Abstract
The rapidly accelerated fibrosarcoma (Raf) kinase is canonically activated by growth factors that regulate multiple cellular processes. In this kinase cascade Raf activation ultimately results in extracellular regulated kinase 1/2 (Erk1/2) activation, which requires Ras binding to the Ras binding domain (RBD) of Raf. We recently reported that all-trans retinoic acid (atRA) rapidly (within minutes) activates Erk1/2 to modulate cell cycle progression in stem cells, which is mediated by cellular retinoic acid binding protein 1 (Crabp1). But how atRA-bound Crabp1 regulated Erk1/2 activity remained unclear. We now report Raf kinase as the direct target of atRA-Crabp1. Molecularly, Crabp1 acts as a novel atRA-inducible scaffold protein for Raf/Mek/Erk in cells without growth factor stimulation. However, Crabp1 can also compete with Ras for direct interaction with the RBD of Raf, thereby negatively modulating growth factor-stimulated Raf activation, which can be enhanced by atRA binding to Crabp1. NMR heteronuclear single quantum coherence (HSQC) analyses reveal the 6-strand β-sheet face of Crabp1 as its Raf-interaction surface. We identify a new atRA-mimicking and Crabp1-selective compound, C3, that can also elicit such an activity. This study uncovers a new signal crosstalk between endocrine (atRA-Crabp1) and growth factor (Ras-Raf) pathways, providing evidence for atRA-Crabp1 as a novel modulator of cell growth. The study also suggests a new therapeutic strategy by employing Crabp1-selective compounds to dampen growth factor stimulation while circumventing RAR-mediated retinoid toxicity.
Highlights
The rapidly accelerated fibrosarcoma (Raf) kinase is canonically activated by growth factors that regulate multiple cellular processes
We report Raf kinase as the direct target of all-trans retinoic acid (atRA)-Crabp[1] and determine the molecular mechanism of cellular retinoic acid binding protein 1 (Crabp1)’s action, which occurs through direct interaction with the Relative signal of Flag (Ras) binding domain (RBD) of Raf kinase
The classical/canonical mitogen activated protein kinase (MAPK) signaling begins with receptor tyrosine kinase activation on the cell membrane and propagates through Ras-Raf-Mek-Erk for various biological processes such as cell proliferation, cell cycle regulation, cell survival and apoptosis, etc
Summary
Crabp[1] sequentially forms complexes with components of the Raf-Mek-Erk signaling pathway to modulate Erk1/2 phosphorylation. Co-immunoprecipitation demonstrates that Crabp[1] forms stable complexes with cRaf, Mek[2] and Erk1/2 in the presence of atRA (Fig. 1C, comparing lanes 2 and 3). These data suggest that Crabp[1] may serve as a signal scaffold, or allosteric regulator, of cRaf, conveying Raf to Mek and Erk activation by forming an atRA-inducible signalosome activating the final target kinase, Erk1/2. In this in vitro kinase assay, atRA alone, without growth factor stimulation, has no effect on Raf activity (Mek[1] phosphorylation; the 2nd lane).
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