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.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
  2. 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)

References

[1] Tsyba LO, Dergai MV, Skrypkina IYa, Nikolaienko OV, Dergai OV, Kropyvko SV, Novokhatska OV, Morderer DYe, Gryaznova TA, Gubar OS, Rynditch AV. ITSN protein family: regulation of diversity, role in signalling and pathology. Biopolym Cell. 2013; 29(3):244–51.
[2] Pucharcós C, Fuentes JJ, Casas C, de la Luna S, Alcántara S, Arbonés ML, Soriano E, Estivill X, Pritchard M. Alu-splice cloning of human Intersectin (ITSN), a putative multivalent binding protein expressed in proliferating and differentiating neurons and overexpressed in Down syndrome. Eur J Hum Genet. 1999;7(6):704-12.
[3] Wilmot B, McWeeney SK, Nixon RR, Montine TJ, Laut J, Harrington CA, Kaye JA, Kramer PL. Translational gene mapping of cognitive decline. Neurobiol Aging. 2008;29(4):524-41.
[4] Scappini E, Koh TW, Martin NP, O'Bryan JP. Intersectin enhances huntingtin aggregation and neurodegeneration through activation of c-Jun-NH2-terminal kinase. Hum Mol Genet. 2007;16(15):1862-71.
[5] Hunter MP, Russo A, O'Bryan JP. Emerging roles for intersectin (ITSN) in regulating signaling and disease pathways. Int J Mol Sci. 2013;14(4):7829-52.
[6] Ballif BA, Villén J, Beausoleil SA, Schwartz D, Gygi SP. Phosphoproteomic analysis of the developing mouse brain. Mol Cell Proteomics. 2004;3(11):1093-101.
[7] Dephoure N, Zhou C, Villén J, Beausoleil SA, Bakalarski CE, Elledge SJ, Gygi SP. A quantitative atlas of mitotic phosphorylation. Proc Natl Acad Sci U S A. 2008;105(31):10762-7.
[8] Villén J, Beausoleil SA, Gerber SA, Gygi SP. Large-scale phosphorylation analysis of mouse liver. Proc Natl Acad Sci U S A. 2007;104(5):1488-93.
[9] Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Villén J, Li J, Cohn MA, Cantley LC, Gygi SP. Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc Natl Acad Sci U S A. 2004;101(33):12130-5.
[10] Dergai O, Dergai M, Skrypkina I, Matskova L, Tsyba L, Gudkova D, Rynditch A. The LMP2A protein of Epstein-Barr virus regulates phosphorylation of ITSN1 and Shb adaptors by tyrosine kinases. Cell Signal. 2013;25(1):33-40.
[11] Morderer DYe, Nikolaienko OV, Skrypkina IYa, Rymarenko OV, Kropyvko SV, Tsyba LO, Rynditch AV. Ca. calmodulin-dependent phosphorylation of endocytic scaffold ITSN1. Biopolym Cell. 2014; 30(1):74–6.
[12] Nikolaienko O, Skrypkina I, Tsyba L, Fedyshyn Y, Morderer D, Buchman V, de la Luna S, Drobot L, Rynditch A. Intersectin 1 forms a complex with adaptor protein Ruk/CIN85 in vivo independently of epidermal growth factor stimulation. Cell Signal. 2009;21(5):753-9.
[13] Savitski MM, Lemeer S, Boesche M, Lang M, Mathieson T, Bantscheff M, Kuster B. Confident phosphorylation site localization using the Mascot Delta Score. Mol Cell Proteomics. 2011;10(2):M110.003830.
[14] Searle BC. Scaffold: a bioinformatic tool for validating MS/MS-based proteomic studies. Proteomics. 2010;10(6):1265-9.
[15] Pearson RB, Woodgett JR, Cohen P, Kemp BE. Substrate specificity of a multifunctional calmodulin-dependent protein kinase. J Biol Chem. 1985;260(27):14471-6.
[16] Ubersax JA, Woodbury EL, Quang PN, Paraz M, Blethrow JD, Shah K, Shokat KM, Morgan DO. Targets of the cyclin-dependent kinase Cdk1. Nature. 2003;425(6960):859-64.
[17] Rudner AD, Murray AW. Phosphorylation by Cdc28 activates the Cdc20-dependent activity of the anaphase-promoting complex. J Cell Biol. 2000;149(7):1377-90.
[18] Lu KP, Liou YC, Zhou XZ. Pinning down proline-directed phosphorylation signaling. Trends Cell Biol. 2002;12(4):164-72.
[19] Taulés M, Rius E, Talaya D, López-Girona A, Bachs O, Agell N. Calmodulin is essential for cyclin-dependent kinase 4 (Cdk4) activity and nuclear accumulation of cyclin D1-Cdk4 during G1. J Biol Chem. 1998;273(50):33279-86.
[20] Huber RJ, Catalano A, O’Day DH. Cyclin-dependent kinase 5 is a calmodulin-binding protein that associates with puromycin-sensitive aminopeptidase in the nucleus of Dictyostelium. Biochim Biophys Acta. 2013;1833(1):11–20.
[21] Kahl CR, Means AR. Regulation of cyclin D1/Cdk4 complexes by calcium/calmodulin-dependent protein kinase I. J Biol Chem. 2004;279(15):15411-9.
[22] Zhen X, Goswami S, Abdali SA, Gil M, Bakshi K, Friedman E. Regulation of cyclin-dependent kinase 5 and calcium/calmodulin-dependent protein kinase II by phosphatidylinositol-linked dopamine receptor in rat brain. Mol Pharmacol. 2004;66(6):1500-7.
[23] Burkhard P, Stetefeld J, Strelkov SV. Coiled coils: a highly versatile protein folding motif. Trends Cell Biol. 2001;11(2):82-8.
[24] Szilák L, Moitra J, Vinson C. Design of a leucine zipper coiled coil stabilized 1.4 kcal mol-1 by phosphorylation of a serine in the e position. Protein Sci. 1997;6(6):1273-83.
[25] Szilák L, Moitra J, Krylov D, Vinson C. Phosphorylation destabilizes alpha-helices. Nat Struct Biol. 1997;4(2):112-4.
[26] Dunker AK, Brown CJ, Lawson JD, Iakoucheva LM, Obradović Z. Intrinsic disorder and protein function. Biochemistry. 2002;41(21):6573-82.
[27] Iakoucheva LM, Radivojac P, Brown CJ, O'Connor TR, Sikes JG, Obradovic Z, Dunker AK. The importance of intrinsic disorder for protein phosphorylation. Nucleic Acids Res. 2004;32(3):1037-49.
[28] Collins MO, Yu L, Campuzano I, Grant SG, Choudhary JS. Phosphoproteomic analysis of the mouse brain cytosol reveals a predominance of protein phosphorylation in regions of intrinsic sequence disorder. Mol Cell Proteomics. 2008;7(7):1331-48.