RSK promotes G2 DNA damage checkpoint silencing and participates in melanoma chemoresistance.

The incidence of malignant melanoma is growing rapidly worldwide and there is still no effective therapy for metastatic disease. This type of cancer is highly resistant to conventional DNA-damaging chemotherapeutics, and intense research has been dedicated for understanding the molecular pathways underlying chemoresistance. The Ras/mitogen-activated protein kinase (MAPK) signalling pathway is often deregulated in melanoma, which frequently harbours activating mutations in NRAS or BRAF. Herein, we demonstrate that the MAPK-activated protein kinase RSK (p90 ribosomal S6 kinase) contributes to melanoma chemoresistance by altering their response to chemotherapeutic agents. We find that RSK phosphorylates checkpoint kinase 1 (Chk1) at an inhibitory site, Ser280, both in vitro and in vivo. Our results indicate that RSK is the predominant protein kinase operating downstream of mitogens and oncogenes of the Ras/MAPK pathway, and consistent with this, we find that RSK constitutively phosphorylates Chk1 in melanoma. We show that RSK inhibition increases Chk1 activity in response to DNA-damaging agents, suggesting that the Ras/MAPK pathway modulates Chk1 function and the response to DNA damage. Accordingly, we demonstrate that RSK promotes G2 DNA damage checkpoint silencing in a Chk1-dependent manner, and find that RSK inhibitors sensitize melanoma cells to DNA-damaging agents. Together, our results identify a novel link between the Ras/MAPK pathway and the DNA damage response, and suggest that RSK inhibitors may be used to modulate chemosensitivity, which is one of the major obstacles to melanoma treatment.

Characterization of a G protein-activated phosphoinositide 3-kinase in vascular smooth muscle cell nuclei.

Recent studies highlight the existence of an autonomous nuclear polyphosphoinositide metabolism related to cellular proliferation and differentiation. However, only few data document the nuclear production of the putative second messengers, the 3-phosphorylated phosphoinositides, by the phosphoinositide 3-kinase (PI3K). In the present paper, we examine whether GTP-binding proteins can directly modulate 3-phosphorylated phosphoinositide metabolism in membrane-free nuclei isolated from pig aorta smooth muscle cells (VSMCs). In vitro PI3K assays performed without the addition of any exogenous substrates revealed that guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) specifically stimulated the nuclear synthesis of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), whereas guanosine 5'-(beta-thio)diphosphate was ineffective. PI3K inhibitors wortmannin and LY294002 prevented GTPgammaS-induced PtdIns(3,4,5)P(3) synthesis. Moreover, pertussis toxin inhibited partially PtdIns(3,4,5)P(3) accumulation, suggesting that nuclear G(i)/G(0) proteins are involved in the activation of PI3K. Immunoblot experiments showed the presence of Galpha(0) proteins in VSMC nuclei. In contrast with previous reports, immunoblots and indirect immunofluorescence failed to detect the p85alpha subunit of the heterodimeric PI3K within VSMC nuclei. By contrast, we have detected the presence of a 117-kDa protein immunologically related to the PI3Kgamma. These results indicate the existence of a G protein-activated PI3K inside VSMC nucleus that might be involved in the control of VSMC proliferation and in the pathogenesis of vascular proliferative disorders.