Paxillin controls actin stress fiber formation and migration of vascular smooth muscle cells by directly binding to the active Fyn.

Ying Zhang, Hiroko Kishi, Tomoka Morita, Sei Kobayashi

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2021 Dec

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Rho-kinase (ROK)-mediated migration of vascular smooth muscle cells plays a crucial role in cardiovascular diseases. Previously we demonstrated Fyn tyrosine kinase as an upstream molecule of ROK to mediate actin stress fiber formation that plays an important role in cell migration, but the molecular mechanism between the two kinases was unclear. To discover a novel signaling molecule that exists between Fyn and ROK, we identified paxillin acting downstream of the active Fyn by combined use of pulldown assay and mass spectrometry. Immunofluorescence staining confirmed co-localization of Fyn and paxillin at the ends of actin stress fibers in human coronary artery smooth muscle cells (CASMCs). Surface plasmon resonance assay demonstrated direct binding between constitutively active Fyn (CA-Fyn) and N-terminus of paxillin (N-pax). The sphingosylphosphorylcholine (SPC)-induced ROK activation, actin stress fiber formation and cell migration were inhibited by paxillin knockdown, which were rescued by full-length paxillin (FL-pax) but not N-pax. N-pax co-localized with CA-Fyn at the cytosol and overexpression of N-pax inhibited the SPC-induced actin stress fiber formation and cell migration, indicating that the direct binding of FL-pax and CA-Fyn at the ends of actin stress fibers is essential for the ROK-mediated actin stress fiber formation and cell migration. Paxillin, as a novel signalling molecule, mediates the SPC-induced actin stress fiber formation and migration in human CASMCs via the Fyn/paxillin/ROK signalling pathway by direct binding of active Fyn. © 2021 Federation of American Societies for Experimental Biology.

Citation

Ying Zhang, Hiroko Kishi, Tomoka Morita, Sei Kobayashi. Paxillin controls actin stress fiber formation and migration of vascular smooth muscle cells by directly binding to the active Fyn. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2021 Dec;35(12):e22012