Biopolym. Cell. 2016; 32(3):203-221.
Genomics, Transcriptomics and Proteomics
Transcriptional and posttranscriptional regulation of the adaptor/scaffold protein gene ITSN1
1Kropyvko S. V., 1Gubar O. S., 1Gryaznova T. A., 1Morderer D. Ye., 1Gerasymchuk D. O., 2Syvak L. A., 2Grabovoy A. N., 1Rynditch A. V.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
  2. National Cancer Institute
    33/43, Lomonosova Str., Kyiv, Ukraine, 03022


ITSN1 adaptor/scaffold protein takes part in a variety of physiological and pathological cellular processes. It has a complex expression regulation and many protein partners. Aim. Characterization of the ITSN1 functioning and expression control is important for understanding its role in cell. Methods. Bioinformatic analysis, semi-quantitative expression analysis by RT-PCR, immunoprecipitation. Results. We have described and analyzed the ITSN1 promoter regions, detected ITSN1 alternatively spliced isoforms at mRNA and protein levels in different cancer specimens. By means of different bioinformatic servers, we have identified the sites for miRNA binding and analyzed the sites for serine, threonine and tyrosine phosphorylation of the ITSN1 protein. Conclusions. We have obtained new data on the ITSN1 expression in pathology. We have also shown the possibility of ITSN1 expression regulation by miRNA and phosphorylation of serine, threonine and tyrosine.
Keywords: ITSN1, bidirectional promoter, alternative splicing, miRs, phosphorylation


[1] 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.
[2] 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.
[3] 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.
[4] Ma Y, Wang B, Li W, Ying G, Fu L, Niu R, Gu F. Reduction of intersectin1-s induced apoptosis of human glioblastoma cells. Brain Res. 2010;1351:222-8.
[5] Gryaznova T, Kropyvko S, Burdyniuk M, Gubar O, Kryklyva V, Tsyba L, Rynditch A. Intersectin adaptor proteins are associated with actin-regulating protein WIP in invadopodia. Cell Signal. 2015;27(7):1499-508.
[6] Snyder JT, Rossman KL, Baumeister MA, Pruitt WM, Siderovski DP, Der CJ, Lemmon MA, Sondek J. Quantitative analysis of the effect of phosphoinositide interactions on the function of Dbl family proteins. J Biol Chem. 2001;276(49):45868-75.
[7] Hussain NK, Yamabhai M, Ramjaun AR, Guy AM, Baranes D, O'Bryan JP, Der CJ, Kay BK, McPherson PS. Splice variants of intersectin are components of the endocytic machinery in neurons and nonneuronal cells. J Biol Chem. 1999;274(22):15671-7.
[8] Guipponi M, Scott HS, Chen H, Schebesta A, Rossier C, Antonarakis SE. Two isoforms of a human intersectin (ITSN) protein are produced by brain-specific alternative splicing in a stop codon. Genomics. 1998;53(3):369-76.
[9] Tsyba L, Nikolaienko O, Dergai O, Dergai M, Novokhatska O, Skrypkina I, Rynditch A. Intersectin multidomain adaptor proteins: regulation of functional diversity. Gene. 2011;473(2):67-75.
[10] 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.
[11] Kropyvko S, Gerasymchuk D, Skrypkina I, Dergai M, Dergai O, Nikolaienko O, Rynditch A, Tsyba L. Structural diversity and differential expression of novel human intersectin 1 isoforms. Mol Biol Rep. 2010;37(6):2789-96.
[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] Wakano C, Byun JS, Di LJ, Gardner K. The dual lives of bidirectional promoters. Biochim Biophys Acta. 2012;1819(7):688-93.
[14] Yang MQ, Koehly LM, Elnitski LL. Comprehensive annotation of bidirectional promoters identifies co-regulation among breast and ovarian cancer genes. PLoS Comput Biol. 2007;3(4):e72.
[15] Trinklein ND, Aldred SF, Hartman SJ, Schroeder DI, Otillar RP, Myers RM. An abundance of bidirectional promoters in the human genome. Genome Res. 2004;14(1):62-6.
[16] Yang MQ, Elnitski LL. Diversity of core promoter elements comprising human bidirectional promoters. BMC Genomics. 2008;9 Suppl 2:S3.
[17] Yang MQ, Taylor J, Elnitski L. Comparative analyses of bidirectional promoters in vertebrates. BMC Bioinformatics. 2008;9 Suppl 6:S9.
[18] Yang MQ, Elnitski LL. Prediction-based approaches to characterize bidirectional promoters in the mammalian genome. BMC Genomics. 2008;9 Suppl 1:S2.
[19] Hu HY, He L, Khaitovich P. Deep sequencing reveals a novel class of bidirectional promoters associated with neuronal genes. BMC Genomics. 2014;15:457.
