Phosphorylation of ADF/Cofilin Abolishes EGF-induced Actin Nucleation at the Leading Edge and Subsequent Lamellipod Extension

doi:10.1083/jcb.151.5.1119

Published online 20 November 2000. doi:10.1083/jcb.151.5.1119

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© The Rockefeller University Press, 0021-9525/2000/11/1119/ $5.00
The Journal of Cell Biology, Volume 151, Number 5, November 27, 2000 1119-1128

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Phosphorylation of ADF/Cofilin Abolishes EGF-induced Actin Nucleation at the Leading Edge and Subsequent Lamellipod Extension

Noureddine Zebda^a, Ora Bernard^c, Maryse Bailly^a, Susan Welti^a, David S. Lawrence^b, and John S. Condeelis^a
^a Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461
^b Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461
^c Walter and Eliza Hall Institute of Medical Research, 3050 Victoria, Australia

Correspondence to: John S. Condeelis, Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461. Tel:(718) 430-4068 Fax:(718) 430-8996

In metastatic rat mammary adenocarcinoma cells, cell motilitycan be induced by epidermal growth factor. One of the earlyevents in this process is the massive generation of actin barbedends, which elongate to form filaments immediately adjacentto the plasma membrane at the tip of the leading edge. As aresult, the membrane moves outward and forms a protrusion. Totest the involvement of ADF/cofilin in the stimulus-inducedbarbed end generation at the leading edge, we inhibited ADF/cofilin'sactivity in vivo by increasing its phosphorylation level usingthe kinase domain of LIM-kinase 1 (GFP-K). We report here thatexpression of GFP-K in rat cells results in the near total phosphorylationof ADF/cofilin, without changing either the G/F-actin ratioor signaling from the EGF receptor in vivo. Phosphorylationof ADF/cofilin is sufficient to completely inhibit the appearanceof barbed ends and lamellipod protrusion, even in the continuedpresence of abundant G-actin. Coexpression of GFP-K, togetherwith an active, nonphosphorylatable mutant of cofilin (S3A cofilin),rescues barbed end formation and lamellipod protrusion, indicatingthat the effects of kinase expression are caused by the phosphorylationof ADF/cofilin. These results indicate a direct role for ADF/cofilinin the generation of the barbed ends that are required for lamellipodextension in response to EGF stimulation.

Key Words: ADF/cofilin, actin, LIM-kinase, EGF, cell motility

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