Cross-talk between GlcNAcylation and phosphorylation: site-specific phosphorylation dynamics in response to globally elevated O-GlcNAc.
Protein GlcNAcylation serves as a nutrient/stress sensor to modulate the functions of many nuclear and cytoplasmic proteins. O-GlcNAc cycles on serine or threonine residues like phosphorylation, is nearly as abundant, and functions, at least partially, via its interplay with phosphorylation. Here, we describe changes in site-specific phosphorylation dynamics in response to globally elevated GlcNAcylation. By combining sequential phospho-residue enrichment, iTRAQ labeling, and high throughput mass spectrometric analyses, phosphorylation dynamics on 711 phosphopeptides were quantified. Based upon their insensitivity to phosphatase inhibition, we conclude that approximately 48% of these phosphorylation sites were not actively cycling in the conditions of the study. However, increased GlcNAcylation influenced phosphate stoichiometry at most of the sites that did appear to be actively cycling. Elevated GlcNAcylation resulted in lower phosphorylation at 280 sites and caused increased phosphorylation at 148 sites. Thus, the cross-talk or interplay between these two abundant posttranslational modifications is extensive, and may arises both by steric competition for occupancy at the same or proximal sites and by each modification regulating the other's enzymatic machinery. The phosphoproteome dynamics presented by this large set of quantitative data not only delineates the complex interplay between phosphorylation and GlcNAcyation, but also provides insights for more focused investigations of specific roles of O-GlcNAc in regulating protein functions and signaling pathways.
Wang
Zihao
Z
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205-2185, USA.
Gucek
Marjan
M
Hart
Gerald W
GW
D000117:Acetylglucosamine / Q000737:chemistry / Q000378:metabolism*; D000215:Acylation; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D004268:DNA-Binding Proteins / Q000378:metabolism; D007093:Imidazoles; D013058:Mass Spectrometry; D051379:Mice; D041681:NIH 3T3 Cells; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D040901:Proteomics; D016601:RNA-Binding Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014025:Titanium
D004268:DNA-Binding Proteins; D007093:Imidazoles; D010748:Phosphopeptides; D016601:RNA-Binding Proteins; C009495:titanium dioxide; D014025:Titanium; C005954:imidazoleacetic acid; D000117:Acetylglucosamine
Nat Cell Biol. 2006 Oct;8(10):1074-83
16964247
Mol Cell Proteomics. 2004 Nov;3(11):1093-101
15345747
Biochim Biophys Acta. 1999 Oct 18;1472(1-2):71-81
10572927
Cell Signal. 2008 Jan;20(1):94-104
18029144
J Biol Chem. 2002 May 24;277(21):19229-35
11904304
Nucleic Acids Res. 2007 Jul;35(Web Server issue):W707-12
17553828
Proc Natl Acad Sci U S A. 2004 Sep 7;101(36):13132-7
15340146
J Biol Chem. 2000 Jul 21;275(29):21981-7
10806197
Proc Natl Acad Sci U S A. 2002 Apr 16;99(8):5313-8
11959983
Mol Cell Proteomics. 2006 May;5(5):914-22
16452087
Biochem Biophys Res Commun. 2004 Jun 18;319(1):21-6
15158436
Biochemistry. 2000 Sep 26;39(38):11609-20
10995228
J Biol Chem. 1999 Nov 5;274(45):32015-22
10542233
Biochim Biophys Acta. 2004 Jul 6;1673(1-2):13-28
15238246
J Biol Chem. 2004 Sep 10;279(37):38466-70
15247246
J Cell Biochem. 2006 Jan 1;97(1):71-83
16237703
Mol Cell Proteomics. 2007 Aug;6(8):1365-79
17507370
Proc Natl Acad Sci U S A. 2004 Jul 20;101(29):10804-9
15249677
Eur J Biochem. 1999 Jun;262(3):824-31
10411645
Science. 2002 Dec 6;298(5600):1912-34
12471243
J Biol Chem. 2001 Mar 30;276(13):9838-45
11148210
Science. 2001 Mar 23;291(5512):2376-8
11269319
J Am Chem Soc. 2007 Jan 24;129(3):635-44
17227027
Mol Cell Proteomics. 2006 May;5(5):923-34
16452088
Nature. 2007 Apr 26;446(7139):1017-22
17460662
Proc Natl Acad Sci U S A. 2005 Aug 16;102(33):11870-5
16085713