Differential p38 mitogen-activated protein kinase-controlled hypophosphorylation of the retinoblastoma protein induced by nitric oxide in neuroblastoma cells

In this report we show that exogenous NO added to human neuroblastoma NB69 cells inhibits cell proliferation and downregulates the epidermal growth factor receptor (EGFR) and its downstream signaling pathways. These comprise the 3-phosphoinositide-dependent kinase 1/Akt/glycogen synthase kinase-3β pathway, the mitogen-activated protein kinase (MAPK)/extracellular-regulated kinases 1 and 2 pathway, and the phospholipase Cγ pathway. In contrast, NO enhances the EGFR-controlled p38MAPK pathway. We also show that NO enhances the activation of the cAMP-responsive element binding protein, a transcription factor controlled by p38MAPK, as demonstrated using 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole (SB202190), a p38MAPK inhibitor. These processes are accompanied by the NO-mediated hypophosphorylation of the retinoblastoma protein (pRb), preferentially at Ser795 compared to Ser780 and Ser807/811, and the downregulation of p27 KIP1, p21 CIP1/WAF1, and p16 INK4a, although NO downregulated p16 INK4a only when the p38MAPK activity was suppressed. The p38MAPK pathway controls the phosphorylation status of pRb as SB202190 enhances the hypophosphorylation of pRb. We reverted the inhibitory action of NO on EGFR and pRb phosphorylation in living cells using cell-permeable reducing agents, which suggested that reversible S-nitrosation controls these proteins. Our results support the notion that NO negatively modulates the p38MAPK-controlled phosphorylation of pRb, inducing the subsequent arrest of the cell cycle at the G1/S transition. © 2007 Elsevier Inc. All rights reserved.This work was financed by grants (to A.V.) from the Dirección General de Investigación, Ministerio de Educación y Ciencia (SAF2005-00631); the Consejería de Educación de la Comunidad de Madrid (S-BIO-0170-2006); and the European Commission (Contract MRTN-CT-2005-19561). O.G.F. is the recipient of an Undergraduate Excellence's scholarship from the Consejería de Educación de la Comunidad de Madrid, and A.J. is a staff research technician from the CSIC.Peer Reviewe