title: Role of methionine adenosyltransferase α2 and β phosphorylation and stabilization in human hepatic stellate cell trans-differentiation.
abstract: Myofibroblastic trans-differentiation of hepatic stellate cells (HSCs) is an essential event in the development of liver fibrogenesis. These changes involve modulation of key regulators of the genome and the proteome. Methionine adenosyltransferases (MAT) catalyze the biosynthesis of the methyl donor, S-adenosylmethionine (SAMe) from methionine. We have previously shown that two MAT genes, MAT2A and MAT2B (encoding MATα2 and MATβ proteins respectively), are required for HSC activation and loss of MAT2A transcriptional control favors its up-regulation during trans-differentiation. Hence MAT genes are intrinsically linked to the HSC machinery during activation. In the current study, we have identified for the first time, post-translational modifications in the MATα2 and MATβ proteins that stabilize them and favor human HSC trans-differentiation. Culture-activation of human HSCs induced the MATα2 and MATβ proteins. Using mass spectrometry, we identified phosphorylation sites in MATα2 and MATβ predicted to be phosphorylated by mitogen-activated protein kinase (MAPK) family members (ERK1/2, V-Raf Murine Sarcoma Viral Oncogene Homolog B1 [B-Raf], MEK). Phosphorylation of both proteins was enhanced during HSC activation. Blocking MEK activation lowered the phosphorylation and stability of MAT proteins without influencing their mRNA levels. Silencing ERK1/2 or B-Raf lowered the phosphorylation and stability of MATβ but not MATα2. Reversal of the activated human HSC cell line, LX2 to quiescence lowered phosphorylation and destabilized MAT proteins. Mutagenesis of MATα2 and MATβ phospho-sites destabilized them and prevented HSC trans-differentiation. The data reveal that phosphorylation of MAT proteins during HSC activation stabilizes them thereby positively regulating trans-differentiation.
authors.affiliation: Division of Gastroenterology and Liver Diseases, USC Research Center for Liver Diseases, Keck School of Medicine University of Southern California, Los Angeles, California.
authors.forename: Komal
authors.initials: K
authors.lastname: Ramani
authors.forename: Shant
authors.initials: S
authors.lastname: Donoyan
authors.forename: Maria Lauda
authors.initials: ML
authors.lastname: Tomasi
authors.forename: Sunhee
authors.initials: S
authors.lastname: Park
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D054338:Cell Transdifferentiation; D004795:Enzyme Stability; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D020868:Gene Silencing; D055166:Hepatic Stellate Cells / Q000166:cytology* / Q000201:enzymology*; D006801:Humans; D008716:Methionine Adenosyltransferase / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D011485:Protein Binding; D048493:Proto-Oncogene Proteins B-raf / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; C527161:MAT2A protein, human; C527162:MAT2B protein, human; D008716:Methionine Adenosyltransferase; D048493:Proto-Oncogene Proteins B-raf; D048049:Extracellular Signal-Regulated MAP Kinases