Identification of Ca2+/calmodulin-dependent phosphorylation sites of endocytic scaffold ITSN1 by tandem mass spectrometry

Morderer, D. Ye.; Nikolaienko, O. V.; Rynditch, A. V.

doi:10.7124/bc.0008F5

Biopolym. Cell. 2015; 31(5):338-344.

http://dx.doi.org/10.7124/bc.0008F5

Structure and Function of Biopolymers

Identification of Ca²⁺/calmodulin-dependent phosphorylation sites of endocytic scaffold ITSN1 by tandem mass spectrometry

1Morderer D. Ye., 2Nikolaienko O. V., 1Rynditch A. V.

Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
Department of Biomedicine, University of Bergen
91, Jonas Lies vei, Bergen, Norway, 5009

Abstract

ITSN1 is a scaffold protein involved in endocytosis, signal transduction and cytoskeleton regulation. It has been previously shown that ITSN1 undergoes Ca²⁺/calmodulin-dependent phosphorylation in vitro. Aim. We intend to identify these phosphorylation sites. Methods. In vitro kinase reaction; liquid chromatography-tandem mass spectrometry (LC/MS/MS). Results. We identified five sites of Ca²⁺/calmodulin-dependent phosphorylation in the recombinant fragments of ITSN1. Conclusions. We have shown that the ITSN1 coiled-coil region (CCR) and the interdomain linkers between EH2 and CCR, SH3A and SH3B, SH3B and SH3C domains were phosphorylated in a Ca²⁺/calmodulin-dependent manner in vitro.

Keywords: ITSN1, Ca²⁺, phosphorylation, LC/MS/MS

Full text: (PDF, in English)

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