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. 2016 Apr 27;2(5):325–331. doi: 10.1021/acscentsci.6b00024

Figure 3.

Figure 3

Bioorthogonal chemical activation of Src oncogenic variant in living cells. (a) Schematic model for bioorthogonal activation of oncogenic Src variant. Substitution of the catalytic lysine (K295) with TCOK-a on the oncogenic Src mutant (Src-Y527F) generated the chemically caged oncogenic Src variant Src-K295TCOK-Y527F (gray). Decaging of TCOK-a via bioorthogonal cleavage reaction could rescue Src activity (orange), leading to phosphorylation of its downstream oncogenic-related substrates. (b) Increase of Src autophosphorylation and downstream phosphorylation level after Me2Tz-mediated decaging of Src-K295TCOK-Y527F in live HEK293T cells. (c) Representative images of 293A cells expressing the oncogenic Src-Y527F variant which led to cell rounding and detaching. 293A cells expressing the “kinase dead” Src mutant (Src-K295R-Y527F) was used as the control. Scale bars, 5 μm. (d) Representative images of phenotypic changes upon chemical rescue of oncogenic Src. Upon Me2Tz mediated bioorthogonal activation, 293A cells expressing Src-K295TCOK-Y527F began to rapidly round up. Me2Tz, 100 μM; Scale bars, 5 μm.