Interactions of invadopodia scaffold protein TKS4 with intersectin family proteins

  • S. V. Kropyvko
  • T. A. Gryaznova
  • A. V. Rynditch

Abstract

Aim. TKS4 is one of the key proteins of invadopodies, specialized membrane structures that provide invasiveness and metastatic potential of malignant cells. This protein is a substrate for the tyrosine kinase SRC, which is involved in the formation of the membrane bends, affects cellular production of active forms of oxygen, interacts with membrane metallоproteinases causing degradation of the extracellular matrix, but its role in invasive structures has not yet been fully understood. Further study of molecular components of invadopodies and their specific interactions provides better understanding of mechanisms of cellular invasion. Methods. Protein-protein interactions were identified by in vitro precipitation of protein complexes using GST-fused proteins and co-immunoprecipitation. Results. Adapter proteins ITSN1 and ITSN2 are new partners of TKS4. Interactions between intersectins and TKS4 are mediated by the SH3A, SH3C and SH3E domains of ITSN1 or ITSN2. TKS4 phosphorylation on tyrosine residues does not affect interactions between TKS4 and intersectins. Conclusions. In this study, we have showed that TKS4 interacts with endocytic adaptor proteins of the intersectin family, ITSN1 and ITSN2. We have also demonstrated that TKS4 and ITSN1 can form a complex in invasive MDA-MB-231 breast cancer cell line.

Keywords: TKS4, ITSN1, ITSN2, protein-protein interactions.

References

Abram C. L., Seals D. F., Pass I., Salinsky D., Maurer L., Roth T.M., Courtneidge S.A. The Adaptor Protein Fish Associates with Members of the ADAMs Family and Localizes to Podosomes of Src-transformed Cells. J Biol Chem. 2003. Vol. 278, № 19. P. 16844–16851. doi: 10.1074/jbc.M300267200.

Lock P., Abram C.L., Gibson T., Courtneidge S.A. A new method for isolating tyrosine kinase substrates used to identify fish, an SH3 and PX domain-containing protein, and Src substrate. EMBO J. 1998. Vol. 17, № 15. Р. 4346–4357. doi: 10.1093/emboj/17.15.4346.

Buschman M.D., Bromann P.A., Cejudo-Martin P., Wen F., Pass I., Courtneidge S.A. The Novel Adaptor Protein Tks4 (SH3PXD2B) Is Required for Functional Podosome Formation. Mol. Biol. Cell. 2009. Vol. 20, № 5. P. 1302–1311. doi: 10.1091/mbc.E08-09-0949.

Murphy D.A., Courtneidge S.A. The “ins” and “outs” of podosomes and invadopodia: characteristics, formation and function. Nature Reviews. Molecular Cell Biology. 2011. Vol. 12, № 7. P. 413–426. doi: 10.1038/nrm3141.

Kropyvko S.V. New partners of TKS4 scaffold protein. Biopolym. Cell. 2015. Vol. 31, № 5. P. 395–401. doi: http://dx.doi.org/10.7124/bc.0008FC.

Tsyba L.O., Dergai M.V., Skrypkina I.Ya., Nikolaienko O.V., Dergai O.V., Kropyvko S.V., Novokhatska O.V., Mor-derer D.Ye., Gryaznova T.A., Gubar O.S., Rynditch A.V. ITSN protein family: regulation of diversity, role in signalling and pathology. Biopolym. Cell. 2013. Vol. 29, № 3. P. 244–251. doi: 10.7124/bc.00081E.

Gryaznova T., Kropyvko S., Burdyniuk M., Gubar O., Kryklyva V., Tsyba L., Rynditch A. Intersectin adaptor proteins are associated with actin-regulating protein WIP in invadopodia. Cell Signal. 2015. Vol. 27, № 7. P. 1499–1508. doi: 10.1016/j.cellsig.2015.03.006.