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The Journal of Immunology, 2001, 166: 5585-5593.
Copyright © 2001 by The American Association of Immunologists

Fc{alpha} Receptor Cross-Linking Causes Translocation of Phosphatidylinositol-Dependent Protein Kinase 1 and Protein Kinase B{alpha} to MHC Class II Peptide-Loading-Like Compartments1

Mark L. Lang, Li Shen, Hong Gao, William F. Cusack, Gillian A. Lang and William F. Wade2

Department of Microbiology, Dartmouth Medical School, Lebanon, NH 03756


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A20 IIA1.6 B cells cotransfected with Fc{alpha}R and wild-type {gamma}-chain (wt-ITAM (immunoreceptor tyrosine-based activation motif)) or Fc{alpha}R and {gamma}-chain, in which the wt-ITAM was substituted with the Fc{gamma}RIIA ITAM (IIA-ITAM), were used to investigate cell signaling events influencing presentation of Fc{alpha}R-targeted exogenous Ag in the context of MHC class II. wt-ITAM cells presented Fc{alpha}R-targeted OVA more efficiently than IIA-ITAM transfectants to OVA-specific T cell hybridomas. Phosphatidylinositol 3-kinase (PI 3-kinase) inhibition abrogated Ag presentation, suggesting that Fc{alpha}R may trigger a PI 3-kinase-dependent signal transduction pathway, and thus phosphatidylinositol-dependent protein kinase (PDK1) and protein kinase B {alpha} (PKB{alpha}) activation. Cross-linking Fc{alpha}R on wt-ITAM or IIA-ITAM cells triggered equivalent PI 3-kinase-dependent activation of PKB{alpha}. Furthermore, Fc{alpha}R cross-linking triggered recruitment of PDK1 and serine-phosphorylated PKB{alpha} to capped cell surface Fc{alpha}R irrespective of the {gamma}-chain ITAM. Although Fc{alpha}R endocytosis was accompanied by translocation of PDK1 and phospho-PKB{alpha} to Fc{alpha}R-containing vesicles in both transfectants, this was decreased in IIA-ITAM cells, and a significant proportion of PDK1 and PKB{alpha} remained at the plasma membrane. In wt-ITAM cells, PDK1 and serine-phosphorylated PKB{alpha} translocated to lysosomal-associated membrane glycoprotein 1- and cathepsin B-containing vesicles, consistent with MHC class II peptide-loading compartments (MIIC) described by other groups. Our data indicate that translocation of signal transduction mediators to MIIC-like compartments accompanies efficient presentation of receptor-targeted Ag, and suggest a mechanism connecting signaling to the Ag-processing pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The human IgA receptor (Fc{alpha}R) is a 50- to 70-kDa transmembrane glycoprotein expressed primarily by myeloid cells, including neutrophils, monocytes, and macrophages (1, 2). A variety of cellular responses is triggered by cross-linking Fc{alpha}R, including phagocytosis, the oxidative burst, and degranulation in neutrophils and monocytes (3, 4). More recently, attention has turned to endocytosis of Fc{alpha}R-targeted exogenous Ag leading to presentation of Ag-derived peptide on MHC class II (5).

The targeting of exogenous Ag to FcR or B cell Ag receptor (BCR)3 results in presentation of the Ag on class II (6, 7, 8, 9, 10). Presentation of receptor-targeted Ag on MHC class II is preceded by endocytosis of receptor-bound Ag and trafficking of receptor-Ag complexes to late endosomes that contain class II (11, 12, 13, 14). These compartments, variously termed MHC class II peptide-loading compartments (MIIC), are characterized by key markers, including lysosomal-associated membrane glycoprotein (lamp1), class II, and cathepsin B (15). Evidence suggests the MIIC is a specialized compartment in which proteolyzed Ag is loaded onto class II for transport to the cell surface and presentation to CD4+ T cells (15, 16). Siemasko and coworkers (17) demonstrated that induction of an MIIC-like compartment was dependent on BCR-triggered signals, including tyrosine kinase activation, Ca2+ flux, and protein kinase C activation. Additionally, BCR-triggered activation of the tyrosine kinase Syk was shown to be essential for presentation of BCR-targeted Ag (18). However, beyond these observations, the details of the signaling events governing formation and function of the MIIC are uncharacterized.

Control of Ag presentation by signal transduction is a favored hypothesis currently undergoing examination (5, 18, 19). To understand how signal transduction events regulate presentation of Fc{alpha}R-targeted Ag, it is necessary to delineate Fc{alpha}R-triggered signal transduction events and identify those involved in Ag presentation. Association of Fc{alpha}R with the FcR {gamma}-chain to form the trimer Fc{alpha}R/{gamma}{gamma} is a necessary prerequisite for Fc{alpha}R signal transduction (20). Signaling by {gamma}-chain is achieved by the intrinsic tyrosine kinase activation motifs, otherwise known as ITAM (immunoreceptor tyrosine-based activation motif) regions (21). The {gamma}-chain ITAM region consists of two YxxL sequences separated by a spacer region of 7 aa. The tyrosine residues within ITAMs are phosphorylated on cross-linking of {gamma}-chain-associated receptors (22).

The associations between Fc{alpha}R/{gamma}{gamma} and tyrosine kinases Lyn, Syk, and Btk have been known for some time (23, 24). More recently, we have demonstrated that upon cross-linking, Fc{alpha}R immediately partitions into membrane glycolipid rafts with accompanying phosphorylation of {gamma}-chain, Lyn, and Btk (25). Consistent with activation of tyrosine kinases, cross-linking Fc{alpha}R triggers calcium release from intracellular stores (26). Park and coworkers (27) demonstrated that cross-linked Fc{alpha}R/{gamma}{gamma} formed complexes containing proteins such as Grb2, Shc, SHIP, Cbl, and SLP-76 in U937 cells. Furthermore, the Fc{alpha}R-triggered activation of NADPH oxidase in neutrophils is inhibited by PI 3-kinase inhibitors (28). NADPH oxidase activation depends on fusion of vesicles containing oxidase subunits and translocation of subunits to the phagolysosomal membrane (29). Thus, Fc{alpha}R appears capable of driving reorganization of intracellular vesicles via signaling elements downstream of PI 3-kinase. Remodeling of vesicles is also evident in MIIC induction (17).

Because Btk is activated by the lipid product of phosphatidylinositol 3-kinase (PI 3-kinase) (30), our observation that cross-linking Fc{alpha}R triggers Btk phosphorylation (M. L. Lang, L. Shen, and W. F. Wade, unpublished observation) suggested that this may also result in PI 3-kinase-dependent activation of the downstream kinases phosphatidylinositol-dependent protein kinase (PDK1) and protein kinase B{alpha} (PKB{alpha}). Activation of PKB{alpha} by BCR has also been demonstrated (31, 32, 33, 34). Cross-linked BCR in A20 IIA1.6 cells triggers PI 3-kinase-dependent translocation of PKB{alpha} to the plasma membrane and increases phosphorylation of PKB{alpha} within 5 min (33). PKB{alpha} is thought to be involved in regulating vesicle traffic (reviewed in Ref. 35). However, no studies have examined the functions of PKB{alpha} with regard to presentation of Ag, and to date PKB{alpha} activation by Fc{alpha}R has not been investigated.

