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First published online November 21, 2007
doi: 10.1242/10.1242/jcs.03492

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MAP-kinase activity necessary for TGF1-stimulated mesangial cell type I collagen expression requires adhesion-dependent phosphorylation of FAK tyrosine 397

Tomoko Hayashida^1,3,*, Ming-Hua Wu², Amy Pierce^1,, Anne-Christine Poncelet⁴, John Varga² and H. William Schnaper^1,3

¹ Division of Kidney Diseases, Department of Pediatrics
² Division of Rheumatology, Department of Medicine, The Feinberg School of Medicine, Northwestern University, 303 E Chicago Ave, Chicago IL 60611, USA
³ Children's Memorial Research Center, Chicago IL 60614, USA
⁴ University of Washington, School of Medicine, Seattle, WA, USA

^* Author for correspondence (e-mail: hayashida{at}northwestern.edu)

Accepted 24 September 2007

The signals mediating transforming growth factor (TGF)-stimulated kidney fibrogenesis are poorly understood. We previously reported TGF-stimulated, Smad-mediated collagen production by human kidney mesangial cells, and that ERK MAP kinase activity optimizes collagen expression and enhances phosphorylation of the Smad3 linker region. Furthermore, we showed that disrupting cytoskeletal integrity decreases type I collagen production. Focal adhesion kinase (FAK, PTK2) activity could integrate these findings. Adhesion-dependent FAK Y397 phosphorylation was detected basally, whereas FAK Y925 phosphorylation was TGF1-dependent. By immunocytochemistry, TGF1 stimulated the merging of phosphorylated FAK with the ends of thickening stress fibers. Cells cultured on poly-L-lysine (pLL) to promote integrin-independent attachment spread less than those on control substrate and failed to demonstrate focal adhesion (FA) engagement with F-actin. FAK Y397 phosphorylation and ERK activity were also decreased under these conditions. In cells with decreased FAK Y397 phosphorylation from either plating on pLL or overexpressing a FAK Y397F point mutant, serine phosphorylation of the Smad linker region, but not of the C-terminus, was reduced. Y397F and Y925F FAK point mutants inhibited TGF-induced Elk-Gal activity, but only the Y397F mutant inhibited TGF-stimulated collagen-promoter activity. The inhibition by the Y397F mutant or by culture on pLL was prevented by co-transfection of constitutively active ERK MAP kinase kinase (MEK), suggesting that FAK Y397 phosphorylation promotes collagen expression via ERK MAP kinase activity. Finally, Y397 FAK phosphorylation, and both C-terminal and linker-region Smad3 phosphorylation were detected in murine TGF-dependent kidney fibrosis. Together, these data demonstrate adhesion-dependent FAK phosphorylation promoting TGF-induced responses to regulate collagen production.

Key words: TGF, FAK, Smad, Sclerosis, Collagen

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