The activation of Src kinase in cells is strictly controlled by intramolecular inhibitory interactions mediated by SH3 and SH2 domains. They impose structural constraints on the kinase domain holding it in a catalytically non-permissive state. The transition between inactive and active conformation is known to be largely regulated by the phosphorylation state of key tyrosines 416 and 527. Here, we identified that phosphorylation of tyrosine 90 reduces binding affinity of the SH3 domain to its interacting partners, opens the Src structure, and renders Src catalytically active. This is accompanied by an increased affinity to the plasma membrane, decreased membrane motility, and slower diffusion from focal adhesions. Phosphorylation of tyrosine 90 controlling SH3-medited intramolecular inhibitory interaction, analogical to tyrosine 527 regulating SH2-C-terminus bond, enables SH3 and SH2 domains to serve as cooperative but independent regulatory elements. This mechanism allows Src to adopt several distinct conformations of varying catalytic activities and interacting properties, enabling it to operate not as a simple switch but as a tunable regulator functioning as a signalling hub in a variety of cellular processes.

Department of Cell Biology BIOCEV Faculty of Science Charles University Vestec Czech Republic

Imaging Methods Core Facility at BIOCEV Faculty of Science Charles University Vestec Czech Republic

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Prague Czech Republic

Proteomics Core Facility at BIOCEV Faculty of Science Charles University Vestec Czech Republic

doi: 10.1101/2022.08.23.504940 PubMed

Zobrazit více v PubMed

Amata I, Maffei M, Pons M. Phosphorylation of unique domains of Src family kinases. Frontiers in Genetics. 2014;5:181. doi: 10.3389/fgene.2014.00181. PubMed DOI PMC

Benda A, Ma Y, Gaus K. Self-calibrated line-scan STED-FCS to quantify lipid dynamics in model and cell membranes. Biophysical Journal. 2015;108:596–609. doi: 10.1016/j.bpj.2014.12.007. PubMed DOI PMC

Bjorge JD, Bellagamba C, Cheng HC, Tanaka A, Wang JH, Fujita DJ. Characterization of two activated mutants of human pp60c-src that escape c-Src kinase regulation by distinct mechanisms. The Journal of Biological Chemistry. 1995;270:24222–24228. doi: 10.1074/jbc.270.41.24222. PubMed DOI

Boerner RJ, Kassel DB, Barker SC, Ellis B, DeLacy P, Knight WB. Correlation of the phosphorylation states of pp60c-src with tyrosine kinase activity: the intramolecular pY530-SH2 complex retains significant activity if Y419 is phosphorylated. Biochemistry. 1996;35:9519–9525. doi: 10.1021/bi960248u. PubMed DOI

Boggon TJ, Eck MJ. Structure and regulation of Src family kinases. Oncogene. 2004;23:7918–7927. doi: 10.1038/sj.onc.1208081. PubMed DOI

Brábek J, Mojzita D, Novotný M, Půta F, Folk P. The SH3 domain of Src can downregulate its kinase activity in the absence of the SH2 domain-pY527 interaction. Biochemical and Biophysical Research Communications. 2002;296:664–670. doi: 10.1016/s0006-291x(02)00884-7. PubMed DOI

Brábek J, Constancio SS, Shin NY, Pozzi A, Weaver AM, Hanks SK. CAS promotes invasiveness of Src-transformed cells. Oncogene. 2004;23:7406–7415. doi: 10.1038/sj.onc.1207965. PubMed DOI

Chen S, O’Reilly LP, Smithgall TE, Engen JR. Tyrosine phosphorylation in the SH3 domain disrupts negative regulatory interactions within the c-Abl kinase core. Journal of Molecular Biology. 2008;383:414–423. doi: 10.1016/j.jmb.2008.08.040. PubMed DOI PMC

Cifone MA. In vitro growth characteristics associated with benign and metastatic variants of tumor cells. Cancer and Metastasis Review. 1982;1:335–347. doi: 10.1007/BF00124216. PubMed DOI

Cordier F, Wang C, Grzesiek S, Nicholson LK. Ligand-induced strain in hydrogen bonds of the c-Src SH3 domain detected by NMR. Journal of Molecular Biology. 2000;304:497–505. doi: 10.1006/jmbi.2000.4274. PubMed DOI

Cowan-Jacob SW, Fendrich G, Manley PW, Jahnke W, Fabbro D, Liebetanz J, Meyer T. The crystal structure of a c-Src complex in an active conformation suggests possible steps in c-Src activation. Structure. 2005;13:861–871. doi: 10.1016/j.str.2005.03.012. PubMed DOI

Dandoulaki M, Petsalaki E, Sumpton D, Zanivan S, Zachos G. Src activation by Chk1 promotes actin patch formation and prevents chromatin bridge breakage in cytokinesis. The Journal of Cell Biology. 2018;217:3071–3089. doi: 10.1083/jcb.201802102. PubMed DOI PMC

Erpel T, Superti-Furga G, Courtneidge SA. Mutational analysis of the Src SH3 domain: the same residues of the ligand binding surface are important for intra- and intermolecular interactions. The EMBO Journal. 1995;14:963–975. doi: 10.1002/j.1460-2075.1995.tb07077.x. PubMed DOI PMC

Fajer M, Meng Y, Roux B. The Activation of c-Src Tyrosine Kinase: Conformational Transition Pathway and Free Energy Landscape. The Journal of Physical Chemistry. B. 2017;121:3352–3363. doi: 10.1021/acs.jpcb.6b08409. PubMed DOI PMC

Frame MC. Src in cancer: deregulation and consequences for cell behaviour. Biochimica et Biophysica Acta. 2002;1602:114–130. doi: 10.1016/s0304-419x(02)00040-9. PubMed DOI

Friedrich J, Ebner R, Kunz-Schughart LA. Experimental anti-tumor therapy in 3-D: spheroids--old hat or new challenge? International Journal of Radiation Biology. 2007;83:849–871. doi: 10.1080/09553000701727531. PubMed DOI

Gemperle J, Hexnerová R, Lepšík M, Tesina P, Dibus M, Novotný M, Brábek J, Veverka V, Rosel D. Structural characterization of CAS SH3 domain selectivity and regulation reveals new CAS interaction partners. Scientific Reports. 2017;7:8057. doi: 10.1038/s41598-017-08303-4. PubMed DOI PMC

Guarino M. Src signaling in cancer invasion. Journal of Cellular Physiology. 2010;223:14–26. doi: 10.1002/jcp.22011. PubMed DOI

Irby RB, Yeatman TJ. Role of Src expression and activation in human cancer. Oncogene. 2000;19:5636–5642. doi: 10.1038/sj.onc.1203912. PubMed DOI

Janoštiak R, Tolde O, Brůhová Z, Novotný M, Hanks SK, Rösel D, Brábek J. Tyrosine phosphorylation within the SH3 domain regulates CAS subcellular localization, cell migration, and invasiveness. Molecular Biology of the Cell. 2011;22:4256–4267. doi: 10.1091/mbc.E11-03-0207. PubMed DOI PMC

Johnson H, Lescarbeau RS, Gutierrez JA, White FM. Phosphotyrosine profiling of NSCLC cells in response to EGF and HGF reveals network specific mediators of invasion. Journal of Proteome Research. 2013;12:1856–1867. doi: 10.1021/pr301192t. PubMed DOI PMC

Klinghoffer RA, Sachsenmaier C, Cooper JA, Soriano P. Src family kinases are required for integrin but not PDGFR signal transduction. The EMBO Journal. 1999;18:2459–2471. doi: 10.1093/emboj/18.9.2459. PubMed DOI PMC

Koudelková L, Pataki AC, Tolde O, Pavlik V, Nobis M, Gemperle J, Anderson K, Brábek J, Rosel D. Novel FRET-Based Src Biosensor Reveals Mechanisms of Src Activation and Its Dynamics in Focal Adhesions. Cell Chemical Biology. 2019;26:255–268. doi: 10.1016/j.chembiol.2018.10.024. PubMed DOI

Koudelková L, Brábek J, Rosel D. Src kinase: Key effector in mechanosignalling. The International Journal of Biochemistry & Cell Biology. 2021;131:105908. doi: 10.1016/j.biocel.2020.105908. PubMed DOI

Luo W, Slebos RJ, Hill S, Li M, Brábek J, Amanchy R, Chaerkady R, Pandey A, Ham AJL, Hanks SK. Global impact of oncogenic Src on a phosphotyrosine proteome. Journal of Proteome Research. 2008;7:3447–3460. doi: 10.1021/pr800187n. PubMed DOI PMC

Machiyama H, Yamaguchi T, Sawada Y, Watanabe TM, Fujita H. SH3 domain of c-Src governs its dynamics at focal adhesions and the cell membrane. The FEBS Journal. 2015;282:4034–4055. doi: 10.1111/febs.13404. PubMed DOI

Macpherson I, Montagnier L. Agar suspension culture for the selective assay of cells transformed by polyoma virus. Virology. 1964;23:291–294. doi: 10.1016/0042-6822(64)90301-0. PubMed DOI

Maffei M, Arbesú M, Le Roux AL, Amata I, Roche S, Pons M. The SH3 Domain Acts as a Scaffold for the N-Terminal Intrinsically Disordered Regions of c-Src. Structure. 2015;23:893–902. doi: 10.1016/j.str.2015.03.009. PubMed DOI

Meyn MA, III, Wilson MB, Abdi FA, Fahey N, Schiavone AP, Wu J, Hochrein JM, Engen JR, Smithgall TE. Src Family Kinases Phosphorylate the Bcr-Abl SH3-SH2 Region and Modulate Bcr-Abl Transforming Activity. Journal of Biological Chemistry. 2006;281:30907–30916. doi: 10.1074/jbc.M605902200. PubMed DOI

