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Originally published In Press as doi:10.1074/jbc.M414066200 on February 9, 2005

J. Biol. Chem., Vol. 280, Issue 15, 14579-14585, April 15, 2005
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Protein O-GlcNAc Modulates Motility-associated Signaling Intermediates in Neutrophils*

Zachary T. Kneass and Richard B. Marchase{ddagger}

From the Department of Cell Biology, MCLM 690, University of Alabama, Birmingham, Alabama 35294

Received for publication, December 14, 2004 , and in revised form, February 9, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The modification of serine/threonine residues on cytoplasmic and nuclear proteins by N-acetylglucosamine (O-GlcNAc) is suggested to play a role in the regulation of a variety of signal transduction pathways. We have previously shown that glucosamine (GlcNH2), a metabolic precursor of O-GlcNAcylation, increases 2O-GlcNAc and enhances motility in neutrophils. Here, we extend this correlation by showing that a mechanistically distinct means of increasing O-GlcNAc, achieved by inhibition of O-GlcNAc removal with O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), increases basal cellular motility and directional migration induced by the chemoattractant formyl-methionine-leucine-phenylalanine (fMLP). Furthermore, we demonstrate that O-GlcNAc modulates the activities of signaling intermediates known to regulate neutrophil movement. GlcNH2 and PUGNAc increase both the basal and fMLP-induced activity of a central mediator of cellular motility, the small GTPase Rac. Phosphoinositide 3-kinase, an important regulator of Rac activity and neutrophil motility, is shown to regulate the signaling pathway on which GlcNH2 and PUGNAc act. Rac is an important upstream regulatory element in p38 and p44/42 mitogen-activated protein kinase (MAPK) signaling in neutrophils, and these MAPKs are implicated in chemotactic signal transduction. We show that GlcNH2 and PUGNAc treatment increases p42/44 and p38 MAPK activities and that these increases are associated with activation of upstream MAPK kinases. These data indicate that O-GlcNAcylation is an important signaling element in neutrophils that modulates the activities of several critical signaling intermediates involved in the regulation of cellular movement.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Numerous cytoplasmic and nuclear proteins are post-translationally modified by O-linked N-acetylglucosamine in {beta} linkage to Ser/Thr residues (O-GlcNAc)1 (1, 2). The sugar donor for the O-GlcNAc transferase (OGT) that catalyzes this modification is a product of glucose metabolism through the hexosamine biosynthesis pathway (HBP) (3). OGT activity is sensitive to changes in substrate availability such that increases in HBP flux, mediated either through glucose or glucosamine (GlcNH2) administration, lead to increased levels of its substrate, UDP-N-acetylglucosamine, which then drives OGT-mediated O-GlcNAcylation (3–7). The dynamic nature of the O-GlcNAc moiety suggests that it may be functionally analogous to phosphorylation in influencing protein functions such as enzymatic activity, protein-protein interactions, and subcellular localization (1, 8–12). Supporting evidence for such a regulatory role comes from the variety of signaling pathways that are regulated by protein O-GlcNAc. For example, protein O-GlcNAc is associated with transcriptional regulation (13, 14), proteasome-mediated protein degradation (15), insulin signaling (4), and cellular stress signaling (17). Using a neutrophil (polymorphonuclear leukocyte or PMN) model, we recently demonstrated that rapid protein O-GlcNAcylation could occur in response to agonist stimulation through a cell-surface receptor-mediated mechanism (18). These data provided further support for the concept that O-GlcNAc may function in a highly dynamic regulatory capacity analogous to protein phosphorylation.

PMNs are a vital component of the innate immune response to infection and tissue damage. Their effectiveness in this capacity is dependent on the activation of actin-based cytoskeletal machinery that allows for directed migration toward a focus of inflammation. Lipid kinase, protein kinase, and small G protein activities are central mediators of PMN motility; however, we have recently implicated substrate-driven O-GlcNAcylation as an additional modulator of this essential PMN function (18). Specifically, GlcNH2-associated increases in O-GlcNAc were found to correlate with enhanced basal and formyl-Met-Leu-Phe-(fMLP) induced motility.

The intracellular signals that mediate PMN motility involve a complex interconnected signaling network that includes phosphoinositide 3-kinases (PI3Ks), small GTP-binding proteins of the Rho family, and mitogen-activated protein kinases (MAPKs). The generation of PI3K{gamma} knock-out mice and the use of PI3K-specific inhibitors have demonstrated an important role for PI3K and its lipid products in chemotaxis (19–22). Knock-outs have also been generated for the small GTPases Rac1 and Rac2, and these also show significant deficiencies in PMN chemotaxis (23–26). Interestingly, Rac activation may be regulated in large part by a recently identified guanine nucleotide exchange factor, P-Rex1 (27). This Rac guanine nucleotide exchange factor is activated by either phosphatidylinositol phosphates or heterotrimeric G{beta}{gamma} subunits, thus providing for a direct link between PI3K and Rac activation. PMNs lacking Rac are also deficient in p38 and p44/42 activation (23, 26, 28), which is significant because pharmacological MAPK inhibition has indicated that the activities of p38 and p44/42 are important in chemotactic signaling (29–36). This may occur through MAPK-mediated regulation of MAPK-activated protein kinase (35, 37, 38), leukocyte-specific protein 1 (39–41), an actin-binding protein that modulates cell motility, and heat shock protein 27 (HSP27) (42–45), which modulates actin polymerization and cellular migration in a variety of cell types.

