O‐glycosylation of the nuclear forms of Pax‐6 products in quail neuroretina cells

Abstract

Many transcription factors are demonstrated as being glycosylated with O-N-acetylglucosamine (GlcNAc) residue in transfected insect cell lines, but rarely in the original cells. For the first time, we demonstrate the O-GlcNAc modification of the p48/p46 Pax-6 gene (a developmental control gene involved in the eye morphogenesis) products in the quail neuroretina (QNR). In conjunction with a systematic PNGase F treatment, we used wheat germ agglutinin (WGA) binding, in vitro labeling with bovine galactosyltransferase, and labeling of cultured QNR with [14C]GlcNH2. Glycosylated forms of Pax-6 proteins were found in the nucleus of the neuroretina cells. WGA-selected Pax-6 proteins produced in the reticulocyte lysate were able to bind a DNA target, as well as to the unglycosylated form. The O-GlcNAc may, however, modulate protein interactions, mainly with other factors involved in the transcription process. Characterization of products released after reductive alkaline treatment of the proteins clearly demonstrates that N-acetylglucosamine is directly linked to serine or threonine residues. Examination of Pax-6 primary sequence allowed us to determine potential O-GlcNAc attachment sites. Most of these expected glycosylation sites appear to be located on the two DNA binding domains and on the carboxyterminal transactivation domain, while experimental evidence taken from WGA-selected proteins experiment points in favor of a main localization on the paired-box domain.

FLRT2 and FLRT3 act as repulsive guidance cues for Unc5-positive neurons

“…Dashed line in ( C , D ) indicates the region (around the IZ) where the dissection was made. ( F ) Protein lysates of the dissected tissues were subjected to lectin pull down (LPD) and samples were blotted with antibodies against FLRT2 ECD (upper panel) and the VZ marker Pax6 (middle panel) (Walther and Gruss, 1991; Lefebvre et al , 2002). Tubulin in the total cell lysates (TCL) was used as loading control (lower panel).…”

YY1 Is Regulated by O-LinkedN-Acetylglucosaminylation (O-GlcNAcylation)

“…This reversible post-translational modification (20) is present in a variety of proteins, including numerous chromatin-associated proteins and several transcription factors (21). The ubiquitous transcription factors Sp1 (22,23), members of the AP-1 family (22), P53 (24), the serum response factor (SRF) (25), the estrogen receptor (ER), Pax-6 (26), and c-Myc (27) all carry O-GlcNAc residues. A subset of the nuclear RNA polymerase II itself is also O-GlcNAc-modified (28) at the carboxyl-terminal moiety (CTD) of the largest subunit.…”

The tumor suppressor HIC1 (hypermethylated in cancer 1) is O‐GlcNAc glycosylated

“…Flag‐HIC1 proteins expressed in Cos7 cells were enriched using anti‐Flag Igs covalently coupled to agarose beads. After elution with 150 µg·mL −1 of the Flag peptide, the bound proteins were labeled with 50 mU of preauto‐galactosylated bovine galactosyltransferase (Sigma) and 5 µCi of UDP‐[6‐ 3 H]galactose (Amersham; Little Chalfont, Buckinghamshire, UK) at 37 °C for 2 h in Buffer L (56.25 m m HEPES, 11.25 m m MnCl 2 , 250 m m galactose, 12.5 m m adenosine mono‐phosphate, pH 6.0) [9]. Samples were run on an 8% (w/v) SDS/PAGE, and the gel was incubated in Amplify® (Amersham) and then fluorographed.…”

“…Bovine galactosyltransferase is a specific and sensitive probe frequently used in the detection of O ‐GlcNAc residues on cytosolic and nuclear proteins [9,34,35]. Full‐length Flag HIC1 proteins were purified from extracts of transfected Cos7 cells using an anti‐(Flag M2) affinity column.…”

“…It consists of the attachment of a single residue of N ‐acetylglucosamine on serine and threonine of the peptidic backbone. Hundreds of proteins are modified by this type of glycosylation [1], including structural proteins such as keratins [2] and highly numerous neuronal structural proteins such as neurofilaments [3], synapsin [4] or Tau5; proteins playing a role in transcription such as RNA polymerase II [6]; transcription factors such as Elf1 [7], c‐Myc [8], Pax6 [9] or the cAMP response element binding protein (CREB) [10]; corepressors such as mSin3A [11] and even histone deacetylases such as HDAC1 [11]. O ‐GlcNAc is particularly interesting given that this glycosylation is abundant, reversible and highly dynamic; it could compete with phosphorylation on the same or on neighboring amino acids [6,8].…”