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Localization of O-GlcNAc modification on the serum response transcription factor
A unique form of nucleoplasmic and cytoplasmic protein glycosylation, O-linked GlcNAc, has previously been detected, using Gal transferase labeling techniques, on a myriad of proteins (for review see Hart, G. W., Haltiwanger, R. S., Holt, G. D., and Kelly, W. G. (1989a) Annu. Rev. Biochem. 58, 841-8...
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Published in: | The Journal of biological chemistry 1992-08, Vol.267 (24), p.16911-16921 |
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Main Authors: | , , , , , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | A unique form of nucleoplasmic and cytoplasmic protein glycosylation, O-linked GlcNAc, has previously been detected, using
Gal transferase labeling techniques, on a myriad of proteins (for review see Hart, G. W., Haltiwanger, R. S., Holt, G. D.,
and Kelly, W. G. (1989a) Annu. Rev. Biochem. 58, 841-874), including many RNA polymerase II transcription factors (Jackson,
S. P., and Tjian, R. (1988) Cell 55, 125-133). However, virtually nothing is known about the degree of glycosylation at individual
sites, or, indeed, the actual sites of attachment of O-GlcNAc on transcription factors. In this paper we provide rigorous
evidence for the occurrence and locations of O-GlcNAc on the c-fos transcription factor, serum response factor (SRF), expressed
in an insect cell line. Fast atom bombardment mass spectrometry (FAB-MS) of proteolytic digests of SRF provides evidence for
the presence of a single substoichiometric O-GlcNAc residue on each of four peptides isolated after sequential cyanogen bromide,
tryptic, and proline specific enzyme digestion: these peptides are 306VSASVSP312, 274GTTSTIQTAP283, 313SAVSSADGTVLK324, and
374DSSTDLTQTSSSGTVTLP391. Using an array of techniques, including manual Edman degradation, aminopeptidase, and elastase digestion,
together with FAB-MS, the major sites of O-GlcNAc attachment were shown to be serine residues within short tandem repeat regions.
The highest level of glycosylation was found on the SSS tandem repeat of peptide (374-391) which is situated within the transcriptional
activation domain of SRF. The other glycosylation sites observed in SRF are located in the region of the protein between the
DNA binding domain and the transcriptional activation domain. Glycosylation of peptides (274-283) and (313-324) was found
to occur on the serine in the TTST tandem repeat and on serine 316 in the SS repeat, respectively. The lowest level of glycosylation
was recovered in peptide (306-312) which lacks tandem repeats. All the glycosylation sites identified in SRF are situated
in a relatively short region of the primary sequence close to or within the transcriptional activation domain which is distant
from the major sites of phosphorylation catalyzed by casein kinase II. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/S0021-9258(18)41871-6 |