Extracellular signals controlling gene expression ultimately converge upon the cell nucleus. Selective nuclear transport plays a key role in regulating the levels and activities of transcription factors, nuclear kinases, steroid hormone receptors and replication factors. Although targeting sequences for nuclear import, export and subnuclear targeting have been defined, these processes remain poorly understood. The nuclear pore complex (NPC) mediates transport across the nuclear envelope. This bidirectional transport process is regulated, in part, by the ubiquitous intracellular mediators GTP and Ca+2, suggesting a coordinated regulation of nuclear transport and other signal transduction pathways. We identified calmodulin as an activator of nuclear transport and suggested that it plays a role in the regulation of transport during cell activation. Thus, both the GTP-dependent molecular switch, Ran, and the Ca+2 dependent switch, calmodulin, may function as regulators of nuclear transport. These studies have led to the proposal that calmodulin and Ran are molecular switches which form a gradient across the nuclear membrane and regulate the stability of transport complexes.Post-translational modifications of nuclear pore components are another means of regulating nuclear transport and gene expression. The Nuclear pore complex contains numerous proteins termed nucleoporins bearing both phosphate and O-GlcNAc attached to Ser and Thr residues. We cloned the cDNA and gene encoding the major nucleoporin p62. By producing p62 in Escherichia coli, we produced a substrate suitable for examining the post translational modification of p62 in vitro and for purification of the O-linked GlcNAc transferase. We have studied the glycosylation and phosphorylation of nuclear pore proteins extensively. This post-translational modification is also present on RNA polymerase II, and numerous polymerase II transcription factors. Addition and removal of O-GlcNAc are dynamic processes occurring in the cytoplasm and nucleoplasm. In may ways, O-GlcNAc addition is analogous to protein phosphorylation. O-GlcNAc is transferred to proteins from UDP-GlcNAc, a sugar nucleotide whose levels are regulated by the hexosamine biosynthetic pathway. The hexosamine biosynthetic pathway is a cellular sensor of energy availability and mediates the effects of glucose on expression of a number of gene products including leptin, the product of the ob gene. Most importantly, the accumulation of UDP-GlcNAc in skeletal muscle, an index of the flux through the hexosamine pathway, correlates highly with the degree of insulin resistance. Based on the substrates modified by O-GlcNAc, we have proposed that O-GlcNAc is directly involved in glucose sensing and the modulation of nuclear transport and transcription . We recently cloned the cDNA encoding the O-linked GlcNAc transferase (OGT) and demonstrated that OGT is a highly conserved protein with multiple tetratricopeptide (TPR) repeats. Current effortsare directed at site-directed mutagenesis of the OGT coding sequence. In addition, our recent identification of the cDNA and gene encoding O-linked GlcNAc transferase should facilitate dissection of the enzyme?s potential role in a novel glycan dependent signal transduction pathway that appears to be disregulated in diseases such as diabetes mellitus. - O-linked GlcNAc Nuclear Pore Nuclear Transport Nuclear Import Nuclear Export Glycobiology HIV Rev HIV Tat
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