In light of our recent study correlating GlcNH2-induced increases in O-GlcNAc with enhanced PMN motility (18), the goal of this study was to examine the impact of O-GlcNAc on motility-associated signal transduction. Importantly, we also sought to confirm that protein O-GlcNAcylation was indeed the mechanism through which GlcNH2 alters PMN function by examining whether an alternate, unrelated means of increasing O-GlcNAc has effects similar to those seen with GlcNH2. We considered this essential because GlcNH2 may have metabolic effects unrelated to protein O-GlcNAcylation (3, 46–48). Therefore we compared the effect of O-GlcNAcylation induced by GlcNH2 to that induced by the O-GlcNAcase inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) (49–51). By using these mechanistically unrelated methods of manipulating the enzymes responsible for O-GlcNAc cycling, we demonstrate that O-GlcNAc modulates neutrophil motility. In accordance with its effects on motility, we show that protein O-GlcNAc increases the amount of GTP-bound, or activated, Rac. Furthermore, O-GlcNAc-associated changes in cell motility are shown to be wortmannin sensitive. Increases in O-GlcNAc are also associated with increased activity of the MAPKs p38 and p44/42, and of the MAPK kinases (MKKs) MKK3/6 and MEK1/2. In addition to confirming that protein O-GlcNAcylation modulates motility and motility-associated signaling, our data support the idea that O-GlcNAc is an important signaling mechanism in PMNs akin to phosphorylation. Furthermore, our results are of general interest because they define a novel axis of O-GlcNAc-modulated signal transduction that involves Rac/PI3K/MAPK, each of these being important signaling intermediates in a variety of systems and cellular processes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—PUGNAc was purchased from Carbogen. Wortmannin and fMLP were purchased from Calbiochem. GTP{gamma}S and GDP were from Upstate. Hanks' buffered salt solution was purchased from Cellgro. Anti-actin antibody was purchased from Abcam. Anti-rabbit IgG and anti-mouse IgG were from Bio-Rad. All other reagents and chemicals were from Sigma unless otherwise indicated.

PMN Isolation—Whole blood from volunteers was obtained by venipuncture and layered onto a double discontinuous gradient formed with equal volumes of Histopaque-1077 (Sigma) over Histopaque-1119 (Sigma) (52). The blood was centrifuged at 700 x g for 30 min. Granulocytes were collected from the 1077/1119 interface and washed in Hanks' salt solution buffered with 10 mM HEPES, pH 7.4 (Hanks' buffered salt solution). Contaminating red blood cells were lysed by incubation in 0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM EDTA for 5 min at 37 °C. The granulocytes were then washed twice in Hanks' buffered salt solution and resuspended in Hanks' buffered salt solution. Cell viability was assessed through trypan blue (Sigma) exclusion.

Immunoblotting—For protein O-GlcNAc immunoblots 2 x 106 PMNs were treated as indicated and directly lysed in 5x sample buffer (0.3 M Tris-HCl, 5% SDS, 50% glycerol, 0.025% bromphenol blue, 5% mercaptoethanol) and boiled for 5 min. 7.5 x 105 cell equivalents per lane (~60 µg of protein) were separated by SDS-PAGE (53) and transferred to Immobilon-P (Millipore). Immunoblotting was performed using a rapid immunodetection method for Immobilon-P (Millipore TN051). Briefly, the membranes were equilibrated in methanol and air-dried. The dry membrane was incubated with a 1:1000 dilution of anti-O-GlcNAc antibody CTD110.6 (54) (a kind gift from Mary Ann Accavitti) in 1% casein, phosphate-buffered saline (Pierce) with 0.01% Tween 20 for 2 h and then washed three times in phosphate-buffered saline. The membrane was then incubated with a 1:5000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgM (Calbiochem) in 1% casein/phosphate-buffered saline with 0.01% Tween 20 for 1 h. After further washing in phosphate-buffered saline the immunoblots were developed with enhanced chemiluminescence (SuperSignal West Pico; Pierce). For Rac and Ras immunoblots, the affinity purified products from the Rac- and Ras-GTP assays and the total input lysates (see below) were separated by SDS-PAGE, transferred to Immobilon-P, and then immunoblotted with Rac1- and Ras-specific antibodies (Upstate) according to the antibody manufacturer's directions. The immunoblots were visualized using enhanced chemiluminescence. The activities of p38, p44/42, MKK3/6, and MEK1/2 were assessed by phosphorylation state-specific antibodies (Cell Signaling Technology). 2 x 106 PMNs were treated as indicated and then directly lysed in 5x sample buffer followed by boiling for 5 min. 7.5 x 105 cell equivalents per lane (~60 µg of protein) were separated by SDS-PAGE and transferred to Immobilon-P. Immunoblotting was performed according to the antibody manufacturer's directions and visualized by enhanced chemiluminescence.

