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

Adrian Fischer,Bettina Warscheid,Wilfried Weber,Gerald Radziwill

doi:10.1038/srep38155

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.

Highlights

Cells process numerous signals, originating from internal biological events or the environment to generate the appropriate cellular response
Analysing C2 skeletal muscle cells and MCF7 breast cancer cells we demonstrate that CNK1 expression and CNK1-mediated signalling decides on proliferation versus differentiation in a cell type- and cell stage-dependent manner
To study the biological impact of oligomeric CNK1 uncoupled from upstream signals, we generated optoCNK1 based on CNK1 fused to PHR-cryptochrome 2 (CRY2) (CNK1-CRY2) (Fig. 1A)

Summary

Introduction

Cells process numerous signals, originating from internal biological events or the environment to generate the appropriate cellular response. We chose an optogenetic approach to precisely control the oligomerization state of CNK1 to study CNK1-mediated signalling uncoupled from upstream signalling induced in a time-resolved manner. PHR-CRY2 (abbreviated hereafter as CRY2) oligomerises within seconds upon exposure to blue light of 460 nm wavelength and dissociates within minutes in the dark[13,14,15] This approach has been successfully used to induce signalling by CRY2-mediated oligomerization of chimeric RAF proteins or chimeric fibroblast growth factor receptors (FGFR)[16,17,18] and by indirect oligomerization of endogenous receptor tyrosine kinases including FGFR, platelet-derived growth factor receptor (PDGFR) or integrins[19]. Analysing C2 skeletal muscle cells and MCF7 breast cancer cells we demonstrate that CNK1 expression and CNK1-mediated signalling decides on proliferation versus differentiation in a cell type- and cell stage-dependent manner

Methods

Results

Conclusion