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Role of O ‐linked β‐ N ‐acetylglucosamine modification in the subcellular distribution of alpha4 phosphoprotein and Sp1 in rat lymphoma cells
Author(s) -
Dauphinee Shauna M.,
Ma Marlene,
Too Catherine K.L.
Publication year - 2005
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.20508
Subject(s) - phosphoprotein , cytosol , chemistry , subcellular localization , nuclear transport , phosphorylation , nuclear export signal , cell nucleus , small interfering rna , microbiology and biotechnology , transfection , biochemistry , enzyme , cytoplasm , biology , gene
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 β‐ N ‐acetylglucosamine ( O ‐GlcNAc) moieties has been implicated in the nuclear transport of some proteins, including transcription factor Sp1. The nucleocytoplasmic enzymes O ‐β‐ N ‐acetylglucosaminyltransferase (OGT) and O ‐β‐ 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 ≤ 0.05) 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 ≤ 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. © 2005 Wiley‐Liss, Inc.