Motility Assays—2 x 105 PMNs were pretreated as indicated, washed, resuspended in Dulbecco's modified Eagle's medium, 5% bovine serum albumin and then placed in the upper filter plate of a 96-well MultiScreen-MIC chemotaxis plate (Millipore) with 3-µm membrane pores. The lower receiver plate contained Dulbecco's modified Eagle's medium and 100 µM fMLF where indicated. Basal motility was assessed in the absence of fMLF. The assays were performed in a 37 °C incubator for 45 min. The filter plate was then carefully removed and migrating cells counted by microscopic examination of receiver plates (55). The data were normalized to the mean value of the control to provide a percent of the control value in each experimental set.

Rac and Ras Activity Assays—Rac and Ras activities were assessed by the affinity capture of Rac- and Ras-GTP (56, 57). 107 PMNs were treated as indicated and then lysed by the addition of 5x Mg2+ lysis/wash buffer (125 mM HEPES, pH 7.5, 750 mM NaCl, 5% Igepal CA-630, 50 mM MgCl2, 5 mM EDTA, 10% glycerol). The lysates were cleared of insoluble debris by spinning at 14,000 x g for 10 min at 4 °C. ~30 µgof the resulting supernatant was mixed with 5x sample buffer, boiled for 5 min, and then set aside for determination of total (input) Rac or Ras by SDS-PAGE and immunoblot analysis as described above. The remainder of each supernatant was incubated with 5 µg of an agarose conjugate of either PAK-1 PBD or Raf-1 RBD (Upstate), for Rac- and Ras-GTP assays, respectively, for 1 h at 4 °C with gentle rocking. The beads were then washed 3 times with 1x Mg2+ lysis/wash buffer and finally resuspended in 2x sample buffer and boiled. The resulting affinity purified products, active Rac or Ras, were then analyzed by SDS-PAGE and immunoblot as described above. GTP{gamma}S- and GDP-loaded positive and negative controls, respectively, were prepared as described above, except that before the addition of beads to the supernatant, the supernatants were first incubated with either 100 µM GTP{gamma}S or 1 mM GDP for 30 min at 30 °C with gentle agitation, followed by the addition of 60 mM MgCl2 to stop the loading.

Statistics—All graphical data are represented as mean values with error bars of ±2 S.E. Significance was assessed with Student's t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
O-(2-Acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate Increases Protein O-GlcNAc and PMN Motility—PUGNAc is a widely used pharmacological inhibitor of the enzyme responsible for the removal of O-GlcNAc residues, neutral {beta}-N-acetylglucosaminidase (O-GlcNAcase) (49–51). It has proven to be a valuable tool for increasing protein O-GlcNAc in a variety of systems (16, 49, 58, 59). As we have previously shown that GlcNH2 effectively drives protein O-GlcNAcylation in PMNs through a substrate driven, or metabolic, mechanism involving the HBP (18) we assessed whether PUGNAc could serve as an alternative and complementary means of increasing O-GlcNAc levels.

Treatment with 100 µM PUGNAc for selected times significantly increased protein-associated O-GlcNAc as determined by immunoblot analyses of whole cell extracts with the O-GlcNAc-specific antibody CTD110.6 (Fig. 1A). A variety of proteins with molecular masses above ~50 kDa displayed increased O-GlcNAc levels that were proportional to the duration of PUGNAc treatment, beginning within 30 min and increasing to maximal levels of O-GlcNAcylation by 2 h. Interestingly, the pattern of O-GlcNAcylation was grossly similar to that induced by GlcNH2 (18). There were no changes to cell viability, as assessed by trypan blue exclusion, at any of the time points examined (data not shown).



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  		FIG. 1.
PUGNAc increases protein O-GlcNAc and PMN motility. PUGNAc was used to inhibit O-GlcNAcase activity (thereby preventing the enzymatic removal O-GlcNAc) and thus increase protein O-GlcNAc levels. A, immunoblots of whole cell lysates were performed using the anti-O-GlcNAc antibody CTD110.6. PMNs were left untreated (C) or were treated with 100 µM PUGNAc for 30, 60, 120, or 180 min. B, basal PMN motility was assessed in the absence of chemotactic factor in Boyden chamber-type migration assays (n ≥ 3 for all conditions). PMNs were left untreated (Control) or were treated with 100 µM PUGNAc for 30, 60, 90, or 180 min. Significant (p < 0.05 as evaluated by Student's t test) increases in motility were observed for 90 and 180 min of PUGNAc treatment when compared with control. C, chemotaxis was assessed in Boyden chamber-type migration assays (n ≥ 3 for all conditions). PMNs were not pretreated (Control and fMLP) or were pretreated with 100 µM PUGNAc for 30, 60, 90, 120, or 180 min. 100 nM fMLP was used to initiate chemotaxis in the PUGNAc-pretreated samples and the sample labeled fMLP. The control sample was not stimulated with chemotactic factor. Significant (p < 0.05 as evaluated by Student's t test) increases in motility were observed for 90, 120, and 180 min of PUGNAc pretreatment when compared fMLP alone (fMLP without pretreatment).