[20] Uesaka M, Nishimura O, Go Y, Nakashima K, Agata K, Imamura T. Bidirectional promoters are the major source of gene activation-associated non-coding RNAs in mammals. BMC Genomics. 2014;15:35.
[21] Kropyvko SV, Tsyba LO, Skrypkina IYa, Rynditch AV. Identification and functional analysis of an alternative promoter of human intersectin 1 gene. Biopolym Cell. 2010;26(2):115–20.
[22] Kozak M. Pushing the limits of the scanning mechanism for initiation of translation. Gene. 2002;299(1-2):1-34.
[23] Clamp M, Fry B, Kamal M, Xie X, Cuff J, Lin MF, Kellis M, Lindblad-Toh K, Lander ES. Distinguishing protein-coding and noncoding genes in the human genome. Proc Natl Acad Sci U S A. 2007;104(49):19428-33.
[24] Chen M, Manley JL. Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches. Nat Rev Mol Cell Biol. 2009;10(11):741-54.
[25] Ghigna C, Valacca C, Biamonti G. Alternative splicing and tumor progression. Curr Genomics. 2008;9(8):556-70.
[26] Irie F, Yamaguchi Y. EphB receptors regulate dendritic spine development via intersectin, Cdc42 and N-WASP. Nat Neurosci. 2002;5(11):1117-8.
[27] Nishimura T, Yamaguchi T, Tokunaga A, Hara A, Hamaguchi T, Kato K, Iwamatsu A, Okano H, Kaibuchi K. Role of numb in dendritic spine development with a Cdc42 GEF intersectin and EphB2. Mol Biol Cell. 2006;17(3):1273-85.
[28] Pechstein A, Bacetic J, Vahedi-Faridi A, Gromova K, Sundborger A, Tomlin N, Krainer G, Vorontsova O, Schäfer JG, Owe SG, Cousin MA, Saenger W, Shupliakov O, Haucke V. Regulation of synaptic vesicle recycling by complex formation between intersectin 1 and the clathrin adaptor complex AP2. Proc Natl Acad Sci U S A. 2010;107(9):4206-11.
[29] Dergai M, Skrypkina I, Dergai O, Tsyba L, Novokhatska O, Filonenko V, Drobot L, Rynditch A. Identification and characterization of a novel mammalian isoform of the endocytic adaptor ITSN1. Gene. 2011;485(2):120-9.
[30] Quesnel-Vallières M, Irimia M, Cordes SP, Blencowe BJ. Essential roles for the splicing regulator nSR100/SRRM4 during nervous system development. Genes Dev. 2015;29(7):746-59.
[31] Tsyba L, Skrypkina I, Rynditch A, Nikolaienko O, Ferenets G, Fortna A, Gardiner K. Alternative splicing of mammalian Intersectin 1: domain associations and tissue specificities. Genomics. 2004;84(1):106-13.
[32] Tsyba L, Gryaznova T, Dergai O, Dergai M, Skrypkina I, Kropyvko S, Boldyryev O, Nikolaienko O, Novokhatska O, Rynditch A. Alternative splicing affecting the SH3A domain controls the binding properties of intersectin 1 in neurons. Biochem Biophys Res Commun. 2008;372(4):929-34.
[33] Novokhatska O, Dergai M, Tsyba L, Skrypkina I, Filonenko V, Moreau J, Rynditch A. Adaptor proteins intersectin 1 and 2 bind similar proline-rich ligands but are differentially recognized by SH2 domain-containing proteins. PLoS One. 2013;8(7):e70546.
[34] Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell. 2005;120(1):15-20.
[35] Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136(2):215-33.
[36] Ørom UA, Nielsen FC, Lund AH. MicroRNA-10a binds the 5'UTR of ribosomal protein mRNAs and enhances their translation. Mol Cell. 2008;30(4):460-71.
[37] Lin DH, Yue P, Zhang C, Wang WH. MicroRNA-194 (miR-194) regulates ROMK channel activity by targeting intersectin 1. Am J Physiol Renal Physiol. 2014;306(1):F53-60.
[38] Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell. 2005;120(1):15-20.
[39] Agarwal V, Bell GW, Nam JW, Bartel DP. Predicting effective microRNA target sites in mammalian mRNAs. Elife. 2015;4.
[40] Enright AJ, John B, Gaul U, Tuschl T, Sander C, Marks DS. MicroRNA targets in Drosophila. Genome Biol. 2003;5(1):R1.
[41] Betel D, Wilson M, Gabow A, Marks DS, Sander C. The resource: targets and expression. Nucleic Acids Res. 2008;36(Database issue):D149-53.
