Phosphorylation of RAF kinase dimers drives conformational changes that facilitate transactivation

TY - JOUR

T1 - Phosphorylation of RAF kinase dimers drives conformational changes that facilitate transactivation

AU - Jambrina, Pablo G.

AU - Rauch, Nora

AU - Pilkington, Ruth

AU - Rybakova, Katja

AU - Nguyen, Lan K.

AU - Kholodenko, Boris N.

AU - Buchete, Nicolae Viorel

AU - Kolch, Walter

AU - Rosta, Edina

PY - 2016/1/18

Y1 - 2016/1/18

N2 - RAF kinases are key players in the MAPK signaling pathway and are important targets for personalized cancer therapy. RAF dimerization is part of the physiological activation mechanism, together with phosphorylation, and is known to convey resistance to RAF inhibitors. Herein, molecular dynamics simulations are used to show that phosphorylation of a key N-terminal acidic (NtA) motif facilitates RAF dimerization by introducing several interprotomer salt bridges between the αC-helix and charged residues upstream of the NtA motif. Additionally, we show that the R-spine of RAF interacts with a conserved Trp residue in the vicinity of the NtA motif, connecting the active sites of two protomers and thereby modulating the cooperative interactions in the RAF dimer. Our findings provide a first structure-based mechanism for the auto-transactivation of RAF and could be generally applicable to other kinases, opening new pathways for overcoming dimerization-related drug resistance. Back to back: Biochemical kinase assays and structural evidence from MD simulations reveal a key electrostatic role for the phosphorylated NtA motif in RAF kinase transactivation. Interprotomer salt bridges and a conserved tryptophan residue located at the N-terminal end of the kinase domain play crucial roles by connecting the R-spines of the two protomers, and phosphorylation leads to important structural changes in the highly conserved HRD motif.

AB - RAF kinases are key players in the MAPK signaling pathway and are important targets for personalized cancer therapy. RAF dimerization is part of the physiological activation mechanism, together with phosphorylation, and is known to convey resistance to RAF inhibitors. Herein, molecular dynamics simulations are used to show that phosphorylation of a key N-terminal acidic (NtA) motif facilitates RAF dimerization by introducing several interprotomer salt bridges between the αC-helix and charged residues upstream of the NtA motif. Additionally, we show that the R-spine of RAF interacts with a conserved Trp residue in the vicinity of the NtA motif, connecting the active sites of two protomers and thereby modulating the cooperative interactions in the RAF dimer. Our findings provide a first structure-based mechanism for the auto-transactivation of RAF and could be generally applicable to other kinases, opening new pathways for overcoming dimerization-related drug resistance. Back to back: Biochemical kinase assays and structural evidence from MD simulations reveal a key electrostatic role for the phosphorylated NtA motif in RAF kinase transactivation. Interprotomer salt bridges and a conserved tryptophan residue located at the N-terminal end of the kinase domain play crucial roles by connecting the R-spines of the two protomers, and phosphorylation leads to important structural changes in the highly conserved HRD motif.

KW - kinases

KW - molecular dynamics

KW - oncogenic signaling

KW - phosphorylation

KW - RAF kinase

UR - http://www.scopus.com/inward/record.url?scp=84954377294&partnerID=8YFLogxK

U2 - 10.1002/anie.201509272

DO - 10.1002/anie.201509272

M3 - Article

AN - SCOPUS:84954377294

SN - 1433-7851

VL - 55

SP - 983

EP - 986

JO - ANGEWANDTE CHEMIE-INTERNATIONAL EDITION

JF - ANGEWANDTE CHEMIE-INTERNATIONAL EDITION

IS - 3

ER -