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

Biochem Biophys Res Commun. 2010 May 28;396(2):376-81. doi: 10.1016/j.bbrc.2010.04.100. Epub 2010 Apr 21.

Abstract

We have recently reported that N-myc downstream-regulated gene 1 (NDRG1)/Ca(2+)-associated protein with a molecular mass of 43kDa (Cap43) suppresses angiogenesis and tumor growth of pancreatic cancer through marked decreases in both the expression of CXC chemokines and phosphorylation of a NF-kappaB signaling molecule, inhibitor of kappaB kinase (IkappaBalpha). 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 CapDelta2 [deletion of amino acids (AA) 130-142] and CapDelta4 [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 Groalpha/CXCL1 and ENA-78/CXCL5, whereas no or low suppression was observed in cell expressing the CapDelta5 mutant [deletion of AA 326-350] and CapDelta6 mutant [deletion of AA 326-394]. We further introduced mutations at the serine and threonine sites at 328 [T328A], 330 [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. IkappaBalpha was found to be specifically phosphorylated by this double mutant [S330A, T346A] and the CapDelta5 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-kappaB signaling pathway and CXC chemokine expression in pancreatic cancer cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • Cell Line, Tumor
  • Chemokines, CXC / biosynthesis*
  • Humans
  • Immediate-Early Proteins / metabolism*
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • NF-kappa B / metabolism
  • Neovascularization, Pathologic / genetics
  • Neovascularization, Pathologic / metabolism*
  • Pancreatic Neoplasms / blood supply
  • Pancreatic Neoplasms / metabolism*
  • Pancreatic Neoplasms / pathology
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / metabolism*
  • Sequence Deletion
  • Serine / genetics
  • Serine / metabolism
  • Threonine / genetics
  • Threonine / metabolism

Substances

  • Cell Cycle Proteins
  • Chemokines, CXC
  • Immediate-Early Proteins
  • Intracellular Signaling Peptides and Proteins
  • N-myc downstream-regulated gene 1 protein
  • NF-kappa B
  • Threonine
  • Serine
  • Protein-Serine-Threonine Kinases
  • serum-glucocorticoid regulated kinase