Biopolym. Cell. 2009; 25(5):424-427.
Short Communications
ITSN1 and Ruk/CIN85 colocalized to clathrin-coated pits in MCF-7 cells
1Nikolaienko O. V., 1Skrypkina I. Ya., 1Tsyba L. O., 2Drobot L. B., 1Rynditch A. V.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
  2. Palladin Institute of Biochemistry, NAS of Ukraine
    9, Leontovycha Str., Kyiv, Ukraine, 01601


Activation of receptor tyrosine kinases (RTK) by corresponding ligands results not only in signal propagation, but also initiates a number of processes, such as clathrin-mediated endocytosis, which precisely regulate biological outcome. These processes are tightly controlled by coordinated action of a plethora of proteins – enzymes, scaffolds and inhibitory molecules. An example of an endocytic accessory protein that also functions in cell signaling is provided by intersectin 1 (ITSN1). Previously we have shown that ITSN1 forms a complex with adaptor protein Ruk/CIN85 and ubiquitin ligase Cbl-b, which are implicated in down regulation of RTK. The present study aimed to determine the subcellular localization of ITSN1-Ruk/CIN85 complexes relatively to clathrin light chain and Cbl-b. Methods. Transient transfection of MCF-7 breast adenocarcinoma cells with the constructs containing Omni-tagged intersectin 1 and clathrin light chain fused with mCherry fluorescent protein was utilized to determine subcellular localization by direct or indirect immunofluorescence. Results. We found that Ruk/CIN85-ITSN1 complexes partially colocalized with Cbl-b and clathrin light chain in MCF-7 cells. Conclusions. In our report we provide experimental evidence that ITSN1-Ruk/CIN85 complexes exist in pre-assembled state with Cbl-b and are targeted to clathrin-coated pits in MCF-7 cells.
Keywords: intersectin, Ruk/CIN85, Cbl-b, clathrin-coated pits, immunofluorescence


[1] Sengar A., Wang W., Bishay J., Cohen S., Egan S. The EH and SH3 domain Ese proteins regulate endocytosis by linking to dynamin and Eps15 EMBO J 1999 18, N 5:1159–1171.
[2] Predescu S., Predescu D., Knezevic I., Klein I., Malik A. Intersectin- 1s regulates the mitochondrial apoptotic pathway in endothelial cells J. Biol. Chem 2007 282, N 23 P. 17166–17178.
[3] Mohney R., Das M., Bivona T., Hanes R., Adams A., Philips M., O'Bryan J. Intersectin activates Ras but stimulates transcription through an independent pathway involving JNK J. Biol. Chem 2003 278, N 47:47038–47045.
[4] Hussain N., Jenna S., Glogauer M., Quinn C., Wasiak S., Guipponi M., Antonarakis S., Kay B., Stossel T., Lamarche- Vane N., McPherson P. Endocytic protein intersectin-l regulates actin assembly via Cdc42 and N-WASP Nat. Cell Biol 2001 3, N 10:927–932.
[5] 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, N 5:753–759.
[6] Tong X., Hussain N., de Heuvel E., Kurakin A., Abi-Jaoude E., Quinn C., Olson M., Marais R., Baranes D., Kay B., McPherson P. The endocytic protein intersectin is a major binding partner for the Ras exchange factor mSos1 in rat brain EMBO J 2000 19, N 6:1263–1271.
[7] Nikolaienko O., Skrypkina I., Dergay O., Matskova L., Tsyba L., Dergay M., Kropyvko S., Vingberg G., Rynditch A. Cbl family proteins – new partners of intersectin 1 Collection of scientific papers «Factors of experimental evolution of organisms». Kyiv: Logos, 2006 Vil. 3:122–127.
[8] Martin N., Mohney R., Dunn S., Das M., Scappini E., O'Bryan J. Intersectin regulates epidermal growth factor receptor endocytosis, ubiquitylation, and signaling Mol. Pharmacol 2006 70, N 5:1643–1653.
[9] Hussain N., Yamabhai M., Ramjaun A., Guyi A., Baranesi D., O'Bryan J., Der C., Kay B., McPherson P. Splice variants of intersectin are components of the endocytic machinery in neurons and nonneuronal cells J. Biol. Chem 1999 274, N 22:15671–15677.
[10] Thomas S., Ritter B., Verbich D., Sanson C., Bourbonniere L., McKinney R., McPherson P. Intersectin regulates dendritic spine development and somatodendritic endocytosis but not synaptic vesicle recycling in hippocampal neurons J. Biol. Chem 2009 284, N 18:12410–12419.
[11] Havrylov S., Ichioka F., Powell K., Borthwick E., Baranska J., Maki M., Buchman V. Adaptor protein Ruk/CIN85 is associated with a subset of COPI-coated membranes of the Golgi complex Traffic 2008 9, N 5:798–812.
[12] Zhang J., Zheng X., Yang X., Liao K. CIN85 associates with endosomal membrane and binds phosphatidic acid Cell Res 2009 19, N 6:733–746.
[13] Sciaky N., Presley J., Smith C., Zaal K., Cole N., Moreira J., Terasaki M., Siggia E., Lippincott-Schwartz J. Golgi tubule traffic and the effects of brefeldin A visualized in living cells J. Cell Biol 1997 139, N 5:1137–1155.
[14] Szymkiewicz I., Kowanetz K., Soubeyran P., Dinarina A., Lipkowitz S., Dikic I. CIN85 participates in Cbl-b-mediated down-regulation of receptor tyrosine kinases J. Biol. Chem 2002 277, N 42:39666–39672.
[15] Kowanetz K., Szymkiewicz I., Haglund K., Kowanetz M., Husnjak K., Taylor J., Soubeyran P., Engstrom U., Ladbury J., Dikic I. Identification of a novel proline-arginine motif involved in CIN85-dependent clustering of Cbl and downregulation of epidermal growth factor receptors J. Biol. Chem 2003 278, N 41:39735–39746.
[16] Dikic I. CIN85/CMS family of adaptor molecules FEBS Lett 2002 529, N 1:110–115.