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The Journal of Immunology, 1999, 163: 5435-5443.
Copyright © 1999 by The American Association of Immunologists

Phosphorylation of the Invariant Chain by Protein Kinase C Regulates MHC Class II Trafficking to Antigen-Processing Compartments

Howard A. Anderson*, Daniel T. Bergstralh*, Tatsuyoshi Kawamura{dagger}, Andrew Blauvelt{dagger} and Paul A. Roche1,*

* Experimental Immunology Branch and {dagger} Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 4 Kawamura, T., M. Qalbani, J. M. Orenstein, and A. Blauvelt. 1999. Human mono-cyte-derived dendritic cells propogated in the presence of GM-CSF, IL-4, and TGF-ß1 morphologically, phenotypically, and functionally resemble resident epidermal Langerhans cells Submitted for publication.


   Abstract
Top
 Abstract
Introduction
 Methods and Materials
 Results
 Discussion
 References
 
The invariant chain (Ii) plays a critical role in the transport of newly synthesized class II molecules to endosomal Ag-processing compartments. Of the two major isoforms of human Ii, only Ii-p35 is phosphorylated in vivo, and inhibiting Ii phosphorylation inhibits the trafficking of newly synthesized class II molecules to Ag-processing compartments. We now report that a member of the protein kinase C family of serine/threonine kinases is responsible for the constitutive phosphorylation of 50% of the total cellular pool of Ii-p35 in a wide variety of APCs, including B lymphocytes, PBMC, immature dendritic cells, and mature dendritic cells. Stimulation of protein kinase C activity in APCs significantly enhanced the kinetics of degradation of class II-associated Ii in Ag-processing compartments and the binding of antigenic peptides to these class II molecules. In cells expressing an Ii-phosphorylation mutant, trafficking of class II molecules to endosomes was impaired and Ii proteolysis was inhibited, demonstrating a direct effect of Ii phosphorylation on MHC class II trafficking. These results demonstrate that phosphorylation of Ii in APCs alters the kinetics of trafficking of newly synthesized class II molecules to lysosomal Ag-processing compartments.


   Introduction
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 Abstract
 Introduction
Methods and Materials
 Results
 Discussion
 References
 
Association with the invariant chain (Ii)2 is essential for newly synthesized class II molecules to acquire a diverse repertoire of peptides for presentation to CD4+ T cells (reviewed in Refs. 1 and 2). During synthesis, Ii assembles into trimers that recruit and assist in the proper folding of three class II {alpha}ß dimers, generating a functional nonameric complex that is subsequently transported from the endoplasmic reticulum (ER) (3, 4, 5, 6). Ii association with class II molecules prevents peptide interaction with the class II peptide binding groove during passage through the secretory pathway (7, 8), and recognition of targeting motifs in the Ii cytoplasmic domain diverts class II {alpha}ßI complexes from the trans-Golgi network to late endosomal/prelysosomal Ag-processing compartments (9, 10). Within these compartments, Ii is subsequently removed from class II molecules by a series of proteolytic cleavages, allowing the accessory protein HLA-DM to catalyze the release of a class II-associated Ii peptide fragment from the class II peptide binding groove (reviewed in Ref. 11). The removal of the class II-associated Ii peptide ultimately results in the association of high-affinity peptides with class II molecules and the subsequent movement of class II-peptide complexes to the cell surface.

In human cells, two alternative Ii isoforms function in targeting class II molecules to endosomal compartments. In addition to the predominant 33-kDa isoform of Ii (Ii-p33), a 35-kDa isoform (Ii-p35) is generated by the use of an alternative upstream translational initiation site (12). Therefore, the two Ii isoforms are identical in all respects except that the Ii-p35 isoform contains an additional 16 amino acids at the cytoplasmic amino terminus. Thus, amino acids 1–16 are cytosolic and unique to the Ii-p35 isoform. In various class II-expressing cell types, the Ii-p35 isoform represents ~20% of the total Ii pool, although the ability of Ii to form heterotrimers results in a significant fraction of the total pool of class II {alpha}ßI complexes possessing at least one Ii-p35 polypeptide (4, 5)3.

