Regulation of calcium/calmodulin-dependent kinase IV by O-GlcNAc modification

Abstract

Similar to phosphorylation, GlcNAcylation (the addition of O-GlcNAc to Ser(Thr) residues on polypeptides) is an abundant, dynamic, and inducible post-translational modification. GlcNAcylated proteins are crucial in regulating virtually all cellular processes, including signaling, cell cycle, and transcription. Here we show that calcium/calmodulin-dependent kinase IV (CaMKIV) is highly GlcNAcylated in vivo. In addition, we show that upon activation of HEK293 cells, hemagglutinin-tagged CaMKIV GlcNAcylation rapidly decreases, in a manner directly opposing its phosphorylation at Thr-200. Correspondingly, there is an increase in CaMKIV interaction with O-GlcNAcase during CaMKIV activation. Furthermore, we identify at least five sites of GlcNAcylation on CaMKIV. Using site-directed mutagenesis, we determine that the GlcNAcylation sites located in the active site of CaMKIV can modulate its phosphorylation at Thr-200 and its activity toward cAMP-response element-binding transcription factor. Our results strongly indicate that the O-GlcNAc modification participates in the regulation of CaMKIV activation and function, possibly coordinating nutritional signals with the immune and nervous systems. This is the first example of an O-GlcNAc/phosphate cycle involving O-GlcNAc transferase/kinase cross-talk.

FIGURE 1.

CaMKIV is GlcNAcylated. A–D, lysates from HEK293 cells transfected with empty HA plasmid or HA-CaMKIV were immunoprecipitated for HA. A, subjected to galactosyltransferase labeling in the presence of UDP-[³H]galactose for autoradiography. B, labeled in the presence of UDP-GalNAz and reacted with TAMRA alkyne for detection by in-gel fluorescence. C, treated with β-elimination. IP, immunoprecipitation; IB, immunoblot. D, treated with γ-phosphatase (γ-PPase) or GlcNAcase prior to immunoblotting for O-GlcNAc and HA. A, prior to autoradiography, the gel was stained for total protein using Coomassie Brilliant Blue G-250 (CBB G250). B, TAMRA fluorescence was detected in-gel prior to staining for total protein using SYPRO Ruby. E, rat cerebellum extract was immunoprecipitated for CaMKIV, subjected to galactosyltransferase labeling in the presence of UDP-GalNAz, reacted with biotin alkyne, and immunoblotted for CaMKIV or biotin (using streptavidin-horseradish peroxidase). F, lysates from Jurkat cells were immunoprecipitated for CaMKIV or using nonspecific (ns) mouse antibodies and immunoblotted for O-GlcNAc or CaMKIV.

FIGURE 2.

GlcNAcylation of CaMKIV opposes its phosphorylation at Thr-200 . A, lysates from HEK293 cells transfected with HA-CaMKIV and treated with 1 μm ionomycin for the indicated times were immunoprecipitated (IP) for HA and immunoblotted (IB) for O-GlcNAc, Thr(P)-200 (pT200), and HA. B, relative fold change in Thr(P)-200/HA signal (diamonds) and O-GlcNAc/HA signal (circles) during ionomycin treatment (normalized to 0 h time point). C, lysates from HEK293 cells transfected with HA-CaMKIV and treated with or without 1 μm ionomycin for 2 min were immunoprecipitated for HA and immunoblotted for O-GlcNAcase, Thr(P)-200, and HA. D, relative fold change in O-GlcNAcase/HA signal with ionomycin treatment (normalized to untreated control). E, lysates from HEK293 cells transfected with HA-CaMKIV wild type, HA-CaMKIV T200A, or HA-CaMKIV T200E were immunoprecipitated for HA and immunoblotted for O-GlcNAc and HA. F, relative fold change in O-GlcNAc/HA signal for T200A and T200E mutants (normalized to wild-type control). Asterisk is used to note p < 0.05 for samples compared with control or 0-h time point.

FIGURE 3.

Identification of GlcNAcylation sites on CaMKIV. A, work flow scheme for labeling and enrichment of GlcNAcylated CaMKIV peptides for O-GlcNAc site identification by liquid chromatography-MS/MS. B, MS/MS spectra for unambiguous assignment of Ser-137. C, Ser-189. D, Ser-356 as GlcNAcylation sites in vivo. E, diagram of CaMKIV displaying location of known phosphorylation sites (red), GlcNAcylation sites (blue), kinase domain (filled black box), autoinhibitory domain (open box), and Ca²⁺/CaM-binding domain (filled gray box).

FIGURE 4.

Removal of O-GlcNAc sites on CaMKIV reduces its GlcNAcylation levels. A, lysates from HEK293 cells transfected with HA-CaMKIV wild type or its indicated mutants were immunoprecipitated (IP) for HA and immunoblotted (IB) for O-GlcNAc and HA. B, relative fold change in O-GlcNAc/HA signal for each CaMKIV mutant (normalized to wild-type (WT) control). Asterisk is used to note p < 0.05 for samples compared with control.

FIGURE 5.

Removal of O-GlcNAc sites on CaMKIV alters Thr-200 phosphorylation during ionomycin treatment. A, lysates from HEK293 cells transfected with HA-CaMKIV wild type or its indicated mutants and treated with 1 μm ionomycin for 2 min were immunoprecipitated (IP) for HA and immunoblotted (IB) for Thr(P)-200 (pT200) or HA. B, relative fold change in Thr(P)-200/HA signal for each CaMKIV mutant (normalized to wild-type control treated with ionomycin). As a control, a duplicate sample transfected with HA-CaMKIV wild type (WT) was left untreated to show the magnitude of Thr(P)-200 induced by ionomycin treatment. Asterisk is used to note p < 0.05 for samples compared with control.

FIGURE 6.

Locations of the GlcNAcylation sites on a predicted CaMKIV structure and within its active site cleft. A, predicted structure of the CaMKIV kinase domain (residues 45–311) based on homology modeling using CaMKIG as template. B, area shown within dotted box is magnified. B, stereo image of active site cleft, displaying residues known to be important for ATP binding (Lys-75 in cyan and Asp-185 in magenta), the phosphorylation site on the activation loop (Thr-200 in orange), and the identified GlcNAcylation sites (Thr-57, Ser-58, and Ser-189 in green). Distances shown are in angstroms and were calculated using MacPyMol. C, lysates from HEK293 cells transfected with HA-CaMKIV wild type, T57A/S58A, or S189A were tested for the ability to bind ATP resin. ATP-resin bound proteins were immunoblotted (IB) for HA.

FIGURE 7.

Removal of O-GlcNAc sites on CaMKIV alters its kinase activity. A, lysates from HEK293 cells transfected with HA-CaMKIV wild type or its indicated mutants were immunoprecipitated (IP) for HA and assayed for kinase activity toward recombinant GST-CREB. CBB, Coomassie Brilliant Blue; IB, immunoblot. B, relative fold change in CREB phosphorylation for each CaMKIV mutant (normalized to wild-type (WT) control). C and D, same as A and B but after treatment with ionomycin for 2 min. E and F, same as A and B but after treatment with ionomycin for 5 min. As a negative control, empty vector was included with this time point. Asterisk is used to note p < 0.05 for samples compared with control. Note: because A, C, and E represent different exposure times, samples between time points cannot be directly compared with one another.