In this study, we present the novel finding that PDK1 and PKB{alpha} are translocated to intracellular vesicles during endocytosis of cross-linked Fc{alpha}R. Furthermore, PI 3-kinase-dependent activation of PKB{alpha} results in relocalization of PDK1 and PKB{alpha} from the cytosol to the plasma membrane and then to MIIC when the signal transduction cascade is generated by a wild-type {gamma}-chain (wt-ITAM). We also examined cells in which Fc{alpha}R was cotransfected with an altered {gamma}-chain (IIA-ITAM) in which the wt-ITAM was replaced by the ITAM from the IgG receptor, Fc{gamma}RIIa. The IIA-ITAM has a 12-aa spacer region between the two YxxL phosphorylation sequences compared with 7 aa in the wt-ITAM (36). We show that IIA-ITAM {gamma}-chain does not trigger efficient presentation of Ag by class II, but does translocate PDK1 and PKB{alpha} to the plasma membrane. However, in IIA-ITAM transfectants, there is reduced association of PDK1 and PKB{alpha} with the MIIC. This study suggests that subcellular localization of PKB{alpha} may play a role in presentation of Fc{alpha}R-targeted Ag.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and transfections

Cells of the A20 IIA1.6 B cell line are FcR negative (37). IIA1.6 cells transfected with Fc{alpha}R and {gamma}-chain were a gift from Dr. M. Van Egmond (University Hospital Utrecht, Utrecht, The Netherlands). The pCAV/Fc{alpha}R cDNA construct was a gift from Dr. C. Maliszewski (Immunex, Seattle, WA). PNUT/{gamma}-chain constructs encoded for {gamma}-chain containing the wt C-terminal sequence (YTGLNTRSQETYYETLKEHPPQ), or {gamma}-chain with this region substituted with the Fc{gamma}RIIa C-terminal sequence (YMTLNPRAPTDDDKNIYLTLPPNDHVNSNN) (36). The major differences between the two ITAM structures are the lengths of the spacer regions between the phosphorylation motifs (underlined). The pNUT vector allows selection using 2 µM methotrexate, and the pCAV vector allows selection using 0.2 µM G418. Bulk cultures of transfectants expressing Fc{alpha}R and {gamma}-chain were cultured in RPMI medium supplemented with 10% FBS, 40 µg/ml gentamicin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 2 µM methotrexate. OVA-specific T cell hybridomas DO-11-10, DO-18.6, and DO-54.8 were a gift from Dr. P. Marrack (National Jewish Center, Denver, CO) and were cultured in DMEM supplemented with nonessential amino acids (Life Technologies, Grand Island, NY), 1 mM sodium pyruvate, 2 mM L-glutamine, 0.75 mg/ml dextrose, 0.85 mg/ml sodium bicarbonate, 50 µg/ml gentamicin, and 10% FBS. The IL-2-dependent T cell line HT-2 was cultured in the same medium supplemented with 5 U/ml IL-2. Human peripheral blood monocytes were obtained by centrifugation of whole blood over Ficoll-Hypaque (Pharmacia-Biotech, Uppsala, Sweden). The mononuclear cell layer was withdrawn and washed three times in PBS. Experiments were performed on crude mononuclear cell fractions (~10% CD163+/CD89+ cells) because purification of monocytes by adherence or cold aggregation could potentially activate integrin-dependent kinase pathways that include PKB{alpha} activation (38).

Abs and fluorochromes

All anti-Ig Abs (including FITC-, indocarbocyanine 3 (Cy3)-, indocarbocyanine 5 (Cy5)-, and HRP-conjugated Abs) were purchased from Jackson ImmunoResearch (West Grove, PA), unless indicated. Fc{alpha}R was ligated with the anti-Fc{alpha}R mAb My43 (mouse IgM produced in our laboratory) (39) and cross-linked with either a FITC-conjugated rabbit anti-mouse IgM (RAM) or FITC-conjugated donkey anti-mouse IgM. Anti-PDK1 and anti-PKB{alpha} are goat polyclonal IgG Abs and were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphoserine 473 PKB{alpha} (sheep IgG) and anti-cathepsin B (rabbit IgG) were obtained from Upstate Biotechnology (Lake Placid, NY). Anti-lamp1 (rat IgG2a) was purchased from PharMingen (San Diego, CA). Isotype control goat serum IgG, sheep serum IgG, and rabbit serum IgG were obtained from Sigma (St. Louis, MO). Blocking human IgG (Cohn fraction) for experiments with monocytes was also obtained from Sigma.

Ag presentation assays

OVA (Worthington Biochemical, Lakewood, NJ) was derivatized with nitro-iodophenol caproate-o-succinimide (NIP; Genosys, The Woodland, TX) to give an average of three NIP haptens per OVA. For derivatization, NIP was dissolved in dimethyl formamide and added to OVA dissolved in borate-buffered saline, pH 8.3. After 1.5 h, the mixture was dialyzed into PBS, pH 7.4. Soluble IgA-OVA complexes (IgA-OVA) were made by mixing chimeric human IgA2 anti-NIP (gift from Drs. R. Jefferies and D. M. Goodall, University of Birmingham, Edgebaston, U.K.) with NIP-derivatized OVA at a molar ratio of 3:1. A20 IIA1.6 cells (2 x 105 cells in 100 µl) were cultured in duplicate with 100 µl IgA anti-NIP/NIP-OVA complexes (IgA-OVA) or OVA alone and OVA-specific T cells (5 x 105 cells in 100 µl) for 20 h, after which supernatants were removed and frozen. Three different OVA-specific T cell hybridomas were used (DO-11-10, DO-18.6, DO-54.8). Ag presentation was measured by assaying ability of serial dilutions of supernatant to promote survival of IL-2-dependent HT-2 T cells (40). Results are expressed as mean of duplicates in representative experiments. In this assay, titers that differ by 4-fold or more are significantly different (41, 42). For inhibitor studies, Fc{alpha}R-transfected cells were pretreated with PI 3-kinase inhibitors for 30 min before addition of IgA-OVA complexes. After 4-h incubation at 37°C, the inhibitor was removed by washing three times in PBS, and the cells were resuspended in 0.15% w/v paraformaldehyde in PBS for 30 min at 22°C. Cells were then washed three times and resuspended in T cell culture medium for Ag presentation assays.

Preparation of stimulated cell lysates

Fc{alpha}R was cross-linked as described and the cells held at 4°C and lysed or warmed to 37°C for indicated times before lysis. For inhibition studies, cells were pretreated with 100 nM wortmannin (Calbiochem-Novabiochem, La Jolla, CA) for 30 min at 37°C. Cell lysis was achieved by addition of 400 µl of 2x lysis buffer to 400 µl of cell suspension containing 107 cells per sample. Lysis buffer contained 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5 mM Na3VO4, 0.1% v/v 2-ME, 1% v/v Triton X-100, 50 mM NaF, 5 mM sodium pyrophosphate, 10 mM sodium {beta}-glycerophosphate, 1 µM microcystin (Calbiochem-Novabiochem), and complete protease inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN). Cells were treated at 4°C with lysis buffer for 30 min before centrifugation at 13,000 x g for 20 min and collection of supernatants. These samples were stored at -70°C and used within 24 h.

SDS-PAGE and immunoblotting

Cell lysates were resolved by SDS-PAGE electrophoresis under reducing conditions. Proteins were transferred to nitrocellulose membranes and incubated overnight at 4°C with 5% nonfat dry milk and 0.5% Tween 20 in PBS. Membranes were incubated with Abs to PKB{alpha} (0.2 µg/ml) or phosphoserine 473 PKB{alpha} (0.5 µg/ml) for 2 h at room temperature, and washed six times for 5 min in PBS and transferred to tubes containing 3% nonfat dry milk/0.05% Tween 20 in PBS. HRP-conjugated, anti-goat, or sheep IgG Ab was added at a 1/5000 dilution (0.2 µg/ml), and membranes were incubated for an additional 2 h at room temperature. Membranes were then washed six times for 5 min in PBS. Proteins were detected by ECL (Amersham-Pharmacia Biotech, Piscataway, NJ).

