Biopolym. Cell. 2014; 30(1):37-41.
Structure and Function of Biopolymers
Interaction between adaptor proteins Ruk/CIN85 and Tks4 in normal and tumor cells of different tissue origins
1Bazalii A. V., 1Samoylenko A. A., 1Petukhov D. M., 2Rynditch A. V., 3Redowicz M.-J., 1Drobot L. B.
  1. Palladin Institute of Biochemistry, NAS of Ukraine
    9, Leontovycha Str., Kyiv, Ukraine, 01601
  2. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
  3. Nencki Institute of Experimental Biology, Polish Academy of Sciences
    3, Pasteur Str., Warszawa, Poland, 02-093

Abstract

Aim. To study the interaction of adaptor protein Ruk/CIN85 SH3 domains with endogenous adaptor protein Tks4 in normal and tumor cells of different tissue origins. Methods. GST in vitro pull-down assay was performed using total cell lysates of cell lines of different origins. Results. Using GST in vitro pull-down assay, we have determined that SH3A domain of adaptor protein Ruk/CIN85 precipitated full-length form of adaptor protein Tks4 (Mr 120 kDa) from lysates of human breast (MCF-7, MDA-MB-231), melanoma (MM-4), colon (HT-29, DLD-1) tumor cells as well as from lysates of mouse Lewis lung carcinoma cells (LLC) and fibroblasts (NIH 3T3). It has been also revealed that all Ruk/CIN85 SH3 domains (A, B and C) with high efficiency precipitate the additional forms of Tks4 with Mr 75, 90 and 160 kDa from lysates of human colon carcinoma cells and mouse fibroblasts. The molecular nature of new multiple forms of Tks4 has not been determined to date. Conclusions. New multiple molecular forms of adaptor protein Tks4 with Mr 75, 90 and 160 kDa, able to interact with high affinity with SH3 domains of Ruk/CIN85, were identified using GST in vitro pull-down assay. The data obtained suggest that interaction between Ruk/CIN85 SH3 domains with Tks4 endogenous forms is determined by cellular context while a level of this interaction can be regulated in the course of physiological cellular responses.
Keywords: adaptor proteins, Ruk/CIN85, Tks4, SH3 domains, GST in vitro pull-down assay

References

[1] Pawson T. Dynamic control of signaling by modular adaptor proteins. Curr Opin Cell Biol 2007; 19(2):112–16.
[2] Buschman MD, Bromann PA, Cejudo-Martin P, Wen F, Pass I, Courtneidge SA. The novel adaptor protein Tks4 (SH3PXD2B) is required for functional podosome formation. Mol Biol Cell. 2009; 20(5):1302–11.
[3] Gout I, Middleton G, Adu J, Ninkina NN, Drobot LB, Filonenko V, Matsuka G, Davies AM, Waterfield M, Buchman VL. Negative regulation of PI 3-kinase by Ruk, a novel adaptor protein. EMBO J. 2000; 19(15):4015–25.
[4] Courtneidge SA. Cell migration and invasion in human disease: the Tks adaptor proteins. Biochem Soc Trans. 2011; 40(1): 129–32.
[5] Havrylov S, Redowicz MJ, Buchman VL. Emerging roles of Ruk/ CIN85 in vesicle-mediated transport, adhesion, migration and malignancy. Traffic. 2010; 11(6):721–31.
[6] Havrylov S, Rzhepetskyy Y, Malinowska A, Drobot L, Redowicz MJ. Proteins recruited by SH3 domains of Ruk/CIN85 adaptor identified by LC-MS/MS. Proteome Sci. 2009; 7:21.
[7] Samoylenko A, Vynnytska-Myronovska B, Byts N, Kozlova N, Basaraba O, Pasichnyk G, Palyvoda K, Bobak Y, Barska M, Mayevska O, Rzhepetsky Y, Shuvayeva H, Lyzogubov V, Usenko V, Savran V, Volodko N, Buchman V, Kietzmann T, Drobot L. Increased levels of the HER1 adaptor protein Rukl/ CIN85 contribute to breast cancer malignancy. Carcinogenesis. 2012; 33(10): 1976–84.
[8] Rzepetsky YuA, Samoylenko AA, Kukharenko OP, Mikhalap SV, Sidorenko SP, Hausser A, Drobot LB. Protein kinase D interacts with adaptor protein Ruk/CIN85 and phosphory lates it. Stud biol. 2009; 3(3):17–28.
[9] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259): 680–85.
[10] Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA. 1979; 76(9):4350–54.
[11] Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG. et al. The sequence of the human genome. Science. 2001; 291 (5507):1304–51.