Biopolym. Cell. 2016; 32(1):26-33.
Structure and Function of Biopolymers
Colocalization of cortactin and PH domain of BCR in HEK293T cells and its potential role in cell signaling.
1Gurianov D. S., 1Antonenko S. V., 1Telegeev G. D.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680


Chromosomal translocation t(9;22)(q34;q11) leads to the generation of different types of the BCR-ABL fusion protein and cause different types of leukemias. The generated fusion proteins differ by the presence or absence of certain domains of BCR – C2, PH, and DH. The mass-spectrometric analysis identified 23 possible interaction partners of PH domain of BCR. Among them is cortactin (CTTN), which is a multidomain protein responsible for the actin branching during endocytosis. The activation of cortactin occurs after phosphorylation by SRC kinase. However, it is unknown whether ABL kinase can phosphorylate and activate CTTN in the same manner. Aim. To demonstrate whether CTTN and PH domain of BCR colocalize in HEK293T cells, to analyze possible phosphorylation sites for ABL kinase in the cortactin and to make a comparative prediction for SRC kinase. Methods. Cells HEK293T were transfected with the engineered pECFP-C3-CTTN and pmCitrine-C1-PH using a cationic polymer transfection and evaluated with fluorescent microscopy. Putative phosphorylation sites of CTTN by ABL and SRC kinases were predicted by GPS 2.1 software. Results. PH domain and CTTN were expressed in HEK293T cells and show specific cellular colocalization. The phoshorylation sites for Abl kinase (Y384, Y396, Y409, Y416, Y427, Y433, and Y449) were detected in the proline-rich region of CTTN and they match corresponded sites predicted for SRC kinase. Conclusions. We have shown that CTTN has cytoplasmic localization and PH domain of BCR localized predominantly but not exclusively in the nucleus. Their partial colocalization occurs in specific dot-like perinuclear regions. A potential phosphorylation of CTTN by ABL may be one of the activation pathways during leukemogenesis.
Keywords: CML, BCR-ABL, PH domain, CTTN


[1] Hecht F, Morgan R, Schrier SL, Adams J, Sandberg AA. The Philadelphia (Ph) chromosome in leukemia. I. A new mechanism due to interstitial deletion and insertion in chronic myelocytic leukemia. Cancer Genet Cytogenet. 1985;14(1-2):3-10.
[2] Telegeev GD, Dubrovska AN, Dybkov MV, Maliuta SS. Influence of BCR/ABL fusion proteins on the course of Ph leukemias. Acta Biochim Pol. 2004;51(3):845-9.
[3] Telegeev GD, Dybkov MV, Dubrovska AN, Miroshnichenko DA, Tyutyunnykova AP, Maliuta SS. Development of molecular oncohematology in Ukraine. Biopolym Cell. 2013; 29(4): 277–82.
[4] Wetzler M, Talpaz M, Van Etten RA, Hirsh-Ginsberg C, Beran M, Kurzrock R. Subcellular localization of Bcr, Abl, and Bcr-Abl proteins in normal and leukemic cells and correlation of expression with myeloid differentiation. J Clin Invest. 1993;92(4):1925-39.
[5] Pane F, Intrieri M, Quintarelli C, Izzo B, Muccioli GC, Salvatore F. BCR/ABL genes and leukemic phenotype: from molecular mechanisms to clinical correlations. Oncogene. 2002;21(56):8652-67.
[6] Cerione RA, Zheng Y. The Dbl family of oncogenes. Curr Opin Cell Biol. 1996;8(2):216-22.
[7] Aghazadeh B, Zhu K, Kubiseski TJ, Liu GA, Pawson T, Zheng Y, Rosen MK. Structure and mutagenesis of the Dbl homology domain. Nat Struct Biol. 1998;5(12):1098-107.
[8] Lemmon MA. Pleckstrin homology domains: not just for phosphoinositides. Biochem Soc Trans. 2004;32(Pt 5):707–11.
[9] Lemmon MA, Ferguson KM. Signal-dependent membrane targeting by pleckstrin homology (PH) domains. Biochem J. 2000;350 Pt 1:1-18.
[10] Lemmon MA, Ferguson KM, Abrams CS. Pleckstrin homology domains and the cytoskeleton. FEBS Lett. 2002;513(1):71-6.
[11] Miroshnychenko D, Dubrovska A, Maliuta S, Telegeev G, Aspenström P. Novel role of pleckstrin homology domain of the Bcr-Abl protein: analysis of protein-protein and protein-lipid interactions. Exp Cell Res. 2010;316(4):530-42.
[12] Zhu J, Zhou K, Hao JJ, Liu J, Smith N, Zhan X. Regulation of cortactin/dynamin interaction by actin polymerization during the fission of clathrin-coated pits. J Cell Sci. 2005;118(Pt 4):807-17.