PKB in vitro activity assay

The PKB{alpha} in vitro kinase assay kit was purchased from Upstate Biotechnology, and the assay was performed according to manufacturer’s instructions. Briefly, anti-PKB{alpha} Ab coupled to protein G-Sepharose (Amersham-Pharmacia Biotech) was used to immunoprecipitate PKB{alpha} from the cell lysates. This Ab recognizes an epitope in the pleckstrin homology domain of PKB{alpha}. After washing, immunoprecipitates were incubated with reaction buffer containing a protein kinase A inhibitor peptide, a substrate peptide modeled on the target sequence of PKB{alpha} on GSK3{alpha}, and 10 µCi [{gamma}-32P]ATP (DuPont-NEN, Boston, MA). After transfer of the 32P-phosphate group to the substrate peptide, immunoprecipitates were removed by centrifugation and TCA added to samples before blotting onto phosphocellulose and washing in 0.75% phosphoric acid, followed by drying in acetone. The phosphocellulose was then added to scintillation vials and counted in a Beckman LS 600IC scintillation counter. Nonspecific backgrounds were assessed by immunoprecipitation of cell lysates with protein G-Sepharose coupled to an isotype control Ab (sheep IgG), and performing the assay with these samples. Background values were routinely <1000 cpm/106 cells and were subtracted from all experimental values before analysis.

Endocytosis of Fc{alpha}R

Cells (106 cells/sample) were incubated with 1 µg of anti-Fc{alpha}R mAb (My43) Ab for 1 h at 4°C before washing three times in ice-cold PBS. Cells were then incubated with 1 µg of FITC-conjugated RAM for 1 h at 4°C before washing three times in PBS. Cells were resuspended in a minimal volume of ice-cold medium (50 µl), then warmed to 37°C by addition of 1 ml medium at 37°C for 30 min. Endocytosis was stopped by addition of 10 ml of ice-cold paraformaldehyde in PBS and incubation at 4°C for 30 min. Cells were then washed by centrifugation and resuspended in PBS. For experiments with monocytes, Fc{alpha}R cross-linking was performed by the same method as for A20 cells, but in the presence of 3 mg/ml human IgG to block binding of Abs to Fc{gamma}R expressed on monocytes.

Intracellular staining

Cells were permeabilized at room temperature by resuspending in 1 ml 0.5% w/v saponin, 0.1% w/v BSA, and 0.1% w/v NaN3 in PBS, and incubating at room temperature for 20 min. Cells were then washed by centrifugation and resuspended in permeabilization buffer containing anti-PDK1 or anti-phosphoserine 473 PKB Abs, as appropriate. Cells were incubated at 4°C for 45 min at room temperature before washing three times in permeabilization buffer and resuspending in buffer containing Cy3-conjugated rabbit anti-goat or rabbit anti-sheep Abs, as required. After incubation for an additional 45 min, cells were washed three times and resuspended in 1 ml PBS. Isotype controls were performed by substitution of anti-PDK1 or anti-PKB with 1 µg of nonspecific sheep or goat IgG, as required. For experiments with monocytes, intracellular staining was performed as above, in the presence of 3 mg/ml human IgG.

Laser-scanning confocal fluorescence microscopy

One million cells were added to chamber slides (Fisher Biosciences, Pittsburgh, PA) previously coated with 0.1 mg/ml poly(L-lysine) and allowed to adhere by overnight incubation at 4°C. Nonadhered cells were removed by gentle rinsing with PBS. Coverslips were then mounted using Prolong Antifade (Molecular Probes, Eugene, OR), and cells were analyzed with a Bio-Rad (Richmond, CA) MRC1024 laser-scanning confocal system equipped with Kry/Arg laser and beam splitter to allow simultaneous two-color and three-color imaging. Images were analyzed using Adobe PhotoShop 4.0 software (Mountain View, CA). Cells with endocytosed Fc{alpha}R were assessed for plasma membrane-associated or vesicle-associated PDK1 or phospho-PKB{alpha}. The distribution of PDK1 or phospho-PKB{alpha} at t = 2 min, in which membrane staining only was observed, was used as the standard for membrane-associated PDK1 or PKB{alpha}. No PDK1 or PKB{alpha} was seen in an organized intracellular location at this time point (2 min). During the subsequent signaling time course, any cells showing the staining pattern seen for 2 min were scored as having membrane-associated PDK1 or phospho-PKB{alpha}. Cells exhibiting staining that was in the interior of the cell (condensed into vesicle-like structures) in the absence of peripheral staining at 30 min were scored as having vesicle-associated fluorescence. Cells with vesicle (MIIC)-associated PDK1 or PKB{alpha} were described as Fc{alpha}R colocalized when fluorochrome distribution overlapped, giving yellow fluorescence rather than separate green (FITC), or red (Cy3) fluorescence.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Presentation of Fc{alpha}R-targeted exogenous Ag to CD4+ T cells

To determine whether altering the structure of the {gamma}-chain ITAM resulted in differences in the ability of Fc{alpha}R/{gamma}-chain cotransfectants to mediate presentation of Fc{alpha}R-targeted OVA, we compared Ag presentation by wt-ITAM and IIA-ITAM transfectants (Fig. 1Go). Fc{alpha}R-targeted OVA was presented to DO–11-10 T cell hybridomas more effectively by wt-ITAM than by IIA-ITAM cells (Fig. 1Goa). Similar data were obtained with OVA-specific DO-18.6 cells (Fig. 1Gob) and DO-54.8 cells (Fig. 1Goc). IL-2 titers induced were consistently 4- to 8-fold higher in the wt-ITAM cells than in the IIA-ITAM cells. No IL-2 titer was detectable in nontransfected parent A20 cells (not shown). Treatment of wt-ITAM or IIA-ITAM cells with IgA or OVA alone at the concentration used for Fc{alpha}R-targeted OVA did not result in Ag presentation (not shown). This suggests that a property associated with the ITAM, such as signal transduction, may modulate presentation of Fc{alpha}R-targeted Ag.



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  		FIGURE 1. Differential presentation of Fc{alpha}R-targeted Ag by wt-ITAM compared with IIA-ITAM cells. Cells were treated with IgA/OVA complexes at concentrations shown (OVA) for 20 h in culture with a, DO-11-10; b, DO-18.6; c, DO-54.8 T cells. Supernatants were collected and cultured with HT-2 T cells, as described in Materials and Methods. Data are represented as IL-2 titers produced by OVA-dependent HT-2 T-cells. •, wt-ITAM cells; {circ}, IIA-ITAM cells. Each graph is representative of three similar experiments performed in duplicate.

 
Fc{alpha}R-triggered presentation of exogenous Ag is abrogated by PI 3-kinase inhibition

To address whether there is a requirement for PI 3-kinase-triggered signal transduction in Fc{alpha}R-mediated Ag presentation, wt-ITAM transfectants were pretreated with the PI 3-kinase inhibitor LY294002 before incubation with IgA anti-NIP/NIP-OVA complexes and fixation (Fig. 2Go). LY294002, a reversible PI 3-kinase inhibitor, was removed by washing before incubation of Ag-treated, fixed, wt-ITAM transfectants with DO-11-10 T cell hybridomas to eliminate the possibility of inhibition of PI 3-kinase in the T cells. Treatment with LY294002 inhibited Fc{alpha}R-mediated presentation of Ag in a dose-dependent manner, suggesting that activation of PI 3-kinase and perhaps downstream effectors are necessary for presentation of Ag on class II. DMSO, the carrier for LY294002, had no effect on Ag presentation.



