Focal adhesion kinase (FAK) is essential for vascular endothelial growth factor-A (VEGFA)/VEGF receptor-2 (VEGFR2)-stimulated angiogenesis and vascular permeability. We have previously noted that presence of the Src homology-2 domain adapter protein B (SHB) is of relevance for VEGFA-stimulated angiogenesis in a FAK-dependent manner. The current study was conducted in order address the temporal dynamics of co-localization between these components in HEK293 and primary lung endothelial cells (EC) by total internal reflection fluorescence microscopy (TIRF). An early (<2.5 min) VEGFA-induced increase in VEGFR2 co-localization with SHB was dependent on tyrosine 1175 in VEGFR2. VEGFA also enhanced SHB co-localization with FAK. FAK co-localization with VEGFR2 was dependent on SHB since it was significantly lower in SHB deficient EC after VEGFA addition. Absence of SHB also resulted in a gradual decline of VEGFR2 co-localization with FAK under basal (prior to VEGFA addition) conditions. A similar basal response was observed with expression of the Y1175F-VEGFR2 mutant in wild type EC. The distribution of focal adhesions in SHB-deficient EC was altered with a primarily perinuclear location. These live cell data implicate SHB as a key component regulating FAK activity in response to VEGFA/VEGFR2.

Department of Immunology Genetics and Pathology Uppsala University 75108 Uppsala Sweden

Department of Medical Cell Biology Uppsala University Box 571 75123 Uppsala Sweden

Institute of Molecular Genetics of the CAS 14220 Prague Czech Republic

Present address Department of Immunology Genetics and Pathology Uppsala University 75108 Uppsala Sweden

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Ferrara N. Vascular endothelial growth factor: basic science and clinical progress. Endocr. Rev. 2004;25:581–611. doi: 10.1210/er.2003-0027. PubMed DOI

Heidenreich R., Machein M., Nicolaus A., Hilbig A., Wild C., Clauss M., Plate K.H., Breier G. Inhibition of solid tumor growth by gene transfer of VEGF receptor-1 mutants. Int. J. Cancer. 2004;111:348–357. doi: 10.1002/ijc.20260. PubMed DOI

Heidenreich R., Murayama T., Silver M., Essl C., Asahara T., Rocken M., Breier G. Tracking adult neovascularization during ischemia and inflammation using Vegfr2-LacZ reporter mice. J. Vasc. Res. 2008;45:437–444. doi: 10.1159/000126106. PubMed DOI

Claesson-Welsh L., Welsh M. VEGFA and tumour angiogenesis. J. Intern. Med. 2013;273:114–127. doi: 10.1111/joim.12019. PubMed DOI

Testini C., Smith R.O., Jin Y., Martinsson P., Sun Y., Hedlund M., Sainz-Jaspeado M., Shibuya M., Hellstrom M., Claesson-Welsh L. Myc-dependent endothelial proliferation is controlled by phosphotyrosine 1212 in VEGF receptor-2. EMBO Rep. 2019 doi: 10.15252/embr.201947845. PubMed DOI PMC

Chen X.L., Nam J.O., Jean C., Lawson C., Walsh C.T., Goka E., Lim S.T., Tomar A., Tancioni I., Uryu S., et al. VEGF-induced vascular permeability is mediated by FAK. Dev. Cell. 2012;22:146–157. doi: 10.1016/j.devcel.2011.11.002. PubMed DOI PMC

Tavora B., Batista S., Reynolds L.E., Jadeja S., Robinson S., Kostourou V., Hart I., Fruttiger M., Parsons M., Hodivala-Dilke K.M. Endothelial FAK is required for tumour angiogenesis. EMBO Mol. Med. 2010;2:516–528. doi: 10.1002/emmm.201000106. PubMed DOI PMC

Welsh M., Jamalpour M., Zang G., Akerblom B. The role of the Src Homology-2 domain containing protein B (SHB) in beta cells. J. Mol. Endocrinol. 2016;56:R21–R31. doi: 10.1530/JME-15-0228. PubMed DOI

