Abstract
The selective pressure imposed by extrinsic death signals and stressors adds to the challenge of isolating and interpreting the roles of proteins in stress-activated signaling networks. By expressing a kinase with activating mutations and a caged lysine blocking the active site, we can rapidly switch on catalytic activity with light and monitor the ensuing dynamics. Applying this approach to MAP kinase 6 (MKK6), which activates the p38 subfamily of MAPKs, we found that decaging active MKK6 in fibroblasts is sufficient to trigger apoptosis in a p38-dependent manner. Both in fibroblasts and in a murine melanoma cell line expressing mutant B-Raf, MKK6 activation rapidly and potently inhibited the pro-proliferative extracellular signal-regulated kinase (ERK) pathway; to our surprise, this negative cross-regulation was equally robust when all p38 isoforms were inhibited. These results position MKK6 as a new pleiotropic signal transducer that promotes both pro-apoptotic and anti-proliferative signaling, and they highlight the utility of caged, light-activated kinases for dissecting stress-activated signaling networks.
Highlights
Cells respond to their dynamically changing and chemically diverse surroundings through highly regulated, intracellular signal transduction networks
This interaction was first suggested by the evidence that ERK1/2 co-immunoprecipitates with p38 [12]; activation of p38 by constitutively active MKK3 resulted in potent inhibition of extracellular signal–regulated kinase (ERK) and its upstream mitogenactivated protein kinase (MAPK) kinase (MAPKK), mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK)
Protein kinases form the backbone of signal transduction networks, and several genetic and proteomic approaches have been developed and refined to study their regulation and function [63,64,65]
Summary
Cells respond to their dynamically changing and chemically diverse surroundings through highly regulated, intracellular signal transduction networks. Whereas the ERK cascade controls cell proliferation in response to growth factor stimulation [3, 4], JNK and p38 are considered stress-activated protein kinases (SAPKs) that commonly promote apoptosis and other responses to environmental insults and inflammatory cytokines [5,6,7] Consistent with these opposing roles that influence normal tissue development, homeostasis, and immune responses, aberrations in MAPK signal. Various strategies have been implemented to address the limitations outlined above, most commonly by inducing the association or dissociation of a protein-protein interaction via the addition of a small-molecule dimerizer or through light exposure [19,20,21,22] This strategy involves the translocation of a signaling protein to the plasma membrane to enhance its activity or the release of a protein from a sequestering interaction [23,24,25,26].
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