Control of Src kinase activity with uniRapR domain. (A) Schematic representation of activity control with the uniRapR domain. (B) Root mean square fluctuations of the ATP binding site (gray structure) based on multiple equilibrium DMD simulations for WT Src (black), apo (red), and holo (green) uniRapR-inserted Src (P < 0.01). (C) HEK293T cells expressing the Src-uniRapR-cerulean-myc construct were treated with different concentrations of rapamycin (0–2 µM), and lysates were assayed for expression of the construct with Western blotting by using anti-GFP. The construct was pulled down with anti-myc and mixed with the paxillin substrate in the presence of ATP for 10 min. Reaction suspensions were blotted and probed with anti-myc and anti–pY31-paxillin to confirm binding and phosphorylation of the substrate, respectively. (D) As controls, constitutively active Src (YF) without the uniRapR domain, kinase dead (YF/KD), Y271A and L1polyP Src mutants with the uniRapR domain, and our previous dimerization-based switch were tested. (E) Y271A and L1polyP substitutions shown on the Src-uniRapR model.

Testing uniRapR in different kinases and effects of Src activation in HeLa cells. Immunoprecipitation, in vitro FAK (A), and p38 (B) assays were performed similarly as for Src kinase. (C) Change in cell area of HeLa cells expressing Src (YF)-uniRapR (n = 8/8 cells) or Src (YF/KD)-uniRapR (n = 8/8 cells). (D) HeLa cells expressing Src-uniRapR (YF)-cerulean demonstrate spreading after the addition of rapamycin.

Activation of Src induces cell changes in zebrafish epidermal cells. (A) Synthetic transposase mRNA was coinjected with the Tol2 Krt4 Src (YF)-uniRapR-cerulean plasmid into one-cell zebrafish embryos, resulting in mosaic expression of Src-uniRapR in the epidermis. Epidermal cells with characteristic flat honeycomb morphology were selected and imaged before (B and F) and after (C and G) 16 h of rapamycin treatment. (C) Epidermal cells expressing Src (YF)-uniRapR-cerulean in zebrafish embryos exposed to 10 μM rapamycin become rounded and undergo dynamic cell shape changes. White arrows indicate an epidermal cell before and after each treatment. Control epidermal cells expressing Src (YF)-uniRapR-cerulean in vehicle (i.e., DMSO) (D and E) or expressing the kinase-dead construct Src (YF/KD)-uniRapR-cerulean in rapamycin (G) have a static morphology and do not undergo dynamic cell shape changes. (Scale bar: 30 μm.)

Testing different lengths of double linkers between uniRapR subdomains. (A) Double linker connecting the subdomains-A and -B are shown in red and cyan. The linker with asterisks is the one used in the current version of uniRapR. (B) Src-uniRapR-cerulean-myc constructs were expressed in HEK293T cells. Cell lysates were blotted with anti-GFP to confirm the expression of the construct. In dimerization-based switch (rapR), coexpressed FRB was also tested with anti-GFP. Cell lysates were pulled down with anti-myc and mixed with substrate paxillin in the presence of ATP for 10 min. Reaction suspension was blotted with anti-myc to confirm the binding, with anti–pY31-paxilin to confirm the phosphorylation of the substrate. Linker 2 is the one required for functional uniRapR. (C) The design with truncated linker 2 (linker 1) does not provide control over Src.

A different design with different connections between secondary structure components (A) does not provide any control over FAK (B).