[42] Morderer DYe, Nikolaienko OV, Rynditch A. V. Identification of Ca2+. calmodulin-dependent phosphorylation sites of endocytic scaffold ITSN1 by tandem mass spectrometry. Biopolym Cell. 2015;31(5):338–44.
[43] 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.
[44] 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.
[45] 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.
[46] 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.
[47] Lienhard GE. Non-functional phosphorylations? Trends Biochem Sci. 2008;33(8):351-2.
[48] Szilák L, Moitra J, Krylov D, Vinson C. Phosphorylation destabilizes alpha-helices. Nat Struct Biol. 1997;4(2):112-4.
[49] 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.
[50] Xavier CP, Rastetter RH, Blömacher M, Stumpf M, Himmel M, Morgan RO, Fernandez MP, Wang C, Osman A, Miyata Y, Gjerset RA, Eichinger L, Hofmann A, Linder S, Noegel AA, Clemen CS. Phosphorylation of CRN2 by CK2 regulates F-actin and Arp2/3 interaction and inhibits cell migration. Sci Rep. 2012;2:241.
[51] Pechstein A, Gerth F, Milosevic I, Jäpel M, Eichhorn-Grünig M, Vorontsova O, Bacetic J, Maritzen T, Shupliakov O, Freund C, Haucke V. Vesicle uncoating regulated by SH3-SH3 domain-mediated complex formation between endophilin and intersectin at synapses. EMBO Rep. 2015;16(2):232-9.
[52] Sharma K, D'Souza RC, Tyanova S, Schaab C, Wiśniewski JR, Cox J, Mann M. Ultradeep human phosphoproteome reveals a distinct regulatory nature of Tyr and Ser/Thr-based signaling. Cell Rep. 2014;8(5):1583-94.
[53] Mertins P, Yang F, Liu T, Mani DR, Petyuk VA, Gillette MA, Clauser KR, Qiao JW, Gritsenko MA, Moore RJ, Levine DA, Townsend R, Erdmann-Gilmore P, Snider JE, Davies SR, Ruggles KV, Fenyo D, Kitchens RT, Li S, Olvera N, Dao F, Rodriguez H, Chan DW, Liebler D, White F, Rodland KD, Mills GB, Smith RD, Paulovich AG, Ellis M, Carr SA. Ischemia in tumors induces early and sustained phosphorylation changes in stress kinase pathways but does not affect global protein levels. Mol Cell Proteomics. 2014;13(7):1690-704.
[54] Yi T, Zhai B, Yu Y, Kiyotsugu Y, Raschle T, Etzkorn M, Seo HC, Nagiec M, Luna RE, Reinherz EL, Blenis J, Gygi SP, Wagner G. Quantitative phosphoproteomic analysis reveals system-wide signaling pathways downstream of SDF-1/CXCR4 in breast cancer stem cells. Proc Natl Acad Sci U S A. 2014;111(21):E2182-90.
[55] Mertins P, Qiao JW, Patel J, Udeshi ND, Clauser KR, Mani DR, Burgess MW, Gillette MA, Jaffe JD, Carr SA. Integrated proteomic analysis of post-translational modifications by serial enrichment. Nat Methods. 2013;10(7):634-7.
[56] Klammer M, Kaminski M, Zedler A, Oppermann F, Blencke S, Marx S, Müller S, Tebbe A, Godl K, Schaab C. Phosphosignature predicts dasatinib response in non-small cell lung cancer. Mol Cell Proteomics. 2012;11(9):651-68.
[57] Lundby A, Secher A, Lage K, Nordsborg NB, Dmytriyev A, Lundby C, Olsen JV. Quantitative maps of protein phosphorylation sites across 14 different rat organs and tissues. Nat Commun. 2012;3:876.
[58] Schreiber TB, Mäusbacher N, Kéri G, Cox J, Daub H. An integrated phosphoproteomics work flow reveals extensive network regulation in early lysophosphatidic acid signaling. Mol Cell Proteomics. 2010;9(6):1047-62.
[59] Han G, Ye M, Liu H, Song C, Sun D, Wu Y, Jiang X, Chen R, Wang C, Wang L, Zou H. Phosphoproteome analysis of human liver tissue by long-gradient nanoflow LC coupled with multiple stage MS analysis. Electrophoresis. 2010;31(6):1080-9.
[60] Mayya V, Lundgren DH, Hwang SI, Rezaul K, Wu L, Eng JK, Rodionov V, Han DK. Quantitative phosphoproteomic analysis of T cell receptor signaling reveals system-wide modulation of protein-protein interactions. Sci Signal. 2009;2(84):ra46.
[61] Schweppe DK, Rigas JR, Gerber SA. Quantitative phosphoproteomic profiling of human non-small cell lung cancer tumors. J Proteomics. 2013;91:286-96.
