Phosphorylated YDXV motifs and Nck SH2/SH3 adaptors act cooperatively to induce actin reorganization

Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, M5G 1X5, Canada; Department of Molecular and Medical Genetics, 4388 Medical Sciences Building, 1 King's College Circle, University of Toronto, Toronto, Ontario, M5S 1A8, Canada; Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada; Department of Microbiology and Immunology, Lyman Duff Medical Building, 3775 University St., McGill University, Montreal, Quebec, H3A 2B4, Canada; Department of Biochemistry, Schulich School of Medicine, Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario N6A 5C1, Canada; Institute of Medical Science, 7213 Medical Sciences Building, 1 King's College Circle, University of Toronto, Toronto, Ontario M5S 1A8, Canada; Division of Nephrology, St. Michael's Hospital, 30 Bond Street, Toronto, Ontario M5B 1W8, Canada

^* To whom correspondence should be addressed. Email: pawson{at}mshri.on.ca.

	Abstract

We have analyzed the means by which the Nck family of adaptor proteins couple adhesion proteins to actin reorganization. The nephrin adhesion protein is essential for the formation of actin-based foot processes in glomerular podocytes. Clustering of nephrin induces its tyrosine phosphorylation, Nck recruitment, and sustained localized actin polymerization. Any one of three phosphorylated (p)YDXV motifs on nephrin is sufficient to recruit Nck through its SH2 domain, and induce localized actin polymerization at these clusters. Similarly, Nck SH3 mutants in which only the second or third SH3 domain is functional can mediate nephrin-induced actin polymerization. However, combining such nephrin and Nck mutants attenuates actin polymerization at nephrin-Nck clusters. We propose that the multiple Nck SH2-binding motifs on nephrin and the multiple SH3 domains of Nck act cooperatively to recruit a high local concentration of effectors at sites of nephrin activation that is required to initiate and maintain actin polymerization in vivo. We also find that YDXV motifs in the Tir protein of enteropathogenic E. coli and nephrin are functionally interchangeable, indicating that Tir reorganizes the actin cytoskeleton by molecular mimicry of nephrin-like signaling. Together, these data identify pYDXV/Nck signaling as a potent and portable mechanism for physiological and pathological actin regulation.