Involvement of Fyn in stress fiber formation
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Slide 1 :
Involvement of Fyn tyrosine kinase in actin stress fiber formation in fibroblasts Dan Xu1, Hiroko Kishi1, Hozumi Kawamichi1, Katsuko Kajiya1, Yuichi Takada1,2, Sei Kobayashi1,2 1.Dept. Mol. Physiol. & Med. Bioreg., Grad. Sch. Med. Yamaguchi Univ, Ube, Japan. 2. Kobayashi Project, Innovation Plaza Hiroshima, Higashi-hiroshima, JST, Japan.
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Background Signaling pathway of stress fiber formation It is known that Rho and its effector Rho-kinase play a critical role in stress fiber formation induced by LPA or SPC. However, except for Rho, the upstream stimulatory mediator for the Rho-kinase-mediated stress fiber formation is still unknown. Src family protein tyrosine kinases (SrcPTKs) SrcPTKs control diverse aspects of cell behaviour, including cell migration and survival. Furthermore, it is thought to inhibit actin cytoskeleton rearrangement. c-Src, one of the members of SrcPTKs, is known to inhibit stress fiber formation by blocking the Rho-Rho-kinase pathway. However, stimulatory signaling molecules are still not found.
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Frame M.C., Nat Rev Mol Cell Biol, 2002 Rho-kinase (ROCK) Actin stress fibers Rho ·GTP (active) Chang, J.H. J Cell Biol, 1995 c-Src (a member of SrcPTKs) ? Stimulatory signaling molecules are still not found. SrcPTKs control diverse aspects of cell behaviour
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Abnormal vascular contraction Fyn tyrosine kinase is an important signaling molecule in SPC-ROCK pathway vasospasm ROCK Nakao, F., et al, Circ Res. 2002
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The novel finding of this study Fyn acts as a novel signaling molecule to stimulate stress fiber formation mediated by ROCK in fibroblasts. (Xu, D., et al, FEBS lett. 2007) Previously it is reported that c-Src inhibits stress fiber formation. (Chang, J.H. J Cell Biol, 1995)
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Pharmacological treatments Fyn siRNAs (mFyn1, mFyn2) Fyn constructs (plasmid DNA) Methods and Results (1)
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? ROCK is involved in stress fiber formation induced by SPC/LPA. 1-1. Effect of ROCK on stress fiber (SF) formation induced by SPC or LPA Control SPC LPA No Inhibitor Y27632 ROCK specific inhibitor Y27632 Stress fiber formation-------inhibited ROCK activation---------------inhibited MYPT1: myosin phosphatase target subunit1 ROCK activation was assayed by MYPT1 phosphorylation. SF SF
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? SrcPTKs are involved in stress fiber formation induced by SPC/LPA. SrcPTKs inhibitors (PP1, PP2 inactive analogue) inhibited Not inhibited inhibited 1-2. Effects of SrcPTKs on stress fiber formation induced by SPC or LPA SF SF
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Control SPC LPA No Inhibitor EPA Fyn activation assay + + + + ? Fyn stimulates stress fiber formation induced by SPC/LPA. 1-3. Effects of Fyn on stress fiber formation induced by SPC or LPA Fyn translocation inhibitor EPA (eicosapentaenoic acid) Stress fiber formation-------inhibited Fyn activation------------------inhibited SF SF
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Pharmacological treatments Fyn siRNAs (mFyn1, mFyn2) Fyn constructs (plasmid DNA) Methods and Results (2)
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Fyn siRNA target sequence region poly A site siRNA: hFyn1 mFyn2 mFyn1 ORF (534a.a.) 227 1831 (QIAGEN) (Nippon EGT) (RNAi)
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Time-dependency Specificity Knocked down !!! 2-1. Fyn siRNAs-mediated knockdown of Fyn protein
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2-2. Fyn siRNAs blocked the formation of stress fibers induced by SPC or LPA Fyn siRNAs Not inhibited inhibited SF SF
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Pharmacological treatments Fyn siRNAs (mFyn1, mFyn2) Fyn constructs (plasmid DNA) Methods and Results (3)
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Wild Type (active) Constitutively active Fyn (ca-Fyn, Y530F) Structures of Fyn constructs Dominant negative Fyn (dn-Fyn, K298M)
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?-myc F-actin Merge mock + SPC dn-Fyn + SPC WT-Fyn + SPC 3-1. The overexpression of dn-Fyn inhibited the formation of stress fibers induced by SPC * * * * * * SF (Fyn overexpression)
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mock + SPC dn-Fyn + LPA WT + LPA ?-myc F-actin Merge 3-2. The overexpression of dn-Fyn resulted in the loss of stress fibers in response to LPA stimulation * * * * * * SF
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Line a Line b ?-myc F-actin Merge ca-Fyn ca-Fyn + Y27632 (ROCK inhibitor) * * * * * * ca-Fyn (green) F-actin (red) Profile analysis 3-3. The overexpression of ca-Fyn induced stress fiber formation and Fyn may be in the upstream of ROCK
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7. The possibility of Fyn in the upstream of ROCK on stress fiber formation. 3-4. Fyn is associated with focal adhesion complex in stress fiber formation Line c * * * * FA: focal adhesion FAK: focal adhesion kinase ca-Fyn (green) F-actin (red) FAK (blue) Profile analysis (ca-Fyn overexpression) FA FA SF
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SPC NIH3T3 Fibroblast Stress Fiber Filopodia SPC, but not LPA, induced filopodia-like protrusion formation in NIH3T3 cells LPA Filopodia-like protrusions
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ROCK inhibitor: Y27632 SrcPTK inhibitor: PP2 siRNAs: Control MAPK1 mFyn1 mFyn2 Filopodia-like protrusion formation Inhibitors and siRNAs didn’t affect SPC-induced filopodia-like protrusion formation Fyn PP2 Stress fiber formation SPC ROCK Y27632 ? Fyn and ROCK were not related
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Summary: Proposed scheme for the signaling pathway of Fyn leading to actin cytoskeleton reorganization. Cell migration Wound healing Epithelial adhesion Cell contraction Intracellular tension Cell shape & locomotion Fyn and ROCK were not related
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