Figure 8.
Schematic illustration of engineered synthetic Src kinases. (a) Schematic illustration of the synthetic Src kinase work concept. In a similar way as in Fig. 1 a and Fig. 4 c, synthetic kinase uses GFP to bring a constitutively active kinase domain (Src KD) in close proximity to a fluorescent fusion substrate protein (target). The persistence of the kinase domain around the fluorescent fusion protein allows for efficient phosphorylation (P) of the target. In the absence of the GFP-target, dGBP1 nanobody is destabilized, and the whole nanobody-kinase fusion protein is targeted for degradation. (b) The structure of Drosophila Src42A protein. The C-terminal part of Drosophila Src42A (amino acid 214–517) spanning the ATP-binding site, Src catalytic domain, and the crucial regulatory tyrosine residue Y511 was used for synthetic kinase constructs shown in (c–e). (c) Linear representation of dGBP1-HA::Src Y400E, dGBP1-HA::Src Y400D and dGBP1-HA::SrcDead in which N-terminal dGBP1 with HA tag (black squares) was fused with C-terminal part of Src shown in b. (d) Linear representation of Src Y400E, Src Y400D, and SrcDead for which the exact same part of Src42A was used as for the constructs presented in c. These constructs lack the dGBP1 binder, and an in-frame START codon (ATG) was added N-terminally. (e) Linear representation of Src Y400E::dGBP1-HA, Src Y400D::dGBP1-HA, SrcDead::dGBP1-HA and Src::dGBP1-HA in which the order is changed: the dGBP-1-HA binder was fused C-terminally to the Src part and an in-frame START codon (ATG) was added N-terminally. In Src::dGBP-1-HA the regulatory tyrosine Y400 is left unmutated. SrcDead constructs contain a K276M single amino acid substitution (M) that abolishes catalytic activity. Numbers refer to amino acid positions from N-terminus (N) to C-terminus (C).