Role of O-linked beta-N-acetylglucosamine modification in the subcellular distribution of alpha4 phosphoprotein and Sp1 in rat lymphoma cells.
The mTOR alpha4 phosphoprotein is a prolactin (PRL)-downregulated gene product that is found in the nucleus of PRL-dependent rat Nb2 lymphoma cells. Alpha4 lacks a nuclear localization signal (NLS) and the mechanism of its nuclear targeting is unknown. Post-translational modification by O-linked beta-N-acetylglucosamine (O-GlcNAc) moieties has been implicated in the nuclear transport of some proteins, including transcription factor Sp1. The nucleocytoplasmic enzymes O-beta-N-acetylglucosaminyltransferase (OGT) and O-beta-N-acetylglucosaminidase (O-GlcNAcase) adds or remove O-GlcNAc moieties, respectively. If O-GlcNac moieties contribute to the nuclear targeting of alpha4, a decrease in O-GlcNAcylation (e.g., by inhibition of OGT) may redistribute alpha4 to the cytosol. The present study showed that alpha4 and Sp1 were both O-GlcNAcylated in quiescent and PRL-treated Nb2 cells. PRL alone or PRL + streptozotocin (STZ; an O-GlcNAcase inhibitor) significantly (P <or=.05) increased the O-GlcNAc/alpha4 ratio above that in control quiescent cells. However, PRL + alloxan (ALX; an OGT inhibitor) or ALX alone did not decrease O-GlcNAcylation of alpha4 below that of controls and alpha4 remained nuclear. In comparison, PRL (+/-ALX/STZ) greatly increased Sp1 protein levels, caused a significant decrease in the GlcNAc/Sp1 ratio (P <or=0.05, n = 3) as compared to controls and partially redistributed Sp1 to the cytosol. Finally, a 50% downregulation of OGT gene expression by small interfering RNA (i.e., siOGT) partially redistributed both alpha4 and Sp1 to the cytosol. The alpha4 protein partner PP2Ac had no detectable O-GlcNAc moieties and its nuclear distribution was not affected by siOGT. In summary, alpha4 and Sp1 contained O-GlcNAc moieties, which contributed to their nuclear targeting in Nb2 cells.
Dauphinee
Shauna M
SM
Department of Biochemistry and Molecular Biology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada.
Ma
Marlene
M
Too
Catherine K L
CK
D000117:Acetylglucosamine / Q000737:chemistry* / Q000378:metabolism*; D000118:Acetylglucosaminidase / Q000235:genetics / Q000378:metabolism; D000496:Alloxan / Q000378:metabolism; D000818:Animals; D045744:Cell Line, Tumor; D020868:Gene Silencing; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008223:Lymphoma / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D011388:Prolactin / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D016329:Sp1 Transcription Factor / Q000737:chemistry* / Q000378:metabolism*; D013311:Streptozocin / Q000378:metabolism
D007527:Isoenzymes; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D016329:Sp1 Transcription Factor; D013311:Streptozocin; D000496:Alloxan; D011388:Prolactin; D010749:Phosphoprotein Phosphatases; D000118:Acetylglucosaminidase; D000117:Acetylglucosamine