Ii-p35 plays an important role in coordinating the assembly and transport of newly synthesized Ii with class II in the ER. In the absence of class II molecules, both isoforms of human Ii are largely retained in the ER due to an arginine-based retention motif present in the Ii-p35 cytosolic domain (13). However, the association of Ii with class II molecules results in the masking of the ER retention motif in Ii-p35 (3, 14, 15), thereby allowing the efficient transport of class II {alpha}ßI complexes containing all isoforms of Ii out of the ER.

In addition to its role in regulating the exit of class II molecules from the ER, Ii-p35 can profoundly effect the endosomal localization of class II molecules. We and others have shown a significant fraction of newly synthesized class II molecules associated with the p33 isoform of Ii traffic to the Ag-processing compartment via the cell surface (16, 17, 18, 19), whereas class II molecules associated with Ii-p35 target these endosomal compartments by a strictly intracellular route (17).

We have previously shown that Ii-p35 is phosphorylated throughout the secretory pathway in professional APCs and that phosphorylation occurs on serine 6 and/or serine 8, residues which are not present in Ii-p33 (20). Preventing Ii phosphorylation by using the general serine/threonine kinase inhibitor staurosporine specifically reduced the accumulation of Ii endosomal degradation products and decreased the amount of newly synthesized class II molecules loaded with peptide Ags, demonstrating that phosphorylation plays a role in regulating Ii-class II transport to endosomes.

We now report that Ii-p35 is constitutively phosphorylated in a variety of APCs and that constitutive Ii-p35 phosphorylation is mediated by a member of the protein kinase C (PKC) family of serine/threonine kinases. Augmentation of Ii-p35 phosphorylation by stimulation of PKC activity enhances the accumulation of Ii degradation products and peptide-loading onto newly synthesized MHC class II molecules. In addition, the kinetics of Ii degradation in heterologous cells expressing wild-type or mutant Ii molecules with class II molecules confirmed that the net effect of Ii phosphorylation is to enhance the rate of class II trafficking to Ag-processing compartments.


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

The human B-lymphoblastoid cell line (LCL) JY (HLA-DR4, w6; Ref. 21), HeLa cells, and PBMC were cultured as previously described (20). HeLa cells were transfected with DR{alpha} (in CDM8), DRß (in CDM8), Ii-p33 (in pcDNA3), and Ii-p35 or Ii-p35 (S8A) (in pcDNA3) by calcium phosphate precipitation as previously described (20). Dendritic cells were propagated from adult plastic-adherent PBMC as previously described (22), but with minor modifications. Briefly, cells were cultured for 7 days in the presence of 1000 U/ml recombinant human GM-CSF (Immunex, Seattle, WA), 1000 U/ml recombinant human IL-4 (R&D Systems, Minneapolis, MN), and 1 ng/ml human platelet-derived TGF-ß1 (R&D Systems). At day 7, dendritic cells were harvested, and any contaminating T cells, monocytes, NK cells, and B cells were removed from CD3-CD14-CD16-CD19- cells (i.e., dendritic cells) by immunomagnetic bead separation. By morphologic, phenotypic, and functional criteria, these cells demonstrated classic features of immature Langerhans cell-like dendritic cells (23).4Immature dendritic cells were then cultured for an additional 2–3 days in the presence of either GM-CSF, IL-4, and TGF-ß1 (but with the TGF-ß1 concentration increased to 10 ng/ml to maintain an immature phenotype), or GM-CSF, IL-4, and 30% (v/v) monocyte-conditioned medium (to induce maturation) (24).4 For all subsequent studies, dendritic cells populations were always >98% pure.

Chemicals

Bisindolylmaleimide I-HCl (BIM), 3-iso butyl-1-methylxanthine (IBMX), forskolin, and the cAMP-dependent protein kinase (PKA) inhibitor peptide myristoylated 14–22 amide were purchased from Calbiochem (La Jolla, CA). Staurosporine was purchased from Boehringer Mannheim (Mannheim, Germany). KT5720 was purchased from Alexis Biochemicals (San Diego, CA). PMA was purchased from Life Technologies (Gaithersburg, MD). Leupeptin was obtained from Sigma (St. Louis, MO).

