Reduced O Glycosylation of Sp1 Is Associated with Increased Proteasome Susceptibility

Abstract

Sp1 is a ubiquitously expressed transcription factor that is particularly important for the regulation of TATA-less genes that encode housekeeping proteins. Most growth factors and receptors are also encoded by such genes. Sp1 is multiply O glycosylated by covalent linkage of the monosaccharide N-acetylglucosamine (O-GlcNAc) to serine and threonine residues. Based on an earlier observation that growth factor gene transcription can be regulated by glucose and glucosamine in vascular smooth muscle cells, we determined whether Sp1 glycosylation could be regulated and if this modification altered Sp1 function. We found that Sp1 becomes hyperglycosylated when cells are exposed to 5 mM glucosamine, whereas under glucose starvation, stimulation with cyclic AMP (cAMP) results in nearly complete deglycosylation of this protein. Correlating with this hypoglycosylated state, Sp1 is rapidly proteolytically degraded by an enzyme(s) that can be inhibited by specific proteasome inhibitors, lactacystin and LLnL. Treatment of cells with glucose or glucosamine protects Sp1 from cAMP-mediated degradation, whereas blockade of glucosamine synthesis abrogates glucose but not glucosamine protection. This effect on Sp1 is specific, in that the Stat-3 and E2F transcription factors did not undergo degradation under these conditions. The O-GlcNAc modification of Sp1 may play a role as a nutritional checkpoint. In the absence of adequate nutrition, Sp1 becomes hypoglycosylated and thereby subject to proteasome degradation. This process could potentially result in reduced general transcription, thereby conserving nutrients.

Metabolic Inhibitors of O‐GlcNAc Transferase That Act In Vivo Implicate Decreased O‐GlcNAc Levels in Leptin‐Mediated Nutrient Sensing

“…However, our data provide the first direct correlation between decreased O‐GlcNAc levels and impaired leptin production in vivo. Our findings are in accord with studies showing that reduced O‐GlcNAc lowers levels of Sp1 in cells6f, 13 and that OGT plays a pivitol role in fat tissues 14. Notably, these data also suggest that regulation of leptin by O‐GlcNAc is bidirectional in vivo as overexpression of OGT,3 knockout of OGA,15 and metabolic upregulation of UDP GlcNAc levels16 all increase leptin expression.…”

P38 mitogen-activated protein kinase mediates hexosamine-induced TGFβ1 mRNA expression in human mesangial cells

“…The cellular content of the transcription factor Sp1, involved in the regulation of TGFβ1 transcription [31], are up-regulated by PKC activation [32]. Furthermore hexosamines promote Sp1 O-linked N-acetylglucosamine glycosylation [33], which stabilizes this transcription factor, increasing its cellular concentrations [34]. Several other studies have proposed a role of the HBP in PKC activation in different cell types [19,35,36,37], and PKC has been shown to mediate glucose-induced p38-MAPK activation [38], confirming our results in control transfected cells.…”

Human Sug1/p45 is involved in the proteasome-dependent degradation of Sp1

“…Our preliminary results indicate that hSug1 does not interact with this target domain of Sp1 (results not shown). We have also shown that increased modification of proteins by OGlcNAc is associated with Sp1 stabilization [4] and that nuclear extract from glucosamine-treated cells fails to degrade recombinant Sp1 [33]. However, hSug1 is not modified by O-GlcNAc even in cells that had been treated with glucosamine, and hSug1 cannot restore the activity of the proteasomes derived from glucosamine-treated cells.…”

“…Previous studies have shown that Sp1 is rapidly degraded by the proteasome under the conditions of glucose-starvation and stimulation by cAMP [4]. However, the control of this degradation process is largely unknown.…”

“…Although Sp1 has generally been considered to constitutively regulate gene expression, its activity and cellular content have been shown to be regulated during a variety of cellular processes [2,3]. We have previously shown that Sp1 is rapidly degraded by the proteasome in cells that are both glucose-starved and stimulated with cAMP [4]. Under these conditions, protein modification of Sp1 and other cellular proteins by O-linked Nacetylglucosamine (O-GlcNAc) is markedly reduced.…”