 
These results suggest a rapid rate of intrinsic O-GlcNAc cycling, providing further evidence of the highly dynamic nature of this post-translational modification in PMNs, and indicated that at least some of the same proteins may be O-GlcNAcylated in response to both GlcNH2 and PUGNAc treatments. Furthermore, they indicate that PUGNAc can serve as an effective tool for rapidly inducing increases in O-GlcNAc, and should be useful therefore for furthering our investigation of O-GlcNAc-associated changes in PMN motility.

As mentioned above, we have previously found that GlcNH2 pretreatment increases basal (in the absence of chemoattractant) and fMLP-induced PMN migration in Boyden chamber-type assays and such increases correlate with increases in protein O-GlcNAc (18). Having established that PUGNAc increases O-GlcNAc levels, we sought to test whether PUGNAc similarly increases PMN migration. Cells were pretreated with 100 µM PUGNAc for various durations and were then assayed for cell migration using simple 45-min Boyden chamber-type assays. PUGNAc increased basal cell motility (in the absence of chemoattractant) in a time-dependent manner (Fig. 1B). Changes in basal motility were significant (p < 0.05) at 90 and 180 min of pretreatment. In assays of fMLP-associated chemotaxis (100 nM fMLP), 100 µM PUGNAc pretreatments of 90, 120, and 180 min were associated with significant increases (over the levels induced by fMLP without pretreatment, p < 0.05 where n ≥ 6 for all conditions), of 17, 26, and 42%, respectively, in cell migration (Fig. 1C). Thus the effects of PUGNAc treatment are grossly similar in terms of basal motility and chemotaxis to the aforementioned results obtained using GlcNH2 (18). The finding that two unrelated mechanisms of inducing protein O-GlcNAc similarly enhance motility strongly suggests that O-GlcNAc modulates neutrophil physiology.

PUGNAc and GlcNH2 Increase Both Basal and fMLP-induced Rac Activity in Rac-GTP-specific Assays—We next sought to determine how protein O-GlcNAc might influence cell motility by examining some of the key facets of the signaling system that is thought to regulate the complex process of cellular movement. The small GTPase Rac is a well described regulatory component in chemotactic signaling that appears to have a central role in establishing both polarity and in regulating the actin cytoskeleton (23–26, 60).

We examined whether PUGNAc and GlcNH2, both of which we had now established as modulators of motility, affected Rac1 activity. Active, GTP-bound, Rac was assessed in Rac immunoblots following affinity capture with PAK-1 PBD (56). Using this method, Rac-GTP levels have been shown to rapidly and transiently increase above resting levels when PMNs are stimulated with the chemoattractant fMLP (25, 56). 100 µM PUGNAc and 10 mM GlcNH2 increased basal Rac activity even in the absence of fMLP (Fig. 2A). We then assessed the effect of 100 µM PUGNAc and 10 mM GlcNH2 on the activation of Rac induced by 10, 60, and 180 s of stimulation with 100 nM fMLP (Fig. 2B). GlcNH2 and PUGNAc pretreatment markedly enhanced fMLP-induced Rac activation over at least the first 60 s of stimulation. These results suggest that protein O-GlcNAcylation might modulate cell motility in a Rac-dependent manner.



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  		FIG. 2.
PUGNAc and GlcNH2 increase both basal and fMLP-induced Rac activity in Rac-GTP-specific assays. Active GTP-bound Rac was assessed by affinity purification using the PAK-1 PBD. The specificity of this assay for GTP-bound Rac was established by the in vitro loading of Rac with GTP{gamma}S or GDP prior to affinity purification (inset). A, basal Rac activity in PMNs treated with GlcNH2 or PUGNAc. PMNs were left untreated (C) or were treated with 10 m M GlcNH2 or 100 µM PUGNAc for 15, 45, or 90 min. Total levels of Rac in the pre-assay lysates are indicated as input. B, chemoattractant-induced Rac activity in PMNs pretreated with GlcNH2 or PUGNAc. PMNs were not pretreated (C and the adjacent three lanes labeled 10, 60, and 180 that are without PUGNAc or GlcNH2 labels) or were pretreated with 10 mM GlcNH2 or 100 µM PUGNAc for 90 min. Rac-GTP was assessed after stimulating the cells with 100 nM fMLP for 10, 60, and 180 s. The control sample (C) was not stimulated with chemotactic factor. Total levels of Rac in the pre-assay lysates are indicated as input.