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  		FIGURE 2. PI 3-kinase inhibition prevents Fc{alpha}R-mediated Ag presentation. Cells (wt-ITAM) were treated with LY294002 at concentrations indicated for 30 min at 37°C before addition of anti-NIP-IgA (18 µg/ml) and NIP-OVA (6 µg/ml) and incubation for an additional 4 h at 37°C. Cells were then fixed in ice-cold 0.15% w/v paraformaldehyde in PBS before incubation with DO-11-10 T cells, as described in Materials and Methods. Graph shows IL-2 titers obtained from four Ag presentation assays performed in duplicate ± SD.

 
Fc{alpha}R triggers PI 3-kinase-dependent activation of PKB

Our results suggested that PI 3-kinase activity triggered by cross-linking of Fc{alpha}R with IgA/OVA complexes was involved in the Ag processing and presentation pathway. In both wt-ITAM and IIA-ITAM cells, cross-linking Fc{alpha}R caused similar tyrosine phosphorylation of PI 3-kinase that was detected in raft fractions (Lang et al., unpublished observation). The effect of PI 3-kinase could be direct through phosphatidylinositol 3,4,5-trisphosphate production or indirect through downstream signaling events. We therefore examined the activation of the downstream kinase PKB{alpha} in wt-ITAM and IIA-ITAM transfectants (Fig. 3Goa). PKB{alpha} activity triggered by Fc{alpha}R on wt-ITAM and IIA-ITAM transfectants was similar after 2-min incubation at 37°C, and was inhibited to background levels by preincubation of cells with 100 nM wortmannin. After incubation of cells at 37°C for 30 min, PKB{alpha} activity was observed to have returned to near basal levels (data not shown). This demonstrates that Fc{alpha}R in wt-ITAM and IIA-ITAM cells triggers PI 3-kinase-dependent activation of PKB{alpha}. Whole cell lysates of wt-ITAM and IIA-ITAM cells were immunoblotted for serine 473-phosphorylated PKB{alpha} with an Ab specific for the serine-phosphorylated form (Fig. 3Gob). In wt-ITAM and IIA-ITAM cells, there was similar serine 473 phosphorylation of PKB{alpha} in response to Fc{alpha}R cross-linking. Phosphorylation was detectable after 30 s and sustained over the course of 30 min in both wt-ITAM and IIA-ITAM cells. Densitometry measurements were performed on immunoblots, and the ratio of phospho-PKB{alpha} to total PKB{alpha} was plotted for each time point (Fig. 3Goc). In wt-ITAM and IIA-ITAM cells, similar PKB{alpha} phosphorylation was observed for over a 30-min period, suggesting similar levels of activity in both transfectants.



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  		FIGURE 3. Fc{alpha}R triggers PI 3-kinase-dependent activation of PKB{alpha}. Fc{alpha}R on wt-ITAM and IIA-ITAM cells was cross-linked with My43 (anti-Fc{alpha}R mAb), followed by a RAM IgM (µ-chain specific), or with RAM alone. Cells were either held at 4°C and lysed, or incubated at 37°C for 0.5, 2, 5, 15, or 30 min and lysed. Cells that were incubated at 37°C for 2 min were assayed for PKB{alpha} activity, as described in Materials and Methods. a, Graph shows steady state PKB{alpha} activity, the effect of warming cells to 37°C, the effect of Fc{alpha}R cross-link, and the consequence of 100 nM wortmannin treatment before Fc{alpha}R cross-link. Data show the incorporation of [{gamma}-32P]phosphate into a substrate peptide, and are expressed as cpm/106 cells and show the mean of three independent experiments ± SD. wt-ITAM cells are represented by black bars, and IIA-ITAM cells by gray bars. b, Lysates from cells with Fc{alpha}R cross-linked for indicated time points were immunoblotted for phosphoserine 473 PKB{alpha} or total PKB{alpha}. c, Densitometry of phospho-PKB{alpha} and total PKB{alpha} was performed on immunoblots, and phospho-PKB{alpha}/total PKB{alpha} ratios were plotted. Data show the mean ratios obtained from three individual experiments ± SD.

 
Translocation of PDK1 and phospho-PKB{alpha} to the plasma membrane and to endocytic vesicles after Fc{alpha}R cross-linking and endocytosis

We demonstrated that Fc{alpha}R cross-linking results in PI 3-kinase-dependent activation of PKB{alpha}. However, these data do not show any direct association between PI 3-kinase or its downstream effectors and Ag presentation. Presentation of Fc{alpha}R-targeted Ag is dependent on transport of Ag through the endocytic pathway to class II-containing MIIC-like compartments. Thus, we were interested in the possibility that the subcellular locations of downstream effectors of PI 3-kinase might provide evidence for a link to Ag presentation. We therefore performed experiments in which Fc{alpha}R was cross-linked and cells fixed either before or after 30 min of Fc{alpha}R endocytosis, before permeabilization and counterstaining for PDK1 or serine 473-phosphorylated PKB{alpha}.

When cells were held at 4°C (0 min) and fixed before counterstaining for PDK1, Fc{alpha}R (green) in both wt-ITAM and IIA-ITAM transfectants had a similar distribution on the cell surface (Fig. 4Goa). Fc{alpha}R had a punctate staining pattern that was evenly distributed around the cell periphery. PDK1 (red) was observed to be localized primarily with the plasma membrane and, like Fc{alpha}R, was evenly distributed around the cell periphery (Fig. 4Goa). After incubating cells at 37°C for 2 min before fixation, Fc{alpha}R (green) was observed to have redistributed on the plasma membrane and have a polarized (capped) distribution on wt-ITAM cells and IIA-ITAM cells (Fig. 4Gob). The degree of Fc{alpha}R capping from cell to cell was variable. Therefore, Fc{alpha}R was described as capped when Fc{alpha}R cell surface staining was clearly polarized and concentrated mostly into one sector comprising no more than two-thirds of the cell periphery. In both wt-ITAM and IIA-ITAM cells with capped Fc{alpha}R, PDK1 (red) was observed to have relocalized from an even distribution around the cell periphery to having a polarized distribution like that of Fc{alpha}R. In these cells, the distribution of Fc{alpha}R (green) and PDK1 (red) overlapped such that Fc{alpha}R and PDK1 were colocalized. Previous studies in our laboratory demonstrated that capped Fc{alpha}R colocalizes with glycolipid membrane rafts and that the distribution of the raft glycosphingolipid marker GM-1 corresponded to the capped distribution of capped Fc{alpha}R (25). Based on current knowledge, it is likely that signaling-competent rafts are in the order of 100-nm size and that they are able to cluster together under cross-linking conditions. Occurrence of even larger domains up to 500 nm or more is possible due to gathering of raft clusters under cytoskeletal influence (43). Thus, the large caps observed in this and previous studies may not coincide with a single large cluster of membrane rafts, but may contain several smaller clusters of rafts and associated proteins that cannot be resolved by the confocal microscope. However, these data are consistent with Fc{alpha}R cross-linking causing recruitment of PDK1 to membrane rafts.



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  		FIGURE 4. Differential Fc{alpha}R-triggered relocalization of PDK1 and PKB{alpha}. Fc{alpha}R (green, FITC) on wt-ITAM and IIA-ITAM cells was ligated at 4°C with anti-Fc{alpha}R, then cross-linked with FITC RAM IgM at 4°C, as described in experimental procedures. Cells were held at 4°C and fixed (a, d), incubated for 2 min at 37°C to allow Fc{alpha}R capping (b, e), or incubated for 30 min at 37°C to allow Fc{alpha}R endocytosis (c, f). Cells were then permeabilized with 0.5% w/v saponin in PBS and counterstained for PDK1 (red, Cy3) (a–c) or phospho-PKB{alpha} (red) (d–f). Cells were mounted on slides and analyzed by two-color confocal microscopy. wt-ITAM cells are shown on left, and IIA-ITAM cells are shown on right.

