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