 
Wortmannin Inhibits PUGNAc- and GlcNH2-associated Increases in Basal PMN Motility—Rac activation is closely tied to the lipid kinase activity of PI3K (25, 27). In fact, these signaling intermediates are thought to function together in a critical positive feedback loop that generates signal amplification at the leading edge of a migrating cell (61, 62). We were therefore interested in assessing whether PI3K played a role in O-GlcNAc-associated increases in PMN motility.

We initially examined the effect of PI3K inhibition on GlcNH2-associated increases in basal and fMLP-induced migration (Fig. 3A). PMNs were incubated with 250 nM wortmannin, which has been used extensively as an inhibitor of PI3K activity in PMNs (21, 22), beginning 15 min before the addition of 10 mM GlcNH2. The cells were then incubated with both wortmannin and GlcNH2 (or with buffer alone for the control samples) for 60 min before being assessed for migration in simple 45-min Boyden chamber-type assays either in the absence (basal motility) or presence (chemotaxis) of 100 nM fMLP. Wortmannin treatment did not result in changes to cell viability, as assessed by trypan blue exclusion (data not shown). Wortmannin effectively blunted basal PMN motility in the control and completely blocked the increase seen with GlcNH2. As other studies have shown, wortmannin was only moderately effective at reducing fMLP-induced chemotaxis (63). This indicates that wortmannin-insensitive pathways are important in neutrophil chemotaxis. This is supported by the fact that PI3K{gamma}-deficient murine PMNs still display chemotaxis in response to fMLP even though their ability to produce phosphatidylinositol 3,4,5-trisphosphate (PIP3) is severely compromised (19, 20). Interestingly, GlcNH2 and wortmannin together resulted in a level of response to fMLP that was comparable with the response to fMLP alone, although less than that seen when only fMLP and GlcNH2 were present.



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  		FIG. 3.
Wortmannin inhibits PUGNAc- and GlcNH2-associated increases in PMN motility. Wortmannin was used to inhibit PI3K and assess whether O-GlcNAc-mediated changes in PMN motility depend on PI3K activity. Motility and migration were assessed in Boyden chamber-type migration assays (n ≥ 3 for all conditions). A, the affect of wortmannin on GlcNH2-associated changes in basal and chemoattractant-induced motility. PMNs were pretreated with 250 nM wortmannin and/or 10 mM GlcNH2 as indicated (untreated samples are indicated by – and treated samples by +). The GlcNH2 pretreatments were for 60 min, and when they were concurrent with wortmannin pretreatment, wortmannin was added 15 min before the GlcNH2. The wortmannin alone samples (without GlcNH2) were treated with inhibitor for 75 min. The samples were assayed for basal motility (the absence of fMLP is indicated as –) or chemotaxis in response to 100 nM fMLP (indicated as +). B, the affect of wortmannin on PUGNAc-associated changes in chemoattractant-induced motility. PMNs were pretreated with 250 nM wortmannin and/or 100 µM PUGNAc as indicated (untreated samples are indicated by – and treated samples by +). The PUGNAc pretreatments were for 60 min, and when they were concurrent with wortmannin pretreatment, wortmannin was added 15 min before the PUGNAc. The wortmannin alone samples (without PUGNAc) were treated with inhibitor for 75 min. The samples were assayed for chemotaxis in response to 100 nM fMLP (indicated as +). C, the affect of wortmannin on PUGNAc-associated changes in basal motility. PMNs were treated with 250 nM wortmannin (Wort and + W) and/or 100 µM PUGNAc (PUG). When it was concurrent with wortmannin treatment, wortmannin was added 15 min before the PUGNAc. The PUGNAc treatments were for 30, 60, or 120 min. The control sample (Con) was untreated, and the wortmannin alone sample (Wort) was treated with inhibitor for 45 min. The samples were assayed for migration in the absence of chemoattractant.

 
Next, we examined whether wortmannin could inhibit PUGNAc-associated changes in PMN motility. The cells were treated as detailed above, except that 100 µM PUGNAc was used instead of GlcNH2. PUGNAc, like GlcNH2, produced a level of response to fMLP in the presence of wortmannin that was comparable with fMLP alone (Fig. 3B). In the case of basal motility, wortmannin was effective at reducing the increases associated with 30, 60, and 120 min of PUGNAc pretreatment to control levels of inhibition (wortmannin was added 15 min before each PUGNAc treatment and remained for the entire duration of those treatments) (Fig. 3C). These data suggest that O-GlcNAc is acting to increase basal motility through a pathway that is PI3K-dependent.

GlcNH2 and PUGNAc Increase the Rate of fMLP-induced MAPK and MKK Activation—The MAPK signaling cascade consists of a tripartite signaling hierarchy composed of a MAPK kinase kinase (MKKK), MKK, and MAPK with small G proteins of the Ras family often acting as upstream regulators of MKKKs (64). For example, the interaction of Ras with Raf allows Raf to activate MEK1/2, which then in turn activates p44/42 (65, 66). Rac activity is required for p38 phosphorylation in fMLP-stimulated PMNs (23, 26). The pathway from Rac to p38 is not clear but likely involves MKK3 and an undefined MKKK (67). Rac, through an unknown mechanism, also appears to be a critical necessary regulator of p44/p42 activation in PMNs (23, 26, 28). The activities of p38 and p44/42 have been found to be important for PMN chemotactic signaling (29–36).

We assessed MAPK signaling following pretreatment with GlcNH2 and PUGNAc. MAPK and MKK activation are conveniently measured by immunoblot analysis using phosphorylation state-specific antibodies. PMNs were pretreated with 10 mM GlcNH2 for either 30 or 90 min and then stimulated with 100 nM fMLP for 30, 60, or 90 s (Fig. 4). The GlcNH2 pretreated groups displayed faster rates of MAPK phosphorylation and hence activation. This increase was apparent for MKK3/6-p38 and MEK1/2-p44/42. We did not observe fMLP-induced JNK activation, and GlcNH2 pretreatment did not alter this (data not shown). Similar experiments were carried out using 100 µM PUGNAc (Fig. 5). The PUGNAc-pretreated groups also displayed faster rates of MAPK activation. These data suggest that O-GlcNAcylation positively modulates MAPK activity and, because of the signaling link between Rac and MAPKs, also support the finding that O-GlcNAcylation positively modulates Rac activation.



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  		FIG. 4.
GlcNH2 increases the rate of fMLP-induced MAPK and MKK activation. MAPK and MKK activities from whole cell lysates were assessed by immunoblotting with the indicated phosphospecific antibodies. The control sample (C) was not pretreated and was unstimulated. The cells were either not pretreated (no GlcNH2) or were pretreated with 10 mM GlcNH2 for 30 or 90 min. MAPK activity was assessed at 30, 60, or 90 s of stimulation with 100 nM fMLP. Actin was used to verify equal sample loading.

 



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  		FIG. 5.
PUGNAc increases the rate of fMLP-induced MAPK and MKK activation. MAPK and MKK activities from whole cell lysates were assessed by immunoblotting with the indicated phosphospecific antibodies. The control sample (C) was not pretreated and was unstimulated. The cells were either not pretreated (no PUGNAc) or were pretreated with 100 µM PUGNAc for 30 or 90 min. MAPK activity was assessed at 30, 60, or 90 s of stimulation with 100 nM fMLP. Actin was used to verify equal sample loading.

 
GlcNH2 and PUGNAc Increase Basal Levels of Ras-GTP but Have Little Effect on fMLP-induced Ras Activity—We found that O-GlcNAcylation clearly influences MEK1/2-p44/42 signaling. Although fMLP induces p44/42 MAPK activity in a Rac-dependent manner in PMNs (23, 26, 28), the archetypal upstream G protein regulator of its activity under most circumstances is Ras and not Rac (64–66). We therefore thought that it was important to evaluate whether PUGNAc or GlcNH2 could affect Ras activity.

Active, GTP-bound Ras was assessed in Ras immunoblots following affinity capture with Raf-1 RBD (57). 100 µM PUGNAc and 10 mM GlcNH2 increased basal Ras activity but had little effect on fMLP induced activity (Fig. 6). These data suggest that Rac is a more likely candidate than Ras for modulating the protein O-GlcNAc-associated changes in p44/42 MAPK activity that are induced by chemoattractant. It should be noted, that under some circumstances there is a considerable amount of cross-talk between Rac and Ras (64, 68–70), leaving open the potential for O-GlcNAc to alter Rac activity through Ras or for Rac to cooperate with Ras in signaling p44/42 activation.



