Title : Localization of
O-GlcNAc modification on the serum response transcription
factor
Abstract :
- 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