 
After incubation of cells at 37°C for 30 min, Fc{alpha}R (green) was observed to have become endocytosed from the cell surface to intracellular vesicles in both wt-ITAM and IIA-ITAM cells (Fig. 4Goc). Larger Fc{alpha}R-containing vesicles were observed in wt-ITAM cells than IIA-ITAM cells. This occurs because cross-linked Fc{alpha}R is delivered to large, signaling-induced, aggregated lamp1-positive vesicles (MIIC-like compartments) in wt-ITAM cells, whereas Fc{alpha}R was partially retained in other, as yet unidentified, endocytic compartments in IIA-ITAM cells (5). These data are consistent with previous studies in our laboratory (25), and the findings of Launay et al. (44). Both studies demonstrated that Fc{alpha}R was endocytosed independently of {gamma}-chain association. Launay and coworkers also demonstrated that association of Fc{alpha}R with wt {gamma}-chain was essential for trafficking to degradative compartments in myeloid cells, and that in the absence of {gamma}-chain association, Fc{alpha}R was not transported to these compartments. In wt-ITAM cells, PDK1 was observed to have translocated from the plasma membrane to the large, intracellular vesicles occupied by Fc{alpha}R after 30 min. In IIA-ITAM cells, a significant proportion of PDK1 was localized at the plasma membrane after 30 min, although Fc{alpha}R was endocytosed. In IIA-ITAM cells, a small proportion of the PDK1 colocalized with internalized Fc{alpha}R. Between 50 and 100 wt-ITAM and IIA-ITAM cells were individually analyzed after Fc{alpha}R endocytosis for PDK1 plasma membrane association. We found that wt-ITAM and IIA-ITAM cells had very distinct phenotypes. In wt-ITAM transfectants at 30 min post-Fc{alpha}R ligation, 10% of cells showed PDK1 at the plasma membrane, whereas in IIA-ITAM transfectants, 71% of cells showed PDK1 localization at the plasma membrane.

Cells held at 4°C had very weak intracellular staining for serine 473 phospho-PKB{alpha} (Fig. 4God). This is consistent with our immunoblot data (Fig. 3Gob) that showed very low levels of phospho-PKB{alpha} before warming cells to 37°C. In both wt-ITAM and IIA-ITAM cells, faint phospho-PKB{alpha} staining was observed (red) that was intracellular and did not demonstrate significant colocalization with cell surface Fc{alpha}R (green). Staining for total cellular PKB{alpha} demonstrated that PKB{alpha} was not associated with the plasma membrane in unstimulated cells (not shown). After incubation at 37°C for 2 min, cell surface Fc{alpha}R was capped in wt-ITAM and IIA-ITAM cells, as described (Fig. 4Goe). In these cells, intense phospho-PKB{alpha} staining (red) was observed consistent with PKB{alpha} activation data shown in Fig. 3Go. In both wt-ITAM and IIA-ITAM cells, phospho-PKB{alpha} was localized primarily at the plasma membrane with a polarized distribution that colocalized with capped Fc{alpha}R (green). These data indicate that Fc{alpha}R triggers phosphorylation of PKB{alpha} at sites of Fc{alpha}R capping and is consistent with recruitment of PKB{alpha} to Fc{alpha}R localized at membrane rafts. After 30-min incubation at 37°C, phospho-PKB{alpha} (red) was colocalized with Fc{alpha}R (green) in intracellular vesicles in wt-ITAM cells (Fig. 4Gof). Although by in vitro kinase assay enzymatic activity of PKB{alpha} had returned to basal levels, by 30 min phosphorylation was still apparent. Although phosphorylation is necessary for activity, phosphorylated PKB{alpha} is not necessarily enzymatically active (35).

In contrast to wt-ITAM cells, in IIA-ITAM cells, a signficant proportion of phospho-PKB{alpha} remained at the plasma membrane, although Fc{alpha}R was endocytosed. Between 50 and 100 wt-ITAM and IIA-ITAM cells were analyzed after Fc{alpha}R endocytosis and assessed for phospho-PKB{alpha} plasma membrane association. In wt-ITAM transfectants, 9% of cells showed phospho-PKB{alpha} localization at the plasma membrane after Fc{alpha}R endocytosis, compared with 77% in IIA-ITAM transfectants. The proportion of wt-ITAM and IIA-ITAM cells showing PKB{alpha} localization at the plasma membrane was similar to that observed for PDK1.

Fc{alpha}R targets PKB{alpha} to an MIIC-like compartment in wt-ITAM transfectants

Based on our observation that in wt-ITAM cells, PDK1 and phospho-PKB{alpha} are targeted to an intracellular compartment containing endocytosed Fc{alpha}R, we performed three-color confocal microscopy to determine the subcellular site of Fc{alpha}R colocalization with phospho-PKB{alpha}. wt-ITAM cells were surface stained for Fc{alpha}R and fixed at 4°C before counterstaining for the late endosome and lysosome marker lamp1 (Fig. 5Goa). As described, at 0 min, there was very weak phospho-PKB{alpha} staining (red) that did not colocalize with surface Fc{alpha}R (green) or the several small lamp1 vesicles (blue) that were detected (Fig. 5Goa). There was no detectable overlap of the green, red, or blue signals. After incubating cells at 37°C for 30 min, Fc{alpha}R was endocytosed and colocalized with both phospho-PKB{alpha} and lamp1 vesicles (Fig. 5Gob). The merged image shows that Fc{alpha}R, phospho-PKB{alpha}, and lamp1 are all colocalized (Fig. 5Gob), and furthermore, the aggregated lamp1 vesicles containing Fc{alpha}R and phospho-PKB{alpha} were coincident. Aggregation of lamp1 vesicles is an indicator of MIIC formation (17). Our data suggest that following Fc{alpha}R endocytosis, phospho-PKB{alpha} is translocated to an MIIC-like compartment.



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  		FIGURE 5. Fc{alpha}R targets PKB{alpha} to an MIIC-like compartment in wt-ITAM cells. Fc{alpha}R on wt-ITAM cells was ligated with My43 and cross-linked with FITC donkey anti-mouse IgM (green), as described in Materials and Methods. Cells were held at 4°C (0 min) (a and c) or incubated for 30 min at 37°C to allow Fc{alpha}R endocytosis (b and d). Cells were counterstained for phosphoserine 473 PKB and lamp1 (a and b) or cathepsin B (c and d). Anti-phosphoserine 473 PKB{alpha} was detected with a Cy3-conjugated donkey anti-sheep IgG (red). Anti-lamp1 was detected with a Cy5-conjugated donkey anti-rat IgG (blue).