[13] Cao H, Orth JD, Chen J, Weller SG, Heuser JE, McNiven MA. Cortactin is a component of clathrin-coated pits and participates in receptor-mediated endocytosis. Mol Cell Biol. 2003;23(6):2162-70.
[14] Kirkbride KC, Hong NH, French CL, Clark ES, Jerome WG, Weaver AM. Regulation of late endosomal/lysosomal maturation and trafficking by cortactin affects Golgi morphology. Cytoskeleton (Hoboken). 2012;69(9):625-43.
[15] Tehrani S, Tomasevic N, Weed S, Sakowicz R, Cooper JA. Src phosphorylation of cortactin enhances actin assembly. Proc Natl Acad Sci U S A. 2007;104(29):11933-8.
[16] Head JA, Jiang D, Li M, Zorn LJ, Schaefer EM, Parsons JT, Weed SA. Cortactin tyrosine phosphorylation requires Rac1 activity and association with the cortical actin cytoskeleton. Mol Biol Cell. 2003;14(8):3216-29.
[17] Webb BA, Zhou S, Eves R, Shen L, Jia L, Mak AS. Phosphorylation of cortactin by p21-activated kinase. Arch Biochem Biophys. 2006;456(2):183-93.
[18] Boyle SN, Michaud GA, Schweitzer B, Predki PF, Koleske AJ. A critical role for cortactin phosphorylation by Abl-family kinases in PDGF-induced dorsal-wave formation. Curr Biol. 2007;17(5):445-51.
[19] Lezin G, Kosaka Y, Yost HJ, Kuehn MR, Brunelli L. A one-step miniprep for the isolation of plasmid DNA and lambda phage particles. PLoS One. 2011;6(8):e23457.
[20] Birnboim HC, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979;7(6):1513-23.
[21] Lezin G, Kuehn MR, Brunelli L. Hofmeister series salts enhance purification of plasmid DNA by non-ionic detergents. Biotechnol Bioeng. 2011;108(8):1872-82.
[22] Xue Y, Liu Z, Cao J, Ma Q, Gao X, Wang Q, Jin C, Zhou Y, Wen L, Ren J. GPS 2.1: enhanced prediction of kinase-specific phosphorylation sites with an algorithm of motif length selection. Protein Eng Des Sel. 2011;24(3):255-60.
[23] Ingley E, Hemmings BA. Pleckstrin homology (PH) domains in signal transduction. J Cell Biochem. 1994;56(4):436-43.
[24] Viaud J, Gaits-Iacovoni F, Payrastre B. Regulation of the DH-PH tandem of guanine nucleotide exchange factor for Rho GTPases by phosphoinositides. Adv Biol Regul. 2012;52(2):303-14.
[25] Zheng Y, Zangrilli D, Cerione RA, Eva A. The pleckstrin homology domain mediates transformation by oncogenic dbl through specific intracellular targeting. J Biol Chem. 1996;271(32):19017–20.
[26] Baumeister MA, Rossman KL, Sondek J, Lemmon MA. The Dbs PH domain contributes independently to membrane targeting and regulation of guanine nucleotide-exchange activity. Biochem J. 2006;400(3):563-72.
[27] Worthylake DK, Rossman KL, Sondek J. Crystal structure of the DH/PH fragment of Dbs without bound GTPase. Structure. 2004;12(6):1078-86.
[28] Chhatriwala MK, Betts L, Worthylake DK, Sondek J. The DH and PH domains of Trio coordinately engage Rho GTPases for their efficient activation. J Mol Biol. 2007;368(5):1307-20.
[29] Clark ES, Whigham AS, Yarbrough WG, Weaver AM. Cortactin is an essential regulator of matrix metalloproteinase secretion and extracellular matrix degradation in invadopodia. Cancer Res. 2007;67(9):4227-35.
[30] Weaver AM. Cortactin in tumor invasiveness. Cancer Lett. 2008;265(2):157-66.
[31] Cao H, Weller S, Orth JD, Chen J, Huang B, Chen JL, Stamnes M, McNiven MA. Actin and Arf1-dependent recruitment of a cortactin-dynamin complex to the Golgi regulates post-Golgi transport. Nat Cell Biol. 2005;7(5):483-92.
[32] Levine TP, Munro S. The pleckstrin homology domain of oxysterol-binding protein recognises a determinant specific to Golgi membranes. Curr Biol. 1998;8(13):729-39.
[33] Levine TP, Munro S. Targeting of Golgi-specific pleckstrin homology domains involves both PtdIns 4-kinase-dependent and -independent components. Curr Biol. 2002;12(9):695-704.
[34] Oser M, Mader CC, Gil-Henn H, Magalhaes M, Bravo-Cordero JJ, Koleske AJ, Condeelis J. Specific tyrosine phosphorylation sites on cortactin regulate Nck1-dependent actin polymerization in invadopodia. J Cell Sci. 2010;123(Pt 21):3662-73.