Biopolymers and cell. 2009; 25 (5): 424 - 427

ITSN1 and Ruk/CIN85 colocalized to clathrin-coated pits in MCF-7 cells

O. V. Nikolaienko, I. Ya. Skrypkina, L. O. Tsyba, L. B. Drobot¹, A. V. Rynditch

Institute of molecular biology and genetics NAS of Ukraine
150, Zabolotnogo Str, Kyiv Ukraine, 03680

¹Palladin Institute of Biochemistry NAS of Ukraine
9, Leontovycha Str., Kyiv, Ukraine, 01601

Aim. 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.