Light-activated kinases enable temporal dissection of signaling networks in living cells

Abstract

We report a general strategy for creating protein kinases in mammalian cells that are poised for very rapid activation by light. By photoactivating a caged version of MEK1, we demonstrate the specific, rapid, and receptor independent activation of an artificial subnetwork within the Raf/MEK/ERK pathway. Time-lapse microscopy allowed us to precisely characterize the kinetics of elementary steps in the signaling cascade and provided insight into adaptive feedback and rate-determining processes in the pathway.

Figure 1

Isolating a subnetwork in MAP kinase signaling via genetically encoding a photocaged lysine in MEK1 active site. (a) Schematic of the MAP kinase signaling pathway and the synthetic, photoactivable subnetwork. (b) Caging a near universally conserved lysine (K97) in the MEK1 active site inactivates the enzyme by sterically blocking ATP binding. Decaging with light rapidly removes the caging group and activates the kinase (figures created using Pymol and MEK1 structure PDB: 1S9J). (c) Photocaged lysine 1.

Figure 2

Specific phosphorylation of ERK2 upon photoactivation of the caged MEK1. HEK293ET cells cotransfected with plasmids encoding PCKRS, pyrrolysyl tRNA_CUA, EGFP-ERK2, and either A-MEK1-ΔN-HA (A, lane 2), D-MEK1-ΔN-HA (D, lanes 3−6), or C-MEK1-ΔN-HA (C, lanes 7−10) were grown in medium supplemented with 2 mM of 1 and 0.1% FBS for 24 h. Cells expressing D-MEK1-ΔN-HA and C-MEK1-ΔN-HA were illuminated with a 365 nm LED lamp for 60 s. Cells were lysed 1, 10, and 60 min after illumination. Cell lysates were resolved by SDS-PAGE, followed by immunoblotting (IB) with the indicated antibodies.

Figure 3

EGFP-ERK2 nuclear translocation upon photoactivation of the caged MEK1. (a) Representative EGFP-ERK2 subcellular fluorescence at different time points after photoactivation (2 s, 365 nm, 1 mW/cm²) of coexpressed C-MEK1-DD. Scale bars represent 5 μm (see movie 2). (b) Normalized F(n/c) (ratio of nuclear to cytoplasmic EGFP fluorescence intensities) as a function of time after photoactivation (mean ± SD of 10 independent experiments). The gray line shows the normalized F(n/c) observed when cells are stimulated with EGF (see Figure S6 and movie 1, SI). (c) Scheme of the potential negative feedback within the photoactivatable subnetwork. (d) U0126 (10 μM) was added 8 min after photoactivation of C-MEK1-DD (black line), control experiment without inhibitor addition is gray line (Figure S10 and movie 3, SI ). (e) High-resolution kinetics of translocation following C-MEK1-DD photoactivation (mean ± SD of 10 independent experiments) is shown. Data were fitted with a sigmoidal function (Figure S12 and movie 5, SI).