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  		FIG. 6.
GlcNH2 and PUGNAc increase basal levels of Ras-GTP but have little effect on fMLP-induced Ras activity. Active GTP-bound Ras was assessed by affinity purification using the Raf-1 RBD. The specificity of this assay for GTP-bound Ras was established by the in vitro loading of Ras with GTP{gamma}S or GDP prior to affinity purification (inset). A, basal Ras activity in PMNs treated with GlcNH2 or PUGNAc. PMNs were left untreated (C) or were treated with 10 m M GlcNH2 or 100 µM PUGNAc for 15, 45, or 90 min. Total levels of Ras in the pre-assay lysates are indicated as input. B, chemoattractant-induced Ras activity in PMNs pretreated with GlcNH2 or PUGNAc. PMNs were not pretreated (C and the adjacent two lanes labeled 10 and 60 that are without PUGNAc or GlcNH2 labels) or were pretreated with 10 mM GlcNH2 or 100 µM PUGNAc for 90 min. Ras-GTP was assessed after stimulating the cells with 100 nM fMLP for 10 or 60 s. The control sample (C) was not stimulated with chemotactic factor. Total levels of Ras in the pre-assay lysates are indicated as input.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Although changes in O-GlcNAcylation have been implicated in the pathogenesis of certain disease states, for example, diabetes mellitus (5, 16, 71, 72) and Alzheimer disease (73, 74), the broader role of this ubiquitous post-translational modification in normal cellular function is as yet largely undefined. Previously, we reported that O-GlcNAc could be rapidly induced in PMNs by an extracellular agonist in a receptor-dependent manner and by GlcNH2 through a substrate-driven mechanism involving the HBP (18). In those studies we showed that GlcNH2-induced O-GlcNAcylation was associated with increases in cellular motility and migration. These data suggested that O-GlcNAc might act as a dynamic regulatory signal that modulates PMN physiology. Here we demonstrate for the first time that O-GlcNAcylation modulates the activities of several critical PMN signaling intermediates involved in the regulation of cellular movement.

To expand on and verify the observation that GlcNH2-induced O-GlcNAcylation is associated with altered PMN motility we assessed whether PUGNAc, which increases O-GlcNAc levels independent of the HBP, could serve as an alternative and complementary means of increasing O-GlcNAc and motility in PMNs. PUGNAc increased protein O-GlcNAc beginning within 30 min of treatment indicating that O-GlcNAc cycling is rapid in PMNs. This is an important observation in itself because it provides additional evidence that protein O-GlcNAcylation is a highly dynamic process in these cells and further substantiates the concept that O-GlcNAc may be important to normal PMN physiology. It also indicates that signals that lead to a reduction in O-GlcNAcase activity, in response to agonist, for example, may be an important means of inducing O-GlcNAcylation in these cells in addition to OGT-mediated mechanisms. In agreement with the effects of GlcNH2, we found that PUGNAc increased basal and chemoattractant-induced motility. This solidifies the conclusion that O-GlcNAc modulates neutrophil motility. Although we might expect that there are differences in the specificity of these agents for O-GlcNAc acceptor proteins (because they act through different mechanisms) and that these differences could result in differential O-GlcNAcylation, our data suggest that in the case of motility-associated signaling the same relevant proteins are being O-GlcNAcylated. This is supported by the observation that the overall patterns of O-GlcNAcylation induced by PUGNAc and GlcNH2 are grossly similar. The fact that two dissimilar O-GlcNAc-inducing treatments enhance motility strongly supports a role for O-GlcNAc as the regulatory mechanism in PMN motility.

We next sought to extend our physiological observations into the signaling pathways known to influence motility. The small GTPase Rac is an essential molecular switch in regulating actin assembly and motility in PMNs (23–26, 60). Therefore we examined whether increased O-GlcNAc levels influenced Rac activity. Three types of Rac have been identified (75), although most studies have focused on the roles of Rac1 and Rac2. Rac1 is ubiquitously expressed, whereas Rac2 expression is restricted to hematopoietic cells (75). Although the precise roles of each of these isoforms remain largely undefined, recent evidence indicates that Rac2 may be the primary regulator of actin assembly and the molecular machinery associated with movement, whereas Rac1 may be important for chemoattractant-directed orientation and gradient detection (76). In addition, Rac1 and Rac2 appear to exhibit cross-talk with one another, and their activities appear to be closely associated (77). This would explain why combined Rac1/Rac2 knock-outs demonstrate a significantly worse chemotactic defect than single knock-outs of either Rac1 or Rac2 (23). Our data indicate that O-GlcNAcylation is associated with increases in both basal and chemoattractant-directed Rac1 activity and that GlcNH2 and PUGNAc were equally effective in their effects on Rac activity. Unfortunately we were unable to adequately assess Rac2 activity. Although the Rac2-specific antibodies we used readily detected Rac2 in the input lysates used for Rac-GTP affinity assays they did not detect Rac2-GTP associated with PAK-1 PBD (data not shown). Thus, it remains unclear how Rac2 is affected by O-GlcNAc. Interestingly the effects of increased O-GlcNAcylation on Rac1 were more pronounced during fMLP-induced Rac activation. O-GlcNAc lengthened the active phase of Rac activation, maintaining maximal levels of Rac activity for at least 60 s as opposed to 10 s in the absence of PUGNAc or GlcNH2 pretreatment. Although it did not appear to increase the absolute level of Rac activation. These data suggest that O-GlcNAc acts at or upstream of Rac and represent the first example of O-GlcNAc-associated changes in Rac activity. However, a recent study has shown that increased flux through the HBP could lead to increased Rho activity, although a specific role for O-GlcNAcylation was not assessed (78).