[62] Hsu PP, Kang SA, Rameseder J, Zhang Y, Ottina KA, Lim D, Peterson TR, Choi Y, Gray NS, Yaffe MB, Marto JA, Sabatini DM. The mTOR-regulated phosphoproteome reveals a mechanism of mTORC1-mediated inhibition of growth factor signaling. Science. 2011;332(6035):1317-22.
[63] Gauci S, Helbig AO, Slijper M, Krijgsveld J, Heck AJ, Mohammed S. Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach. Anal Chem. 2009;81(11):4493-501.
[64] Bian Y, Song C, Cheng K, Dong M, Wang F, Huang J, Sun D, Wang L, Ye M, Zou H. An enzyme assisted RP-RPLC approach for in-depth analysis of human liver phosphoproteome. J Proteomics. 2014;96:253-62.
[65] Christensen GL, Kelstrup CD, Lyngsø C, Sarwar U, Bøgebo R, Sheikh SP, Gammeltoft S, Olsen JV, Hansen JL. Quantitative phosphoproteomics dissection of seven-transmembrane receptor signaling using full and biased agonists. Mol Cell Proteomics. 2010;9(7):1540-53.
[66] Olsen JV, Vermeulen M, Santamaria A, Kumar C, Miller ML, Jensen LJ, Gnad F, Cox J, Jensen TS, Nigg EA, Brunak S, Mann M. Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis. Sci Signal. 2010;3(104):ra3.
[67] Kettenbach AN, Schweppe DK, Faherty BK, Pechenick D, Pletnev AA, Gerber SA. Quantitative phosphoproteomics identifies substrates and functional modules of Aurora and Polo-like kinase activities in mitotic cells. Sci Signal. 2011;4(179):rs5.
[68] Wu F, Wang P, Zhang J, Young LC, Lai R, Li L. Studies of phosphoproteomic changes induced by nucleophosmin-anaplastic lymphoma kinase (ALK) highlight deregulation of tumor necrosis factor (TNF)/Fas/TNF-related apoptosis-induced ligand signaling pathway in ALK-positive anaplastic large cell lymphoma. Mol Cell Proteomics. 2010;9(7):1616-32.
[69] Imami K, Sugiyama N, Imamura H, Wakabayashi M, Tomita M, Taniguchi M, Ueno T, Toi M, Ishihama Y. Temporal profiling of lapatinib-suppressed phosphorylation signals in EGFR/HER2 pathways. Mol Cell Proteomics. 2012;11(12):1741-57.
[70] Van Hoof D, Muñoz J, Braam SR, Pinkse MW, Linding R, Heck AJ, Mummery CL, Krijgsveld J. Phosphorylation dynamics during early differentiation of human embryonic stem cells. Cell Stem Cell. 2009;5(2):214-26.
[71] 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.
[72] Herskowitz JH, Seyfried NT, Duong DM, Xia Q, Rees HD, Gearing M, Peng J, Lah JJ, Levey AI. Phosphoproteomic analysis reveals site-specific changes in GFAP and NDRG2 phosphorylation in frontotemporal lobar degeneration. J Proteome Res. 2010;9(12):6368-79.
[73] Xiao K, Sun J, Kim J, Rajagopal S, Zhai B, Villén J, Haas W, Kovacs JJ, Shukla AK, Hara MR, Hernandez M, Lachmann A, Zhao S, Lin Y, Cheng Y, Mizuno K, Ma'ayan A, Gygi SP, Lefkowitz RJ. Global phosphorylation analysis of beta-arrestin-mediated signaling downstream of a seven transmembrane receptor (7TMR). Proc Natl Acad Sci U S A. 2010;107(34):15299-304.
[74] Raijmakers R, Kraiczek K, de Jong AP, Mohammed S, Heck AJ. Exploring the human leukocyte phosphoproteome using a microfluidic reversed-phase-TiO2-reversed-phase high-performance liquid chromatography phosphochip coupled to a quadrupole time-of-flight mass spectrometer. Anal Chem. 2010;82(3):824-32.
[75] Brill LM, Xiong W, Lee KB, Ficarro SB, Crain A, Xu Y, Terskikh A, Snyder EY, Ding S. Phosphoproteomic analysis of human embryonic stem cells. Cell Stem Cell. 2009;5(2):204-13.
[76] Old WM, Shabb JB, Houel S, Wang H, Couts KL, Yen CY, Litman ES, Croy CH, Meyer-Arendt K, Miranda JG, Brown RA, Witze ES, Schweppe RE, Resing KA, Ahn NG. Functional proteomics identifies targets of phosphorylation by B-Raf signaling in melanoma. Mol Cell. 2009;34(1):115-31.