Akerblom B., Zang G., Zhuang Z.W., Calounova G., Simons M., Welsh M. Heterogeneity among RIP-Tag2 insulinomas allows vascular endothelial growth factor-A independent tumor expansion as revealed by studies in Shb mutant mice: Implications for tumor angiogenesis. Mol. Oncol. 2012;6:333–346. doi: 10.1016/j.molonc.2012.01.006. PubMed DOI PMC

Christoffersson G., Zang G., Zhuang Z.W., Vagesjo E., Simons M., Phillipson M., Welsh M. Vascular adaptation to a dysfunctional endothelium as a consequence of Shb deficiency. Angiogenesis. 2012;15:469–480. doi: 10.1007/s10456-012-9275-z. PubMed DOI PMC

Funa N.S., Kriz V., Zang G., Calounova G., Akerblom B., Mares J., Larsson E., Sun Y., Betsholtz C., Welsh M. Dysfunctional microvasculature as a consequence of shb gene inactivation causes impaired tumor growth. Cancer Res. 2009;69:2141–2148. doi: 10.1158/0008-5472.CAN-08-3797. PubMed DOI

Nikpour M., Gustafsson K., Vagesjo E., Seignez C., Giraud A., Phillipson M., Welsh M. Shb deficiency in endothelium but not in leucocytes is responsible for impaired vascular performance during hindlimb ischaemia. Acta Physiol. (Oxf) 2015;214:200–209. doi: 10.1111/apha.12448. PubMed DOI

Zang G., Gustafsson K., Jamalpour M., Hong J., Genove G., Welsh M. Vascular dysfunction and increased metastasis of B16F10 melanomas in Shb deficient mice as compared with their wild type counterparts. BMC Cancer. 2015;15:234. doi: 10.1186/s12885-015-1269-y. PubMed DOI PMC

Zang G., Christoffersson G., Tian G., Harun-Or-Rashid M., Vagesjo E., Phillipson M., Barg S., Tengholm A., Welsh M. Aberrant association between vascular endothelial growth factor receptor-2 and VE-cadherin in response to vascular endothelial growth factor-a in Shb-deficient lung endothelial cells. Cell. Signal. 2013;25:85–92. doi: 10.1016/j.cellsig.2012.09.018. PubMed DOI

Holmqvist K., Cross M., Riley D., Welsh M. The Shb adaptor protein causes Src-dependent cell spreading and activation of focal adhesion kinase in murine brain endothelial cells. Cell. Signal. 2003;15:171–179. doi: 10.1016/S0898-6568(02)00076-1. PubMed DOI

Dergai O.V., Yaruchik A.M., Rynditch A.V. Focal adhesion kinase (FAK1) regulates SHB phosphorylation and its binding with a range of signaling proteins. Biopolym. Cell. 2016;32:34–40. doi: 10.7124/bc.00090A. DOI

Holmqvist K., Cross M.J., Rolny C., Hagerkvist R., Rahimi N., Matsumoto T., Claesson-Welsh L., Welsh M. The adaptor protein shb binds to tyrosine 1175 in vascular endothelial growth factor (VEGF) receptor-2 and regulates VEGF-dependent cellular migration. J. Biol. Chem. 2004;279:22267–22275. doi: 10.1074/jbc.M312729200. PubMed DOI

Alenkvist I., Dyachok O., Tian G., Li J., Mehrabanfar S., Jin Y., Birnir B., Tengholm A., Welsh M. Absence of Shb impairs insulin secretion by elevated FAK activity in pancreatic islets. J. Endocrinol. 2014;223:267–275. doi: 10.1530/JOE-14-0531. PubMed DOI

Gustafsson K., Heffner G., Wenzel P.L., Curran M., Grawe J., McKinney-Freeman S.L., Daley G.Q., Welsh M. The Src homology 2 protein Shb promotes cell cycle progression in murine hematopoietic stem cells by regulation of focal adhesion kinase activity. Exp. Cell Res. 2013;319:1852–1864. doi: 10.1016/j.yexcr.2013.03.020. PubMed DOI