PI3K is an important regulatory intermediate of PMN motility that is closely tied to Rac (25, 27). Type 1 PI3Ks can be activated by cell-surface receptors to produce PIP3 (22), which acts as a membrane-associated docking site for pleckstrin homology domains (79). PI3K activity directs chemoattractant-induced Rac activation, although Rac participates in a positive feedback loop that further induces PI3K and PIP3 accumulation (61, 62). This amplifies the polarized asymmetry of PIP3 and polymerized actin at the leading edge, critical events in motility and directional migration. Exogenous PIP3 is capable of inducing migration and actin polymerization in a wortmannin and Rho family GTPase (presumably Rac)-dependent manner (80), indicating that in the absence of additional stimuli, isolated PI3K and Rac activation may induce changes in cellular motility. Therefore, we examined the role of PI3K in mediating the effects of O-GlcNAcylation on motility. Wortmannin effectively blocked the ability of PUGNAc and GlcNH2 to induce changes in basal motility, or chemokinesis. The ability of wortmannin to effectively block fMLP-associated chemotaxis was reduced in GlcNH2- and PUGNAc-pretreated PMNs. These data are consistent with the notion that O-GlcNAc-mediated changes in motility occur through a mechanism that requires PI3K activity; however, further studies are required to define more precisely the role of PI3K in O-GlcNAc-associated changes in motility.

MAPKs are downstream effectors in the signaling pathways that mediate motility. In PMNs, pharmacological inhibition of p38 and p44/42 MAPKs has been shown to decrease chemotaxis (29–36). Although, the precise role of MAPK activation in regulating motility is unclear, both p38 and p44/42 phosphorylate and activate mitogen-activated protein kinase-activated protein kinase (35, 81), which in turn regulates leukocyte-specific protein 1 (39) and the small heat shock protein HSP27 (82). Leukocyte-specific protein 1 and HSP27 are involved in the regulation of the actin cytoskeleton and motility (39, 41–45). We found that increases in O-GlcNAc lead to significant increases in the rate of MAPK cascade activation with a slight increase in the maximal level of MAPK and MKK phosphorylation. Because Rac regulates the p38 and p44/42 signaling cascades in fMLP-stimulated PMNs, these increases in O-Glc-NAc-associated MAPK activity are consistent with similarly increased levels of active Rac in response to O-GlcNAcylation. Ras, however, is the traditional and best described mediator of MAPK signaling with respect to p44/p42 (64–66). We found that although Ras-GTP was not increased substantially with fMLP stimulation, its basal levels were elevated in association with increased O-GlcNAc levels. This then leaves Rac as the stronger candidate for mediating O-GlcNAc-associated changes in MAPK activity.

Several studies, all performed under different cellular contexts, have recently demonstrated that increases in HBP flux can lead to increased MAPK activation (16, 78, 83–85). In addition, HBP-driven changes in p44/p42 activation have been suggested to occur through a Rho GTPase-mediated mechanism in cultured rat aortic smooth muscle cells (78). Of further significance is the finding that high concentrations of glucose, potentially through the HBP, sensitize vascular smooth muscle cells to serum, inducing chemotaxis via pathways involving PI3K, the Ras superfamily of GTPases and p44/42 MAPK (increased phosphorylation of p38 was also observed but was not associated with glucose-mediated changes in migration) (16). Our data support a role for Rac, a Rho family GTPase, PI3K, and the p38 and p44/42 MAPKs in O-GlcNAc-associated changes in PMN migration. The similarities among these varied studies indicate that there may be a common intermediate signaling mechanism that underlies O-GlcNAc-mediated increases in MAPK activity, small monomeric GTPase activity and motility.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant DK55647 and the Juvenile Diabetes Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 205-934-1294; Fax: 205-934-0950; E-mail: rbmarchase{at}uab.edu.

1 The abbreviations used are: O-GlcNAc, O-linked {beta}-N-acetylglucosamine; OGT, UDP-N-acetylglucosamine:polypeptide O-{beta}-N-acetylglucosaminyltransferase; O-GlcNAcase, neutral {beta}-N-acetylglucosaminidase; GlcNH2, glucosamine; HBP, hexosamine biosynthesis pathway; PUGNAc, O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino-N-phenylcarbamate; PMN, neutrophil or polymorphonuclear leukocyte; fMLP, formyl-methionine-leucine-phenylalanine; PI3K, phosphoinositide 3-kinase; MAPK, mitogen-activated protein kinase; MKK, mitogen-activated protein kinase kinase; MKKK, mitogen-activated protein kinase kinase kinase; HSP27, heat shock protein 27; PAK-1 PBD, p21-binding domain of p21-activated kinase 1; Raf-1 RBD, Ras-binding domain of Raf-1; GTP{gamma}S, guanosine 5'-3-O-(thio)triphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate. Back


   ACKNOWLEDGMENTS
 
We thank Sherry Johnson for administrative support and John Chatham and Pam Bounelis for insightful input. We additionally thank Mary Ann Accavitti and the UAB Epitope Recognition and Immunodetection Core for the production of the CTD110.6 monoclonal antibody.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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