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Sci. Signal., 8 February 2011
Vol. 4, Issue 159, p. ec36
[DOI: 10.1126/scisignal.4159ec36]

EDITORS' CHOICE

Glycosylation Making It Short and Sweet

Elizabeth M. Adler

Science Signaling, AAAS, Washington, DC 20005, USA

Human host cell factor 1 (HCF-1) undergoes proteolytic cleavage to yield noncovalently associated N-terminal (HCF-1N) and C-terminal (HCF-1C) subunits with distinct roles in regulating the cell cycle. Functional activation of the HCF-1C subunit, which acts during M phase, depends on this process of proteolytic maturation, which occurs at six centrally located 26–amino acid HCF-1PRO repeat sequences, each of which contains a glutamic acid adjacent to the cleavage site (see Hanover). Noting the presence of threonine sites crucial for HCF-1 proteolysis in the HCF-1PRO repeats, and that threonines are targets of both phosphorylation and glycosylation, Capotosti et al. wondered if one of these posttranslational modifications might be implicated in this process. They determined that the kinase inhibitor staurosporine failed to inhibit the in vitro cleavage of an HCF-1–derived precursor protein containing three repeats (HCF-1rep123), whereas alloxan, which inhibits the enzyme O-linked β-N-acetylglucosamine transferase (OGT), inhibited in vitro HCF-1rep123 cleavage, and OGT knockdown inhibited its cleavage in human 293 cells. Immunoblot analysis revealed O-GlcNAcylation of the HCF-1rep123 precursor purified from 293 cells, and of its cleavage products, but, unexpectedly, not of a cleavage-resistant mutant form (HCF-1repxxx) in which the glutamic acids at the cleavage sites were replaced with alanines (E10A mutation). Indeed, mass spectrometric and immunoblot analyses showed that O-GlcNAcylation was largely limited to HCF-1N rather than the HCF-1PRO repeats. Immunoblot analyses of immunoprecipitates of full-length HCF-1 and various mutants revealed that OGT bound HCF-1; its binding depended on the HCF-1PRO repeats and was enhanced by the E10A mutation. Recombinant human OGT promoted cleavage of the HCF-1PRO repeat in vitro; cleavage depended on the presence of the donor substrate UDP-GlcNac and was inhibited by alloxan. O-GlcNAcylation was apparent before cleavage, and experiments in which alloxan was added at different time points indicated that O-GlcNAcylation did not promote HCF-1 autoproteolysis but, rather, that OGT possessed proteolytic activity. In invertebrates, HCF-1 is cleaved by Taspase1 and, intriguingly, although a mutant form in which the HCF-1PRO repeats were replaced by Taspase1 cleavage sites underwent proteolysis in 293 cells, it not only was deficient in OGT binding but also failed to mediate HCF-1C M phase functions. Thus, OGT appears to bind to and cleave the HCF-1PRO repeats and glycosylate the HCF-1N subunit, a process crucial for induction of HCF-1C function.

F. Capotosti, S. Guernier, F. Lammers, P. Waridel, Y. Cai, J. Jin, J. W. Conaway, R. C. Conaway, W. Herr, O-GlcNAc transferase catalyzes site-specific proteolysis of HCF-1. Cell 144, 376–388 (2011). [PubMed]

J. A. Hanover, A versatile sugar transferase makes the cut. Cell 144, 321–323 (2011). [PubMed]

Citation: E. M. Adler, Making It Short and Sweet. Sci. Signal. 4, ec36 (2011).



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