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)/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.
Murakami
Yuichi
Y
Department of Pharmaceutical Oncology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
Hosoi
Fumihito
F
Izumi
Hiroto
H
Maruyama
Yuichiro
Y
Ureshino
Hiroki
H
Watari
Kosuke
K
Kohno
Kimitoshi
K
Kuwano
Michihiko
M
Ono
Mayumi
M
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D019743:Chemokines, CXC / Q000096:biosynthesis*; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D009389:Neovascularization, Pathologic / Q000235:genetics / Q000378:metabolism*; D010190:Pancreatic Neoplasms / Q000098:blood supply / Q000378:metabolism* / Q000473:pathology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017384:Sequence Deletion; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D000099184:Serum-Glucocorticoid Regulated Kinases; D000099259:N-myc Downstream-Regulated Gene 1 Protein
D018797:Cell Cycle Proteins; D019743:Chemokines, CXC; D017874:Immediate-Early Proteins; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; D017346:Protein Serine-Threonine Kinases; D012694:Serine; D013912:Threonine; D000099184:Serum-Glucocorticoid Regulated Kinases; D000099259:N-myc Downstream-Regulated Gene 1 Protein