 
To confirm that Fc{alpha}R could target phospho-PKB{alpha} to an MIIC-like compartment, we performed an experiment in which wt-ITAM cells were counterstained for cathepsin B, a distinct marker of the MIIC (Fig. 5Go, c and d). At 0 min, cell surface Fc{alpha}R (green) did not colocalize with the weak phospho-PKB{alpha} signal (red) or cathepsin B (blue) (Fig. 5Goc). This is shown in the merged image in which green, red, and blue signals are all separate. Cathepsin B vesicle distribution was distinct from lamp1 vesicle distribution in unstimulated cells. This is because lamp1 is distributed in late endosomes and lysosomes, but in MIIC after Ag receptor signaling. Cathepsin B resides in a distinct subset of these subcellular compartments, the MIIC (15). After endocytosis of Fc{alpha}R for 30 min at 37°C, Fc{alpha}R colocalized with both phospho-PKB{alpha} and cathepsin B (Fig. 5God). The merged image shows three-color colocalization of Fc{alpha}R, phospho-PKB{alpha}, and cathepsin B. These data show that cross-linking of wt-ITAM-containing {gamma}-chain-associated Fc{alpha}R results in targeting of both Fc{alpha}R and phospho-PKB{alpha} to a cathepsin B-containing compartment, and therefore to an MIIC-like compartment.

Translocation of phospho-PKB{alpha} to MIIC in IgA/OVA-pulsed wt-ITAM cells

In our Ag presentation assays, we pulsed cells with IgA/OVA complexes (Fig. 1Go). We treated cells with IgA/OVA complexes to determine whether the approach used in the Ag presentation assays could result in MIIC formation and translocation of PKB{alpha} to MIIC. We observed that after endocytosis of Fc{alpha}R cross-linked with IgA/NIP-OVA complexes for 30 min at 37°C, Fc{alpha}R was colocalized with aggregated lamp1 vesicles and phosphoserine 473 PKB{alpha} (Fig. 6Go). In unstimulated cells with surface-bound IgA/NIP-OVA, lamp1 vesicles were not aggregated and there was no detectable phosphoserine 473 PKB{alpha} as in the My43 experiments (not shown). These studies show that the data obtained from cross-linking studies with My43 are comparable with that obtained with IgA/OVA complexes.



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  		FIGURE 6. Fc{alpha}R-triggered PKB{alpha} translocation to MIIC occurs in response to IgA-Ag pulsing of wt-ITAM cells. wt-ITAM cells were incubated at 4°C with IgA/NIP-OVA complexes at a 3:1 ratio (2.5 µg IgA/106 cells) for 1 h. Cells were then incubated at 37°C for 30 min before stopping the reaction with ice-cold PBS. Cells were washed by centrifugation before fixation and staining for IgA (green, FITC), phosphoserine 473 PKB (red, Cy3), and lamp1 (blue, Cy5).

 
To address the likelihood that translocation of PKB{alpha} to intracellular vesicles is a phenomenon associated with Ag receptors other than Fc{alpha}R, we cross-linked the BCR that is constitutively expressed on the A20 B cell line and counterstained for phospho-PKB{alpha}, as described. We observed rapid capping of BCR and codistribution of PKB{alpha}, followed by translocation of BCR and PKB{alpha} to the same intracellular compartment (data not shown).

Translocation of phospho-PKB{alpha} to MIIC in peripheral blood monocytes

Our experimental system used A20 mouse B cells transfected with Fc{alpha}R. To demonstrate that Fc{alpha}R in its native context could cause translocation of PKB{alpha} to MIIC, we triggered endocytosis of Fc{alpha}R by human monocytes and counterstained for PKB{alpha} and lamp1 (Fig. 7Go). In unstimulated cells (0 min), we observed a different distribution of lamp1 than that seen for A20 cells (Fig. 7Goa). In monocytes, lamp1 occurred in several small peripheral vesicles, and there appeared to be a degree of colocalization between Fc{alpha}R and lamp1 at 0 min. We observed that cross-linked, endocytosed Fc{alpha}R was transported to lamp1-containing vesicles within 30 min (Fig. 7Gob). In agreement with our studies on transfected A20 cells, Fc{alpha}R targeting to lamp1 vesicles in monocytes was accompanied by vesicle aggregation that was consistent with induction of MIIC-like structures. In the same experiment, we counterstained the monocytes for phosphoserine 473 PKB{alpha}. We observed low basal phospho-PKB{alpha} staining before Fc{alpha}R cross-linking (Fig. 7Goc). On Fc{alpha}R cross-linking, an increase in phospho-PKB{alpha} staining was observed, and there was distinct staining that colocalized with internalized Fc{alpha}R (Fig. 7God). Taken together, these data indicate that constitutively expressed Fc{alpha}R in monocytes is transported to lamp1 vesicles and that this is accompanied by translocation of phospho-PKB{alpha}.



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  		FIGURE 7. Fc{alpha}R-triggered PKB{alpha} translocation to MIIC occurs in monocytes. Fc{alpha}R (green, FITC) was cross-linked on CD163+/CD89+ peripheral blood monocytes at 4°C. Cells were either held at 4°C and fixed (a and c), or incubated at 37°C for 30 min before fixation (b and d). Cells were permeabilized and counterstained for lamp1 (red, Cy3) (a and b). In the same experiment, parallel samples were counterstained for phospho-PKB{alpha} (red, Cy3), as shown (c and d).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our data presented herein, as well as other reports, implicate the involvement of signal transduction events in efficient receptor-mediated Ag presentation (5, 17, 18, 19). Cells expressing Fc{alpha}R and wt {gamma}-chain (wt-ITAM) demonstrated a higher capacity for class II presentation of Fc{alpha}R-targeted Ag than cells expressing Fc{alpha}R and a mutant {gamma}-chain (IIA-ITAM). Interestingly, although IIA-ITAM cells were able to endocytose Fc{alpha}R/Ag complexes equivalently to wt-ITAM cells (5), they did not present Ag efficiently to T cell hybridomas. This suggests that there are differences in the generation of peptide/class II complexes between wt-ITAM and IIA-ITAM cells. IIA-ITAM cells also demonstrated less efficient induction of MIIC-like compartments and reduced Ag catabolism (5), leading us to hypothesize that the defect in their Ag presentation could lie in trafficking or function of intracellular vesicles.

In this study, we demonstrated that PI-3 kinase activity is also required for effective Fc{alpha}R-mediated Ag presentation, pointing to the possible role of PDK1 or PKB{alpha} that are downstream of PI-3 kinase activation. We focused mainly on PKB{alpha} as it is downstream of PDK1. In vitro kinase assays and immunoblotting of cell lysates for serine-phosphorylated PKB{alpha} showed that wt-ITAM and IIA-ITAM cells triggered similar activation of PKB{alpha} throughout the time period (30 min) during which Fc{alpha}R internalization and induction of MIIC occur. Although overall PKB{alpha} activation in wt-ITAM and IIA-ITAM transfectants did not appear different, studies on PKB{alpha} subcellular location revealed differences between wt-ITAM and IIA-ITAM cells. By confocal microscopy, we demonstrated that in wt-ITAM cells, PKB{alpha} was quantitatively translocated first to the plasma membrane, where it was cocapped with Fc{alpha}R. Fc{alpha}R and phospho-PKB{alpha} were then translocated to intracellular compartments containing cathepsin B and lamp1 (standard markers of MIIC compartments) (15, 16). In IIA-ITAM cells, we observed that after localization at the plasma membrane, the major proportion of phospho-PKB{alpha} remained at this site and was not translocated to the MIIC, despite the internalization and transport of Fc{alpha}R to the latter compartment.

