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Human HCF-1 (also referred to as HCFC-1) is a transcriptional co-regulator that undergoes a complex maturation process involving extensive O-GlcNAcylation and site-specific proteolysis. HCF-1 proteolysis results in two active, noncovalently associated HCF-1N and HCF-1C subunits that regulate distinct phases of the cell-division cycle. HCF-1 O-GlcNAcylation and site-specific proteolysis are both catalyzed by O-GlcNAc transferase (OGT), which thus displays an unusual dual enzymatic activity. OGT cleaves HCF-1 at six highly conserved 26 amino acid repeat sequences called HCF-1PRO repeats. Here we characterize the substrate requirements for OGT cleavage of HCF-1. We show that the HCF-1PRO-repeat cleavage signal possesses particular OGT-binding properties. The glutamate residue at the cleavage site that is intimately involved in the cleavage reaction specifically inhibits association with OGT and its bound cofactor UDP-GlcNAc. Further, we identify a novel OGT-binding sequence nearby the first HCF-1PRO-repeat cleavage signal that enhances cleavage. These results demonstrate that distinct OGT-binding sites in HCF-1 promote proteolysis, and provide novel insights into the mechanism of this unusual protease activity.

Human HCF 1 (also referred to as HCFC 1) is a transcriptional co regulator that undergoes a complex maturation process involving extensive O GlcNAcylation and site specific proteolysis. HCF 1 proteolysis results in two active noncovalently associated HCF 1N and HCF 1C subunits that regulate distinct phases of the cell division cycle. HCF 1 O GlcNAcylation and site specific proteolysis are both catalyzed by O GlcNAc transferase (OGT) which thus displays an unusual dual enzymatic activity. OGT cleaves HCF 1 at six highly conserved 26 amino acid repeat sequences called HCF 1PRO repeats. Here we characterize the substrate requirements for OGT cleavage of HCF 1. We show that the HCF 1PRO repeat cleavage signal possesses particular OGT binding properties. The glutamate residue at the cleavage site that is intimately involved in the cleavage reaction specifically inhibits association with OGT and its bound cofactor UDP GlcNAc. Further we identify a novel OGT binding sequence nearby the first HCF 1PRO repeat cleavage signal that enhances cleavage. These results demonstrate that distinct OGT binding sites in HCF 1 promote proteolysis and provide novel insights into the mechanism of this unusual protease activity.