An N-terminal region of Sp1 targets its proteasome-dependent degradation in vitro

The transcription factor Spl is important for the expression of many cellular genes. Previously, it was shown that reduced O-glycosylation of Spl is associated with increased proteasome susceptibility. Spl undergoes proteasome-dependent degradation in cells stressed with glucose deprivation and adenylate cyclase activation, and this process is blocked in cells treated with glucosamine. In this study, using a reconstituted in vitro system, we identified the principal structural determinant in Spl that targets Spl for proteasome-dependent degradation. We found by using deletion analysis that the N-terminal 54 amino acids of Spl is required for Spl degradation. This element can act as an independent processing signal by directing degradation of an unrelated protein Recognition of this Spl element by the proteasome-dependent system is saturable, and ubiquitination of this element is not required for recognition. Time course experiments revealed that Spl degradation is a two-step process. First, a discrete endoproteolytic cleavage occurs downstream of the target region immediately C-terminal to Leu(56). The Spl sequence C-terminal to the cleavage site is subsequently degraded, whereas the N-terminal peptide remains intact. The identification of this Spl degradation-targeting signal will facilitate the identification of the critical proteins involved in the control of Spl proteasome-dependent degradation and the role of O-GlcNAc in this process.