Receptor uptake of Ag and delivery to a class II-containing compartment for processing involve signaling and vesicle fusion events (reviewed in Ref. 14). The current literature clearly suggests a role for PKB{alpha} in regulating vesicle fusion/translocation. Recent evidence presented by Barbier and coworkers (45) demonstrated that fluid-phase endocytosis was increased by expression of a constitutively active PKB{alpha} (CA-PKB{alpha}), or was decreased by expression of a kinase-dead PKB. Furthermore, this study showed that in vitro fusion of Rab5 vesicles with early endosome (EE) was inhibited by kinase-dead PKB{alpha} and increased by CA-PKB{alpha}. This result is consistent with PKB{alpha} having a role in early vesicle fusion, which would ensue with Fc{alpha}R internalization. PKB has also been shown to play a role in vesicle fusion in exocytosis, a related but different vesicle transport system. Stimulation of adipocytes with insulin triggers PKB activation and translocation of the endosomal glucose transporter GLUT4 to the cell surface (46). Expression of a CA-PKB{alpha} triggered GLUT4 translocation in the absence of insulin stimulation. The CA-PKB{alpha}-mediated GLUT4 translocation was inhibited by botulinum toxin, which proteolyzes SNAP23, a key protein in vesicle fusion. This result suggests SNAP23, or a SNAP23-dependent component of the vesicle fusion/docking complex, may be a target for PKB{alpha} regulation.

The role of PKB{alpha} in regulating two different vesicle transport systems raises the possibility that fusion and docking proteins on intracellular vesicles may be among the downstream targets of PKB{alpha} following Fc{alpha}R-mediated Ag internalization. There are at least three possible interpretations of the differential localization of PKB{alpha} that could explain the reduction in Ag presentation by IIA-ITAM transfectants: 1) PKB{alpha} may regulate key proteins that are required for vesicle fusion events, such as EE/EE or EE/late endosome fusion, upstream of delivery of Fc{alpha}R Ag to the MIIC. 2) PKB{alpha} could regulate processes necessary for MIIC immunologic function, such as invariant chain cleavage and/or Ag catabolism and generation of peptide/class II complexes. 3) PKB{alpha} is only diagnostic of poor Ag presentation and does not regulate any function required for it.

Although the data presented in this study support the notion that the receptor-associated PI-3 kinase proximal signaling is causal to the deficiency in Ag presentation by the IIA-ITAM cells, PI-3 kinase activity is not required for formation of MIIC structures. Siemasko and coworkers (17), who first proposed an association between signal transduction and induction of the MIIC, demonstrated that although inhibitors of tyrosine kinases prevented MIIC induction, PI 3-kinase inhibition did not. However, the functional capacity of the MIIC structures was not determined in that study. The observation that inhibition of tyrosine kinases prevented MIIC induction supports our previous work that also implicated tyrosine kinase activation as being critical for optimal MIIC formation and receptor-mediated Ag presentation (5).

Two questions therefore arise from these observations. Are tyrosine kinase and PI-3 kinase activation linked to MIIC formation and function? Are tyrosine kinase and PI-kinase systems linked in a linear fashion or a parallel fashion for optimal Ag presentation? The observation that PI-3 kinase inhibitors do not prevent MIIC formation, whereas tyrosine kinase inhibitors, which ultimately can inhibit PI-3 kinase activity, do inhibit MIIC formation, suggests that these signals are linked in parallel. The earliest events in the progress of ligated Fc{alpha}R through the processing pathway involve passage through Rab5/Rab4-positive vesicles (Lang, Shen, Wade, unpublished observation). A connection between Rab5 and PI 3-kinase has been shown by Christoforidis and coworkers (47), who demonstrated that PI 3-kinases are Rab5 effectors and that blocking PI 3-kinases with Abs could inhibit fusion of early endosomes. Given that PKB is required for fusion of Rab5 vesicles (45), it appears that the PI 3-kinase/PDK1/PKB pathway is a major link between signal transduction and endocytic vesicle function.

It is possible that tyrosine kinases regulate early events that set the stage for correct functioning of PKB{alpha} in the endocytic pathway. Consistent with this idea, we have demonstrated that the tyrosine phosphorylation of Blk, Syk, and Btk is less efficient in IIA-ITAM cells compared with wt-ITAM cells after Fc{alpha}R cross-linking (M. L. Lang et al., unpublished observation). Perhaps in the absence of activation of key tyrosine kinases, a substrate or binding protein is not provided for PKB{alpha} and results in exclusion of PKB{alpha} from endocytic vesicles, thus affecting targeting or subsequent function. It will be important to identify molecules in the endocytic pathway that are candidate substrates for PKB{alpha}, and also to determine how tyrosine kinases might modify PKB{alpha} function to determine how Fc{alpha}R signaling regulates Ag processing.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant RO1AI22816. Back

2 Address correspondence and reprint requests to Dr. William F. Wade, Department of Microbiology and Immunology, Dartmouth Medical School, Lebanon, NH 03756. Back

3 Abbreviations used in this paper: BCR, B cell Ag receptor; CA-PKB{alpha}, constitutively active PKB{alpha}; Cy3, indocarbocyanine 3; EE, early endosome; ITAM, immunoreceptor tyrosine-based activation motif; lamp1, lysosomal-associated membrane glycoprotein; LE, late endosome; MIIC, MHC class II peptide-loading compartment; NIP, nitroiodophenol caproate-o-succinimide; PDK1, phosphatidylinositol-dependent protein kinase; PI 3-kinase, phosphatidylinositol 3-kinase; PKB{alpha}, protein kinase B{alpha}; RAM, rabbit anti-mouse IgM; wt, wild type. Back

