O-Linked β-N-Acetylglucosaminyltransferase Substrate Specificity Is Regulated by Myosin Phosphatase Targeting and Other Interacting Proteins

Gerald W. Hart,Wagner B. Dias,Michael P. Housley,Kaoru Sakabe,Win D. Cheung

doi:10.1074/jbc.m806199200

Gerald W. Hart, Wagner B. Dias + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m806199200

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Abstract

O-GlcNAc-transferase (OGT) substrate specificity is regulated by transiently interacting proteins. To further examine the regulation of OGT, we have identified 27 putative OGT-interacting proteins through a yeast two-hybrid screen. Two of these proteins, Trak1 (OIP106) and O-GlcNAcase, have been shown previously to interact with and regulate OGT. We demonstrate here that MYPT1 and CARM1 also interact with and target OGT. MYPT1 and CARM1 are substrates of OGT in vitro and in vivo. MYPT1 and CARM1 also function to alter OGT substrate specificity in vitro. Furthermore depletion of MYPT1 in Neuro-2a neuroblastoma cells alters GlcNAcylation of several proteins under basal conditions, suggesting that MYPT1 regulates OGT substrate specificity in vivo.

Highlights

In many respects, protein GlcNAcylation is analogous to Ser/ Thr phosphorylation
A primary difference is that, unlike phosphorylation, which is catalyzed by almost 400 Ser/ Thr kinases encoded by the human genome (4), GlcNAcylation is catalyzed by the products of a single human gene (5, 6)
OGT is responsive to a wide range of UDP-GlcNAc concentrations, and the affinity of OGT for peptide substrates is altered with different UDPGlcNAc concentrations (7)

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Neuro-2a murine neuroblastoma cells (ATCC) were grown in Dulbecco’s modified Eagle’s medium (5 mM glucose; Mediatech) containing 10% (v/v) fetal bovine serum (Gemini Bio-Products) and penicillin/streptomycin (Mediatech) in a humidified incubator at 37 °C with 5% CO2. GST was expressed using the pGEX-5x1 plasmid (GE Healthcare) and purified over glutathione-Sepharose (GE Healthcare) according to the manufacturer’s instructions. GST-MYPT1 was expressed and purified as described previously with minor modifications (21). The fractions containing GST-MYPT1 were pooled and desalted into 25 mM Tris-HCl, pH 7.5, 150 mM NaCl. GST-CARM1 was expressed and purified over glutathioneSepharose according to the manufacturer’s instructions with minor modifications. 3 ␮g of ncOGT was assayed toward 1 ␮g of GST, GST-MYPT1, or GST-CARM1 in buffer containing 50 mM Tris-HCl, pH 7.5, 1 ␮g of purified bovine serum albumin, 1 unit of calf intestinal alkaline phosphatase, 0.5 ␮Ci of UDP-[3H]GlcNAc. Reactions were performed at room temperature for 1 h, stopped with Laemmli buffer, and separated by SDS-PAGE. OGT activity measurements from whole cell lysate were performed as described previously with minor modifications (19). The normal experimental deviation across control samples was measured to be Ϯ0.05 log(-fold change) units or ϳ12% and indicated on the plot by a gray bar between Ϫ0.05 and ϩ0.05

RESULTS

DISCUSSION

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