Identification of a co-activator that links growth factor signalling to c-Jun/AP-1 activation

Abstract

The AP-1 transcription factor c-Jun is essential for cellular proliferation in many cell types, but the molecular link between growth factors and c-Jun activation has been enigmatic. In this study we identify a previously uncharacterized RING-domain-containing protein, RACO-1 (RING domain AP-1 co-activator-1), as a c-Jun co-activator that is regulated by growth factor signalling. RACO-1 interacted with c-Jun independently of amino-terminal phosphorylation, and was both necessary and sufficient for c-Jun/AP-1 activation. Growth factor-mediated stimulation of AP-1 was attributable to MEK/ERK-dependent stabilization of RACO-1 protein. Stimulation of the MEK/ERK pathway strongly promoted Lys 63-linked ubiquitylation of RACO-1, which antagonized Lys 48-linked degradative auto-ubiquitylation of the same Lys residues. RACO-1 depletion reduced cellular proliferation and decreased expression of several growth-associated AP-1 target genes, such as cdc2, cyclinD1 and hb-egf. Moreover, transgenic overexpression of RACO-1 augmented intestinal tumour formation triggered by aberrant Wnt signalling and cooperated with oncogenic Ras in colonic hyperproliferation. Thus RACO-1 is a co-activator that links c-Jun to growth factor signalling and is essential for AP-1 function in proliferation.

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Figure 1: RACO-1 binds to c-Jun independently of N-terminal phosphorylation.
Figure 2: RACO-1 is a MEKK1-dependent c-Jun co-activator regulated by MAP kinase signalling pathways.
Figure 3: Growth factor signalling through the MEK pathway stabilizes RACO-1 protein.
Figure 4: MEK signalling promotes the formation of Lys 63-linked ubiquitin chains that antagonize degradative auto-ubiquitylation.
Figure 5: The residues targeted for MEK1-induced ubiquitylation chain elongation are also critical for degradative poly-ubiquitylation.
Figure 6: RACO-1 is an important regulator of cell-cycle progression and proliferation in NIH3T3 fibroblasts.
Figure 7: Transgenic expression of RACO-1 in vivo enhances APCmin+-induced intestinal tumorigenesis and cooperates with oncogenic Ras in colonic hyperproliferation.

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Acknowledgements

We are grateful to N. Ahn, M. Cobb and A. Hock for providing reagents. We thank B. Spencer-Dean, E. Nye and R. Mitter for technical support; the Molecular Biology Core Facility, Paterson Institute for Cancer Research, Manchester, UK for microarray analysis; and H. Walden for critical reading of the manuscript. The London Research Institute is funded by Cancer Research UK.

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C.D and A.C performed all experiments; C.D and A.B designed and analysed all experiments and wrote the manuscript; K.H and J.H generated R26RACO−1 transgenic mice; F.C generated RACO-1 antibodies.

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Correspondence to Axel Behrens.

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Davies, C., Chakraborty, A., Cipriani, F. et al. Identification of a co-activator that links growth factor signalling to c-Jun/AP-1 activation. Nat Cell Biol 12, 963–972 (2010). https://doi.org/10.1038/ncb2098

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