| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 1225956 | Journal of Proteomics | 2012 | 7 Pages |
Cortactin is an F-actin binding protein that functions as a scaffold to regulate Arp2/3 mediated actin polymerization in lamellipodia and invadopodia formation as well as functioning in cell migration and endocytosis of many different cell types. In light of the fact that regulated actin polymerization is critical for many cellular processes we launched a search for novel cortactin interactions with cellular proteins that might indicate heretofore undescribed biological activities supported by cortactin. Using a modified stable isotope labeling in cell culture (SILAC) approach in HEK293 cells and Flag-tagged cortactin (F-cortactin) as bait, we identified a limited set of cortactin interactions including several proteins which have not previously been identified as cortactin associated proteins. Among these were serine/threonine-protein phosphatase 2A subunit beta (PP2A-beta) and RCC2/TD60, a Rac guanine nucleotide exchange factor (GEF) required for completion of mitosis and cytokinesis. The interaction between cortactin and RCC2/TD60 was verified in cell lysates immunoprecitated with anti-RCC2/TD60 antibody. Furthermore, cortactin was localized by immunofluorescence in the equatorial plane of dividing HeLa cells in the region where RCC2/TD60 has previously been localized thus providing support for a complex containing cortactin and RCC2/TD60 complex that may play a functional role in cells undergoing mitosis.
Graphical abstractFigure optionsDownload full-size imageDownload high-quality image (77 K)Download as PowerPoint slideHighlights► SILAC technology improves identification of valid protein-protein interactors in pull-down assays by identifying false positive interactors. ► The analytic approach was used to identify novel interacting proteins with cortactin, a multi-functional, actin binding protein. ► The analysis successfully identified previously characterized interacting protein as well as novel candidate binding proteins. ► This experimental approach identified the cellular scaffold protein cortactin in complex with RCC2/TD60 indicating a possible role in mitosis.
