Identification of sites subjected to serine/threonine phosphorylation by SGK1 affecting N-myc downstream-regulated gene 1 (NDRG1)/Cap43-dependent suppression of angiogenic CXC chemokine expression in human pancreatic cancer cells

We have recently reported that N-myc downstream-regulated gene 1 (NDRG1)/Ca2+-associated protein with a molecular mass of 43 kDa (Cap43) suppresses angiogenesis and tumor growth of pancreatic cancer through marked decreases in both the expression of CXC chemokines and phosphorylation of a NF-kappa B signaling molecule, inhibitor of kappa B kinase (I kappa B alpha). NDRG1/Cap43 is phosphorylated at serine/threonine sites in its C-terminal domain by serum- and glucocorticoid-regulated kinase 1 (SGK1). In this study, we attempted to clarify the domain or site of NDRG1/Cap43 responsible for its suppression of CXC chemokine expression in pancreatic cancer cells. Expression of the deletion constructs Cap Delta 2 [deletion of amino acids (AA) 130-142] and Cap Delta 4 [deletion of AA 180-294] as well as the wild-type full sequence of NDRG1/Cap43 (F-Cap), suppressed the production of CXC chemokines such as Gro alpha/CXCL1 and ENA-78/CXCL5, whereas no or low suppression was observed in cell expressing the Cap Delta 5 mutant [deletion of AA 326-350] and Cap Delta 6 mutant (deletion of AA 326-394]. We further introduced mutations at the serine and threonine sites at 328 [T3284330 [ S330A] and 346 [T346A], which are susceptible to phosphorylation by SGK1, and also constructed double mutants [T328A, S330A], [T328A, T346A] and [S330A, T346A]. Expression of all these mutants, with the exception of [ S330A, T346A], suppressed the production of CXC chemokine to similar levels as their wild-type counterpart. I kappa B alpha was found to be specifically phosphorylated by this double mutant [S330A, T346A] and the Cap Delta 5 mutant at levels comparable to that induced in their wild-type counterpart. Phosphorylation of NDRG1/Cap43 at both serine330 and threonine346 is required for its suppressive action on the NF-kappa B signaling pathway and CXC chemokine expression in pancreatic cancer cells. (C) 2010 Elsevier Inc. All rights reserved.