Gustafsson K., Jamalpour M., Trinh C., Kharas M.G., Welsh M. The Src homology-2 protein Shb modulates focal adhesion kinase signaling in a BCR-ABL myeloproliferative disorder causing accelerated progression of disease. J. Hematol. Oncol. 2014;7:45. doi: 10.1186/1756-8722-7-45. PubMed DOI PMC

Kriz V., Mares J., Wentzel P., Funa N.S., Calounova G., Zhang X.Q., Forsberg-Nilsson K., Forsberg M., Welsh M. Shb null allele is inherited with a transmission ratio distortion and causes reduced viability in utero. Dev. Dyn. 2007;236:2485–2492. doi: 10.1002/dvdy.21257. PubMed DOI

Matsumoto T., Bohman S., Dixelius J., Berge T., Dimberg A., Magnusson P., Wang L., Wikner C., Qi J.H., Wernstedt C., et al. VEGF receptor-2 Y951 signaling and a role for the adapter molecule TSAd in tumor angiogenesis. EMBO J. 2005;24:2342–2353. doi: 10.1038/sj.emboj.7600709. PubMed DOI PMC

Yu Q., Shuai H., Ahooghalandari P., Gylfe E., Tengholm A. Glucose controls glucagon secretion by directly modulating cAMP in alpha cells. Diabetologia. 2019;62:1212–1224. doi: 10.1007/s00125-019-4857-6. PubMed DOI PMC

Gampel A., Moss L., Jones M.C., Brunton V., Norman J.C., Mellor H. VEGF regulates the mobilization of VEGFR2/KDR from an intracellular endothelial storage compartment. Blood. 2006;108:2624–2631. doi: 10.1182/blood-2005-12-007484. PubMed DOI

Kanteti R., Batra S.K., Lennon F.E., Salgia R. FAK and paxillin, two potential targets in pancreatic cancer. Oncotarget. 2016;7:31586–31601. doi: 10.18632/oncotarget.8040. PubMed DOI PMC

Lundby A., Franciosa G., Emdal K.B., Refsgaard J.C., Gnosa S.P., Bekker-Jensen D.B., Secher A., Maurya S.R., Paul I., Mendez B.L., et al. Oncogenic Mutations Rewire Signaling Pathways by Switching Protein Recruitment to Phosphotyrosine Sites. Cell. 2019;179:543–560.e526. doi: 10.1016/j.cell.2019.09.008. PubMed DOI

Jorgensen C., Sherman A., Chen G.I., Pasculescu A., Poliakov A., Hsiung M., Larsen B., Wilkinson D.G., Linding R., Pawson T. Cell-specific information processing in segregating populations of Eph receptor ephrin-expressing cells. Science. 2009;326:1502–1509. doi: 10.1126/science.1176615. PubMed DOI

Takahashi T., Yamaguchi S., Chida K., Shibuya M. A single autophosphorylation site on KDR/Flk-1 is essential for VEGF-A-dependent activation of PLC-gamma and DNA synthesis in vascular endothelial cells. EMBO J. 2001;20:2768–2778. doi: 10.1093/emboj/20.11.2768. PubMed DOI PMC

Pedrosa A.R., Bodrug N., Gomez-Escudero J., Carter E.P., Reynolds L.E., Georgiou P.N., Fernandez I., Lees D.M., Kostourou V., Alexopoulou A.N., et al. Tumor Angiogenesis Is Differentially Regulated by Phosphorylation of Endothelial Cell Focal Adhesion Kinase Tyrosines-397 and -861. Cancer Res. 2019;79:4371–4386. doi: 10.1158/0008-5472.CAN-18-3934. PubMed DOI

Cross M.J., Lu L., Magnusson P., Nyqvist D., Holmqvist K., Welsh M., Claesson-Welsh L. The Shb adaptor protein binds to tyrosine 766 in the FGFR-1 and regulates the Ras/MEK/MAPK pathway via FRS2 phosphorylation in endothelial cells. Mol. Biol. Cell. 2002;13:2881–2893. doi: 10.1091/mbc.e02-02-0103. PubMed DOI PMC