Novel function and intracellular localization of methionine adenosyltransferase 2beta splicing variants.
Human methionine adenosyltransferase 2beta (MAT2beta) encodes for two major splicing variants, V1 and V2, which are differentially expressed in normal tissues. Both variants are induced in human liver cancer and positively regulate growth. The aim of this work was to identify interacting proteins of V1 and V2. His-tagged V1 and V2 were overexpressed in Rosetta pLysS cells, purified, and used in a pulldown assay to identify interacting proteins from human colon cancer cell line RKO cell lysates. The eluted lysates were subjected to Western blot and in solution proteomic analyses. HuR, an mRNA-binding protein known to stabilize the mRNA of several cyclins, was identified to interact with V1 and V2. Immunoprecipitation and Western blotting confirmed their interaction in both liver and colon cancer cells. These variant proteins are located in both nucleus and cytoplasm in liver and colon cancer cells and, when overexpressed, increased the cytoplasmic HuR content. This led to increased expression of cyclin D1 and cyclin A, known targets of HuR. When endogenous expression of V1 or V2 is reduced by small interference RNA, cytoplasmic HuR content fell and the expression of these HuR target genes also decreased. Knockdown of cyclin D1 or cyclin A blunted, whereas knockdown of HuR largely prevented, the ability of V1 or V2 overexpression to induce growth. In conclusion, MAT2beta variants reside mostly in the nucleus and regulate HuR subcellular content to affect cell proliferation.
Xia
Meng
M
Division of Gastroenterology and Liver Diseases, University of Southern California (USC) Research Center for Liver Diseases, USC-UCLA Research Center for Alcoholic Liver and Pancreatic Diseases, Keck School of Medicine USC, Los Angeles, CA 90033, USA.
Chen
Yongheng
Y
Wang
Ling-Chi
LC
Zandi
Ebrahim
E
Yang
Heping
H
Bemanian
Sean
S
Martínez-Chantar
M Luz
ML
Mato
José M
JM
Lu
Shelly C
SC
D017398:Alternative Splicing; D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000201:enzymology; D049109:Cell Proliferation; D019925:Cyclin A / Q000235:genetics; D019938:Cyclin D1 / Q000235:genetics; D003593:Cytoplasm / Q000201:enzymology; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D015972:Gene Expression Regulation, Neoplastic; D056945:Hep G2 Cells; D006801:Humans; D042541:Intracellular Space / Q000201:enzymology*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008716:Methionine Adenosyltransferase / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D011485:Protein Binding; D034622:RNA Interference; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D000954:Antigens, Surface; D019925:Cyclin A; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; D007527:Isoenzymes; D016601:RNA-Binding Proteins; D019938:Cyclin D1; C527162:MAT2B protein, human; D008716:Methionine Adenosyltransferase