RTK signaling regulator SPRY2 associates with endocytic adaptor ITSN1 in vivo

Novokhatska, O. V.; Skrypkina, I. Ya.; Dergai, M. V.; Tsyba, L. O.; Rynditch, A. V.

doi:10.7124/bc.000067

Biopolym. Cell. 2012; 28(4):314-316.

http://dx.doi.org/10.7124/bc.000067

Short Communications

RTK signaling regulator SPRY2 associates with endocytic adaptor ITSN1 in vivo

1Novokhatska O. V., 1Skrypkina I. Ya., 1Dergai M. V., 1Tsyba L. O., 1Rynditch A. V.

State Key Laboratory of Molecular and Cellular Biology
Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680

Abstract

Mitogenic signaling from RTK (receptor tyrosine kinase) is tightly linked to endocytosis that regulates the fidelity of signal transduction. Intersectin 1 (ITSN1) is an evolutionarily conserved adaptor implicated in the earliest stages of clathrin-mediated endocytosis and involved in malignant transformation when overexpressed. The aim of this study was to find proteins that could connect ITSN1 to RTK signaling. Methods. In silico prediction of interaction motifs, expression of proteins in bacterial system and mammalian cell culture, immunoprecipitation, GST pull-down analysis, immunofluorescent studies. Results. Using «Scansite» service a binding of ITSN1 SH3A domain to signaling protein SPRY2 was predicted. For experimental verification of this interaction the GST fusion SH3 domains of ITSN1 were produced and purified from Escherichia coli. GST pull-down analysis showed that SH3A, N-SH3A (neuron-specific variant), SH3C and SH3E domains of ITSN1 were able to pull-down Flag-SPRY2 from cell lysate. Immunofluorescent analysis together with coimmunoprecipitation indicated that Omni- ITSN1 and Flag-SPRY2 were partially colocalized and formed a complex in vivo. Conclusions. Our data showed that endocytic adaptor ITSN1 and signaling protein SPRY2 are associated in vivo presumably via PRD/SH3 interaction.

Keywords: intersectin 1, signaling, SPRY2, SH3 domain

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