-Releasing Factor 2 / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006834:Hydrazines; D007739:L Cells; D008567:Membranes, Artificial; D051379:Mice; D018613:Microscopy, Confocal; D008954:Models, Biological; D017830:Octoxynol / Q000494:pharmacology; D010590:Phalloidine; D010766:Phosphorylation; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050737:Proto-Oncogene Proteins c-crk; D015398:Signal Transduction; D012825:Silicon / Q000737:chemistry; D014443:Tyrosine / Q000378:metabolism
D000199:Actins; C417561:Alexa 488 hydrazide; D024042:Collagen Type I; D003598:Cytoskeletal Proteins; D003902:Detergents; D005456:Fluorescent Dyes; D020735:Guanine Nucleotide-Releasing Factor 2; D006834:Hydrazines; D008567:Membranes, Artificial; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D010590:Phalloidine; D014443:Tyrosine; D017830:Octoxynol; D012825:Silicon