p14ARF triggers G2 arrest through ERK-mediated Cdc25C phosphorylation, ubiquitination and proteasomal degradation.
The Cdc25C phosphatase is a key regulator of mitotic entry which activity is tightly regulated by phosphorylation. In response to DNA damage, phosphorylation at serine 216 induces the cytosolic retention of Cdc25C through 14-3-3 binding. We previously reported the ability of the p14ARF tumor suppressor to induce the accumulation of inactive phospho-Cdc25C(Ser216) protein as well as a decrease of Cdc25C steady state level and correlated these events with a p53-independent G2 arrest. The aim of this study was to investigate the cellular signaling pathways involved in this process. By using specific pharmacological inhibitors, we demonstrate that activation of the ERK1/2 MAP kinases pathway is involved in the p53-independent G2 checkpoint induced by p14ARF Moreover, we show that activated P-ERK1/2 bind and phosphorylate Cdc25C on its ser216 residue following p14ARF expression, thereby identifying Cdc25C as a new ERK1/2 target. Importantly, we further show that phosphorylation at Ser216 by phospho-ERK1/2 promotes Cdc25C ubiquitination and proteasomal degradation, suggesting that Cdc25C proteolysis is required for a sustained G2 arrest in response to p14ARF. Taken together, these results demonstrate that the MAPK ERK signaling pathway contributes to the p53-independent antiproliferative functions of p14ARF. Furthermore, they identify a new mechanism by which phosphorylation at serine 216 participates to Cdc25C inactivation.
Eymin
Beatrice
B
INSERM U578, Groupe de Recherche sur le Cancer du Poumon, Université Joseph Fourier, La Tronche, France. Beatrice.Eymin@ujf-grenoble.fr
Claverie
Paule
P
Salon
Caroline
C
Brambilla
Christian
C
Brambilla
Elisabeth
E
Gazzeri
Sylvie
S
D018797:Cell Cycle Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D016195:G2 Phase / Q000502:physiology*; D015870:Gene Expression; D006801:Humans; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D020935:MAP Kinase Signaling System; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D014407:Tumor Cells, Cultured; D025803:Tumor Suppressor Protein p14ARF / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*; D020687:cdc25 Phosphatases / Q000378:metabolism*
D018797:Cell Cycle Proteins; D025803:Tumor Suppressor Protein p14ARF; D025801:Ubiquitin; D010768:Phosphoserine; D048049:Extracellular Signal-Regulated MAP Kinases; D020930:MAP Kinase Kinase Kinases; C517009:CDC25C protein, human; D020687:cdc25 Phosphatases; D046988:Proteasome Endopeptidase Complex