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.

Resveratrol Affects Protein Kinase C Activity and Promotes Apoptosis in Human Colon Carcinoma Cells

“…Activation of ERK has been considered to be involved in cell proliferation (McCubrey et al, 2007;Abrams et al, 2010); while inhibition of ERK activation with a MEK inhibitor, PD98059, induced apoptosis in a dose-dependent manner in breast cancer cells. The ERK pathway was up-regulated in human cancers, such as colon cancer (Davies et al, 2010), and blockage of the ERK pathway would enhance the induction of apoptosis in cancer cells .…”

Arginine methylation of the c-Jun coactivator RACO-1 is required for c-Jun/AP-1 activation

“…(Davies et al, 2010). Arginine methylation appeared to counteract RACO-1 degradation, as overexpression of myc-PRMT1 led to a considerable increase in RACO-1 protein levels ( Figure 5A) and half-life as determined by CHX chase ( Figure 5B).…”

“…We have previously shown that K48-linked autoubiquitylation is counteracted by Ras/MEK1-induced K63-linked ubiquitylation (Davies et al, 2010). Given that arginine methylation can affect the deposition of other posttranslational modifications on the same protein (Hyllus et al, 2007;Yamagata et al, 2008), we thus investigated the impact of arginine methylation on K63-linked ubiquitylation of RACO-1.…”

“…GFP-RACO-1 was co-immunoprecipitated with Flag-RACO-1, implying that dimerisation does occur ( Figure 1B). Point mutations of the first two cysteine residues of the RACO-1 RING domain (RACO-1 RM) destroy RING integrity (Davies et al, 2010); however, dimer formation was not affected. Thus, the RING domain is dispensable for dimerisation ( Figure 1B).…”

“…RACO-1 is a RING domain-containing ubiquitin E3 ligase and in unstimulated conditions RACO-1 is unstable due to K48-linked autoubiquitylation. Upon activation of the Ras/MEK/ERK pathway, K63-linked ubiquitin chains are attached to the same residues targeted for degradative ubiquitylation, thereby resulting in enhanced protein levels (Davies et al, 2010).…”