Figure 1.
Schematic illustration of engineered kinases. (a) Overview of engineered kinase work concept. A synthetic kinase uses a small fluorescent protein binder (here: vhhGFP4, a GFP nanobody) to bring a constitutively active kinase domain (kinase) in close proximity to a fluorescent fusion protein (target). The persistence of the kinase domain around the fluorescent fusion protein allows for efficient phosphorylation (P) of the target. (b) Schematic illustration of non-muscle myosin II activation and actomyosin contractility. Non-phosphorylated Sqh/Myosin-II regulatory light chain (MRLC) assembles into an inactive compact molecule through a head to tail interaction. Reversible phosphorylation of Sqh at Ser21 (Ser19 in mammalian MRLC) results in myosin II molecule unfolding, allowing association with other myosin II molecules in an anti-parallel fashion and binding to the actin filaments through the head domains. An ATP-dependent conformational change in myosin II drives the actin filament sliding in an anti-parallel manner and results in contraction. (c) The structure of mammalian and Drosophila ROCK proteins. The Drosophila Rok kinase region shares ∼65% identity with the corresponding isolated domain of mammalian ROCK1 (Verdier et al., 2006). (d–f) The N-terminal kinase region (N-Rok) of Drosophila Rok (amino acid 1–452) was used for synthetic kinase constructs shown in (d–f). (d) Linear representation of N-Rok::vhhGFP4, N-RokDead::vhhGFP4, N-Rok::vhhGFP4-HA, N-RokDead::vhhGFP4-HA, vhhGFP4, N-Rok-HA and N-RokDead-HA. (e) Linear representation of N-Rok::dGBP1, in which a destabilized GFP-binding nanobody (dGBP1; Tang et al., 2016) substitutes vhhGFP4 from d. (f) Linear representation of N-Rok::2m22 and N-RokDead::2m22 in which a DARPin recognizing mCherry substitutes vhhGFP4 from d. In some constructs, the human influenza hemagglutinin tag (HA; black squares) was added to the C-terminus of the synthetic kinases to allow detection by immunofluorescence. N-RokDead contains a K116G single amino acid substitution (G) that abolishes catalytic activity. The proteins are aligned vertically with the N-Rok domain. Numbers refer to amino acid positions from N-terminus (N) to C-terminus (C).

Schematic illustration of engineered kinases. (a) Overview of engineered kinase work concept. A synthetic kinase uses a small fluorescent protein binder (here: vhhGFP4, a GFP nanobody) to bring a constitutively active kinase domain (kinase) in close proximity to a fluorescent fusion protein (target). The persistence of the kinase domain around the fluorescent fusion protein allows for efficient phosphorylation (P) of the target. (b) Schematic illustration of non-muscle myosin II activation and actomyosin contractility. Non-phosphorylated Sqh/Myosin-II regulatory light chain (MRLC) assembles into an inactive compact molecule through a head to tail interaction. Reversible phosphorylation of Sqh at Ser21 (Ser19 in mammalian MRLC) results in myosin II molecule unfolding, allowing association with other myosin II molecules in an anti-parallel fashion and binding to the actin filaments through the head domains. An ATP-dependent conformational change in myosin II drives the actin filament sliding in an anti-parallel manner and results in contraction. (c) The structure of mammalian and Drosophila ROCK proteins. The Drosophila Rok kinase region shares ∼65% identity with the corresponding isolated domain of mammalian ROCK1 (Verdier et al., 2006). (d–f) The N-terminal kinase region (N-Rok) of Drosophila Rok (amino acid 1–452) was used for synthetic kinase constructs shown in (d–f). (d) Linear representation of N-Rok::vhhGFP4, N-RokDead::vhhGFP4, N-Rok::vhhGFP4-HA, N-RokDead::vhhGFP4-HA, vhhGFP4, N-Rok-HA and N-RokDead-HA. (e) Linear representation of N-Rok::dGBP1, in which a destabilized GFP-binding nanobody (dGBP1; Tang et al., 2016) substitutes vhhGFP4 from d. (f) Linear representation of N-Rok::2m22 and N-RokDead::2m22 in which a DARPin recognizing mCherry substitutes vhhGFP4 from d. In some constructs, the human influenza hemagglutinin tag (HA; black squares) was added to the C-terminus of the synthetic kinases to allow detection by immunofluorescence. N-RokDead contains a K116G single amino acid substitution (G) that abolishes catalytic activity. The proteins are aligned vertically with the N-Rok domain. Numbers refer to amino acid positions from N-terminus (N) to C-terminus (C).

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal