Optogenetic clustering of CNK1 reveals mechanistic insights in RAF and AKT signalling controlling cell fate decisions

Abstract

Scaffold proteins such as the multidomain protein CNK1 orchestrate the signalling network by integrating and controlling the underlying pathways. Using an optogenetic approach to stimulate CNK1 uncoupled from upstream effectors, we identified selective clusters of CNK1 that either stimulate RAF-MEK-ERK or AKT signalling depending on the light intensity applied. OptoCNK1 implemented in MCF7 cells induces differentiation at low light intensity stimulating ERK activity whereas stimulation of AKT signalling by higher light intensity promotes cell proliferation. CNK1 clustering in response to increasing EGF concentrations revealed that CNK1 binds to RAF correlating with ERK activation at low EGF dose. At higher EGF dose active AKT binds to CNK1 and phosphorylates and inhibits RAF. Knockdown of CNK1 protects CNK1 from this AKT/RAF crosstalk. In C2 skeletal muscle cells CNK1 expression is induced with the onset of differentiation. Hence, AKT-bound CNK1 counteracts ERK stimulation in differentiated but not in proliferating cells. Ectopically expressed CNK1 facilitates C2 cell differentiation and knockdown of CNK1 impaired the transcriptional network underlying C2 cell differentiation. Thus, CNK1 expression, CNK1 clustering and the thereto related differential signalling processes decide on proliferation and differentiation in a cell type- and cell stage-dependent manner by orchestrating AKT and RAF signalling.

Membrane localization of acetylated CNK1 mediates a positive feedback on RAF/ERK signaling

“…Growth factors stimulate the RAF/MEK/ERK and PI3K/AKT pathways, although with different intensities ( 37 ). CNK1 is an effector of growth-stimulated RTKs and promotes ERK and AKT signaling in a mutually exclusive manner ( 16 ). Early in signaling and at low signal intensity, CNK1 forms complexes with CRAF, leading to ERK activation.…”

Optogenetic Approaches for the Spatiotemporal Control of Signal Transduction Pathways

“…Alternating pulses of blue/far-red light with pulses of blue/red light kept optoAKT in its active state while optoSOS repeatedly switched between the active and inactive state [ 5 ]. Similarly, Bugaj and Lim combined PHYB/PIF-based optoSOS and iLID-based optoPI3K [ 68 , 76 ]. Multichromatic approaches enable the analysis of interconnected signaling pathways in a highly dynamic manner to better understand the temporal aspects of the cross-interaction.…”

“…AKT phosphorylation-dependent oligomerization and the subcellular localization control the function of CNK1 [ 77 , 78 ]. In stimulated cells, CNK1 connected AKT and RAF and mediated AKT-dependent phosphorylation and inhibition of RAF [ 68 ]. Depending on the cluster size induced by blue light, CNK1-CRY2 (optoCNK1) stimulated RAF/ERK in case of low light intensity and AKT by higher light intensity.…”

“…OptoCNK1-stimulated AKT counteracted ERK signaling because of the negative feedback of AKT on RAF. In MCF7 cells, optoCNK1 induced ERK-mediated proliferation or AKT-induced differentiation dependent on the light intensity applied to facilitate the control of cell fate decision at the level of AKT and ERK by a single light-controlled signaling protein [ 68 ].…”

Perspectives of RAS and RHEB GTPase Signaling Pathways in Regenerating Brain Neurons

“…Dependent on the light intensity, Opto-CNK1 stimulated RAF/MEK/ERK or AKT signaling in Michigan Cancer Foundation 7 (MCF7, human breast adenocarcinoma cell line) cells. At low light intensity Opto-CNK1 induced differentiation by stimulating ERK activity, whereas triggering AKT signaling stimulated cell proliferation at higher light intensity [245].…”