Morrogh LM, Hinshelwood S, Costello P, Cory GO, Kinnon C. The SH3 domain of Bruton’s tyrosine kinase displays altered ligand binding properties when auto-phosphorylated in vitro. European Journal of Immunology. 1999;29:2269–2279. doi: 10.1002/(SICI)1521-4141(199907)29:07<2269::AID-IMMU2269>3.0.CO;2-#. PubMed DOI

Park H, Wahl MI, Afar DE, Turck CW, Rawlings DJ, Tam C, Scharenberg AM, Kinet JP, Witte ON. Regulation of Btk function by a major autophosphorylation site within the SH3 domain. Immunity. 1996;4:515–525. doi: 10.1016/s1074-7613(00)80417-3. PubMed DOI

Pellman D, Garber EA, Cross FR, Hanafusa H. An N-terminal peptide from p60src can direct myristylation and plasma membrane localization when fused to heterologous proteins. Nature. 1985;314:374–377. doi: 10.1038/314374a0. PubMed DOI

Penuel E, Martin GS. Transformation by v-Src: Ras-MAPK and PI3K-mTOR mediate parallel pathways. Molecular Biology of the Cell. 1999;10:1693–1703. doi: 10.1091/mbc.10.6.1693. PubMed DOI PMC

Pérez Y, Maffei M, Igea A, Amata I, Gairí M, Nebreda AR, Bernadó P, Pons M. Lipid binding by the Unique and SH3 domains of c-Src suggests a new regulatory mechanism. Scientific Reports. 2013;3:1295. doi: 10.1038/srep01295. PubMed DOI PMC

Roskoski R. Src kinase regulation by phosphorylation and dephosphorylation. Biochemical and Biophysical Research Communications. 2005;331:1–14. doi: 10.1016/j.bbrc.2005.03.012. PubMed DOI

Spassov DS, Ruiz-Saenz A, Piple A, Moasser MM. A Dimerization Function in the Intrinsically Disordered N-Terminal Region of Src. Cell Reports. 2018;25:449–463. doi: 10.1016/j.celrep.2018.09.035. PubMed DOI PMC

Tatárová Z, Brábek J, Rösel D, Novotný M. SH3 domain tyrosine phosphorylation--sites, role and evolution. PLOS ONE. 2012;7:e36310. doi: 10.1371/journal.pone.0036310. PubMed DOI PMC

Thomas SM, Brugge JS. Cellular functions regulated by Src family kinases. Annual Review of Cell and Developmental Biology. 1997;13:513–609. doi: 10.1146/annurev.cellbio.13.1.513. PubMed DOI

Wang C, Pawley NH, Nicholson LK. The role of backbone motions in ligand binding to the c-Src SH3 domain. Journal of Molecular Biology. 2001;313:873–887. doi: 10.1006/jmbi.2001.5083. PubMed DOI

Wu Y, Spencer SD, Lasky LA. Tyrosine Phosphorylation Regulates the SH3-mediated Binding of the Wiskott-Aldrich Syndrome Protein to PSTPIP, a Cytoskeletal-associated Protein. Journal of Biological Chemistry. 1998;273:5765–5770. doi: 10.1074/jbc.273.10.5765. PubMed DOI

Wu JC, Chen YC, Kuo CT, Wenshin Yu H, Chen YQ, Chiou A, Kuo JC. Focal adhesion kinase-dependent focal adhesion recruitment of SH2 domains directs SRC into focal adhesions to regulate cell adhesion and migration. Scientific Reports. 2015;5:18476. doi: 10.1038/srep18476. PubMed DOI PMC

Xu W, Harrison SC, Eck MJ. Three-dimensional structure of the tyrosine kinase c-Src. Nature. 1997;385:595–602. doi: 10.1038/385595a0. PubMed DOI

Xu W, Doshi A, Lei M, Eck MJ, Harrison SC. Crystal structures of c-Src reveal features of its autoinhibitory mechanism. Molecular Cell. 1999;3:629–638. doi: 10.1016/s1097-2765(00)80356-1. PubMed DOI

Yeatman TJ. A renaissance for SRC. Nature Reviews. Cancer. 2004;4:470–480. doi: 10.1038/nrc1366. PubMed DOI

Young MA, Gonfloni S, Superti-Furga G, Roux B, Kuriyan J. Dynamic Coupling between the SH2 and SH3 Domains of C-Src and Hck Underlies Their Inactivation by C-Terminal Tyrosine Phosphorylation. Cell. 2001;105:115–126. doi: 10.1016/S0092-8674(01)00301-4. PubMed DOI

Plasma membrane and nuclear phosphatidylinositol 4,5-bisphosphate signalling in cancer

Synthesis and migrastatic activity of cytochalasin analogues lacking a macrocyclic moiety