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J. Biol. Chem., Vol. 283, Issue 14, 9177-9186, April 4, 2008

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Neph1, a Component of the Kidney Slit Diaphragm, Is Tyrosine-phosphorylated by the Src Family Tyrosine Kinase and Modulates Intracellular Signaling by Binding to Grb2^*

Yutaka Harita, Hidetake Kurihara^¶, Hidetaka Kosako, Tohru Tezuka^||, Takashi Sekine, Takashi Igarashi, and Seisuke Hattori^**1

From the Division of Cellular Proteomics (BML) and the ^||Department of Oncology, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, the Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, the ^¶Department of Anatomy, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, and the ^**Department of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan

There are several lines of evidence that the podocyte slit diaphragm (SD), which serves as a structural framework for the filtration barrier in kidney glomerulus, also plays an essential role as a signaling platform. Several SD components including nephrin and TRPC6 are known to be phosphorylated by a Src family tyrosine kinase (SFK), Fyn. Here we have characterized Neph1, another SD component, as a novel substrate of SFK. Fyn interacts with and phosphorylates the cytoplasmic domain of Neph1 in vitro and in intact cells. Peptide mass fingerprinting and site-directed mutagenesis identified several tyrosine phosphorylation sites. In pull-down assays using rat glomerular lysates, Neph1 but not nephrin specifically binds to adaptor protein Grb2 and tyrosine kinase Csk in a phosphorylation-dependent manner. Both tyrosine 637 and 638 of Neph1 are crucial for Neph1-Grb2 binding. Phosphorylation of tyrosine 637 is significantly up-regulated in in vivo models of podocyte injury. Furthermore, Neph1 attenuates ERK activation elicited by Fyn, and this inhibitory effect requires the intact binding motif for the Grb2 SH2 domain. Our results shown here demonstrate that Neph1 is a novel in vivo substrate of SFK and suggest that Neph1 modulates ERK signaling through phosphorylation-dependent interaction with Grb2. Thus, SFK orchestrates a wide spectrum of protein-protein interactions and intracellular signaling networks at SD through tyrosine phosphorylation.

Received for publication, August 29, 2007 , and in revised form, January 16, 2008.

^* This work was supported by the program for the International Research and Educational Institute for Integrated Medical Sciences (IREIIMS). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Fax: 81-3-6409-2073; E-mail: hattoris{at}ims.u-tokyo.ac.jp.

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