Received for publication September 20, 2000. Accepted for publication February 20, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Morton, H. C., M. van Egmond, J. G. J. van de Winkel. 1996. Structure and function of human IgA receptors (Fc{alpha}R). Crit. Rev. Immunol. 16:423.[Medline]
  2. Kerr, M. A., W. W. Stewart, B. C. Bonner, M. R. Greer, S. J. MacKenzie, M. G. Steele. 1995. The diversity of leukocyte IgA receptors. Contrib. Neph. 111:60.
  3. Kerr, M. A.. 1990. The structure and function of human IgA. Biochem. J. 271:285.[Medline]
  4. Shen, L.. 1992. Receptors for IgA on phagocytic cells. Immunol. Res. 11:273.[Medline]
  5. Shen, L., M. van Egmond, K. Siemasko, M. L. Lang, H. Gao, T. Wade, M. R. Clark, J. G. J. van de Winkel, W. F. Wade. 2001. Presentation of ovalbumin internalized via the IgA Fc receptor (CD89) is enhanced through FcR{gamma} chain signaling. Blood 97:205.[Abstract/Free Full Text]
  6. Rock, K. L., B. Benacerraf, A. K. Abbas. 1984. Antigen presentation by hapten-specific B lymphocytes: role of surface immunoglobulin receptors. J. Exp. Med. 160:1002.
  7. Tony, H. P., D. C. Parker. 1985. Major histocompatibility complex-restricted, polyclonal B cell responses resulting from helper T cell recognition of anti-immunoglobulin presented by small B lymphocytes. J. Exp. Med. 161:223.[Abstract/Free Full Text]
  8. Casten, L. A., P. Kaumaya, S. K. Pierce. 1988. Enhanced T cell responses to antigenic peptides targeted to B cell surface Ig, Ia, or class I molecules. J. Exp. Med. 168:171.[Abstract/Free Full Text]
  9. Amigorena, S., C. Bonnerot. 1999. Fc receptor signaling and trafficking: a connection for antigen processing. Immunol. Rev. 172:279.[Medline]
  10. Wagle, N. M., P. Cheng, J. Kim, T. W. Sproul, K. D. Kausch, S. K. Pierce. 2000. B-lymphocyte signaling receptors and the control of class-II antigen processing. Curr. Top. Microbiol. Immunol. 245:106.
  11. Amigorena, S., J. S. Drake, P. Webster, I. Mellman. 1994. Transient accumulation of new class II molecules in a novel endocytic compartment in B lymphocytes. Nature 369:113.[Medline]
  12. Drake, J. R., P. Webster, J. C. Cambier, I. Mellman. 1997. Delivery of B-cell receptor-internalized antigen to endosomes and class II vesicles. J. Exp. Med. 186:1299.[Abstract/Free Full Text]
  13. Mellman, I.. 1996. Endocytosis and molecular sorting. Annu. Rev. Cell. Dev. Biol. 12:575.[Medline]
  14. Watts, C.. 1997. Capture and processing of exogenous antigens for presentation on MHC molecules. Annu. Rev. Immunol. 15:821.[Medline]
  15. Drake, J. R., T. A. Lewis, K. B. Condon, R. N. Mitchell, P. Webster. 1999. Involvement of MIIC-like late endosomes in B cell receptor-mediated antigen processing in murine B cells. J. Immunol. 162:1150.[Abstract/Free Full Text]
  16. Neefjes, J.. 1999. CIIV, MIIC and other compartments for MHC class II loading. Eur. J. Immunol. 29:1421.[Medline]
  17. Siemasko, K., B. J. Eisfelder, E. Williamson, S. Kabak, M. R. Clark. 1998. Cutting edge: signals from the B lymphocyte antigen receptor regulate MHC class II containing late endosomes. J. Immunol. 160:5203.[Abstract/Free Full Text]
  18. Lanker, D., V. Briken, K. Adler, P. Weiser, S. Cassard, U. Blank, M. Viguier, C. Bonnerot. 1998. Syk tyrosine kinase and B cell antigen receptor (BCR) immunoglobulin-{alpha} subunit determine BCR-mediated major histocompatibility complex class II-restricted antigen presentation. J. Exp. Med. 188:819.[Abstract/Free Full Text]
  19. Siemasko, K., B. J. Eisfelder, C. Stebbins, S. Kabak, A. J. Sant, W. Song, M. R. Clark. 1999. Ig{alpha} and Ig{beta} are required for efficient trafficking to late endosomes and to enhance antigen presentation. J. Immunol. 162:6518.[Abstract/Free Full Text]
  20. Morton, H. C., I. E. van den Herik-Oudijk, P. Vossebeld, A. Snijders, A. J. Verhoeven, P. J. A. Capel, J. G. J van de Winkel. 1995. Functional association between the human myeloid immunoglobulin A Fc receptor (CD89) and FcR {gamma}-chain. J. Biol. Chem. 270:29781.[Abstract/Free Full Text]
  21. Flaswinkel, H., M. Barner, M. Reth. 1995. The tyrosine activation motif as a target of protein tyrosine kinases and SH2 domains. Semin. Immunol. 7:21.[Medline]
  22. Pfefferkorn, L. C., G. R. Yeaman. 1994. Association of IgA-Fc receptors (Fc{alpha}R) with Fc{epsilon}RI{gamma}2 subunits in U937 cells: aggregation induces the tyrosine phopshorylation of {gamma}2. J. Immunol. 153:3228.[Abstract]
  23. Gulle, H., A. Samstag, M. M. Eibl, H. M. Wolf. 1998. Physical and functional association of Fc{alpha}R with protein tyrosine kinase Lyn. Blood 91:383.[Abstract/Free Full Text]
  24. Launay, P., A. Lehuen, T. Kawakami, U. Blank, R. C. Monteiro. 1998. IgA Fc receptor (CD89) activation enables coupling to Syk and Btk tyrosine kinase pathways: differential signaling after IFN-{gamma} or phorbol ester stimulation. J. Leukocyte Biol. 63:636.[Abstract]
  25. Lang, M. L., L. Shen, W. F. Wade. 1999. {gamma}-Chain dependent recruitment of tyrosine kinases to membrane rafts by the human IgA receptor Fc{alpha}R. J. Immunol. 163:5391.[Abstract/Free Full Text]
  26. Lang, M. L., M. J. Glennie, M. A. Kerr. 1997. Human neutrophil Fc{alpha}R and Fc{gamma}RIIa but not Fc{gamma}RIIIb generate intracellular calcium signals which trigger the respiratory burst. Biochem. Soc. Trans. 25:333.
  27. Park, R. K., D. I. Kayvon, Y. M. Deo, D. L. Durden. 1999. Role of Src in the modulation of multiple adaptor proteins in Fc{alpha}RI oxidant signaling. Blood 94:2112.[Abstract/Free Full Text]
  28. Lang, M. L., M. A. Kerr. 1997. Human neutrophil Fc{alpha}R and Fc{gamma}R signal through PI 3-kinase to trigger a respiratory burst. Biochem. Soc. Trans. 25:603.
  29. Kobayashi, T., H. Seguchi. 1999. Novel insight into current models of NADPH oxidase regulation, assembly and localization in human polymorphonuclear leukocytes. Histol. Histopathol. 14:1295.[Medline]
  30. Salim, K., M. J. Bottomly, E. Querfurth, M. J. Zvelebil, I. Gout, R. Scaife, R. L. Margolis, R. Gigg, E. I. E. Smith, P. C. Driscoo, et al 1996. Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domain of dynamin and Bruton’s tyrosine kinase. EMBO J. 15:6241.[Medline]
  31. Gold, M. R., M. P. Scheid, L. Santos, M. Dang-Lawson, R. A. Roth, L. Matsuuchi, V. Duronio, D. L. Krebs. 1999. The B cell antigen receptor activates the Akt (protein kinase B)/glycogen synthase kinase-3 signaling pathway via phosphatidylinositol 3-kinase. J. Immunol. 163:1894.[Abstract/Free Full Text]
  32. Craxton, A., A. Jiang, T. Kurosaki, E. A. Clark. 1999. Syk and Bruton’s tyrosine kinase are required for B cell antigen receptor-mediated activation of the kinase Akt. J. Biol. Chem. 274:30644.[Abstract/Free Full Text]
  33. Astoul, E., S. Watton, D. Cantrell. 1999. The dynamics of protein kinase B regulation during B cell antigen receptor engagement. J. Cell Biol. 145:1511.[Abstract/Free Full Text]
  34. Aman, M. J., T. D. Lamkin, H. Okada, T. Kurosaki, K. S. Ravichandran. 1998. The inositol phosphatase SHIP inhibits Akt/PKB activation in B cells. J. Biol. Chem. 273:33922.[Abstract/Free Full Text]
  35. Chan, T. O., S. E. Rittenhouse, P. N. Tsichlis. 1999. AKT/PKB and other D3 phosphoinositide-regulated kinases: kinase activation by phosphoinositide-dependent phosphorylation. Annu. Rev. Biochem. 68:965.[Medline]
  36. Van den Herik-Oudijk, I. E., W. H. M. Ter Bekke, M. J. Tempelman, P. J. A. Capel, J. G. J. Van de Winkel. 1995. Functional differences between two Fc receptor ITAM signaling motifs. Blood 86:3302.[Abstract/Free Full Text]
  37. Jones, B., J. P. Tite, C. A. Janeway. 1986. Different phenotypic variants of the mouse B cell tumor A20/AJ are selected by antigen and mitogen-triggered cytotoxicity of L3T4 positive I-A restricted T cell clones. J. Immunol. 136:384.
  38. Banfic, H., X. Tang, I. H. Batty, C. P. Downes, C. Chen, S. E. Rittenhouse. 1998. A novel integrin-activated pathway forms PKB/Akt-stimulatory phosphatidylinositol 3,4-bisphosphate via phosphatidylinositol 3-phosphate in platelets. J. Biol. Chem. 273:13.[Abstract/Free Full Text]
  39. Shen, L., R. Lasser, M. W. Fanger. 1989. My43, a monoclonal antibody that reacts with human myeloid cells inhibits monocyte IgA bindin