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  • A new regulatory mechanism for Raf kinase activation, retinoic acid-bound Crabp1.

A new regulatory mechanism for Raf kinase activation, retinoic acid-bound Crabp1.

Scientific reports (2019-07-31)
Sung Wook Park, Jennifer Nhieu, Shawna D Persaud, Michelle C Miller, Youlin Xia, Yi-Wei Lin, Yu-Lung Lin, Hiroyuki Kagechika, Kevin H Mayo, Li-Na Wei
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
ISOGRO®-15N Powder -Growth Medium, 98 atom % 15N
Sigma-Aldrich
Monoclonal ANTI-FLAG® M2 antibody produced in mouse, clone M2, purified immunoglobulin (Purified IgG1 subclass), buffered aqueous solution (10 mM sodium phosphate, 150 mM NaCl, pH 7.4, containing 0.02% sodium azide)
Sigma-Aldrich
Retinoic acid, ≥98% (HPLC), powder
Sigma-Aldrich
EGF human, Animal-component free, recombinant, expressed in E. coli, ≥98% (SDS-PAGE), ≥98% (HPLC), suitable for cell culture
Sigma-Aldrich
Duolink® flowPLA Detection Kit - FarRed, Duolink® PLA kit for Flow Cytometry with FarRed Detection
Sigma-Aldrich
Raf-1 RBD Protein, GST, 300 µg, GST fusion-protein, corresponding to the human Ras Binding Domain (RBD, residues 1-149) of Raf-1, expressed in E. coli. with purity 50% at full length molecular weight 42 kDa. For use in Affinity Binding Assays