Role of O-linked β-N-acetylglucosamine modification in the subcellular distribution of alpha4 phosphoprotein and Sp1 in rat lymphoma cells
✍ Scribed by Shauna M. Dauphinee; Marlene Ma; Catherine K.L. Too
- Publisher
- John Wiley and Sons
- Year
- 2005
- Tongue
- English
- Weight
- 247 KB
- Volume
- 96
- Category
- Article
- ISSN
- 0730-2312
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✦ Synopsis
Abstract
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.