GST fusion proteins

The prokaryotic expression vector pTrc 99A containing GST was generously provided by Dr. Piergiuseppe DeBerardinis (Institute of Protein Biochemistry and Enzymology, Napoli, Italy). The cytoplasmic tails of Ii-p33 or Ii-p35 were amplified using sp64 Ii-p33–2xATG (12) as a template by PCR using primers to amplify amino acids 1–32 (for the GST-Ii-p33 construct) or amino acids -16 to 32 (for the GST-Ii-p33 construct). PCR primers were engineered to contain SmaI (forward) and KpnI (backward) sites, and were cloned at the amino terminus of the GST into NcoI(fill)/KpnI restriction sites. The sequence of each fusion protein was confirmed by DNA sequence analysis. GST fusion proteins were expressed in BL21-DES cells and were purified using standard protocols.

In vitro kinase assay

In vitro kinase assays using recombinant PKA catalytic subunit (New England BioLabs, Beverly, MA) were performed in a buffer of 50 mM Tris-HCl, 10 mM MgCl2 (pH 7.5). Kinase assays using rat brain PKC catalytic subunit (Calbiochem) were performed in a buffer of 10 mM HEPES, 10 mM MgCl2, 1 mM CaCl2, 250 µM ATP, 500 µM DTT (pH 7.0). Each reaction was performed by incubating ~1 µg of GST-Ii-p33 or GST-Ii-p35 fusion proteins prebound to glutathione-Sepharose beads with 5 µCi of [{gamma}-32P]ATP in a 20-µl reaction volume for 45 min at 30°C. Fusion proteins were washed two times with PBS and eluted from the glutathione-Sepharose beads by boiling in SDS-PAGE sample buffer. Tryptic phosphopeptides were generated from GST-Ii-p35 proteins phosphorylated with PKA or PKC or from Ii isolated from B-LCL and resolved by TLC and visualized by autoradiography as previously described (20).

Metabolic labeling of cells

Cells were labeled with [35S]methionine of [32P]orthophosphate as previously described (20). HeLa cells were cultured on 10-cm Falcon tissue culture dishes (Becton Dickinson, Franklin Lakes, NJ) and were transfected with class II {alpha}-chain, ß-chain, or Ii cDNAs. After 36 h, the cells were pulse-labeled with 0.25 mCi [35S]methionine and then chased (or not) in complete medium containing excess unlabeled methionine. JY cells were pulse-labeled with [35S]methionine for 30 min and chased (or not) in complete medium containing excess unlabeled methionine for various periods of time. JY cells were also cultured for 3 h in media containing 0.25 mCi [32P]orthophosphate to monitor phosphorylation of Ii-p35.

Immunoprecipitation and electrophoresis

Class II-Ii-p33/p35 were immunoprecipitated from Triton X-100 solubilized cell extracts as previously described (20). Briefly, the anti-Ii-specific mAb Pin1.1 or the anti-class II {alpha}-chain mAb DA6.147 were used to immunoprecipitate MHC class II-Ii complexes as indicated. Immunoprecipitates were resolved by SDS-PAGE or 2D-PAGE (nonequilibrium pH gradient electrophoresis followed by reducing SDS-PAGE) and visualized by fluorography as described (20). The Ii-specific mAb Bu45 and HRP-conjugated goat anti-mouse Ig Ab were used to detect Ii on immunoblots. Immunoblots were quantitated using densitometry, while either phosphorimager or densitometer analyses, as indicated, were used to quantitate radioactive PAGE gels.

Immunofluorescence microscopy

Dendritic cells were attached to poly L-lysine coated cover slips and fixed for 1 h with 2% paraformaldehyde in PBS. The paraformaldehyde was quenched by washing cells with 50 mM NH4Cl in PBS, and the cells were permeabilized in PBS containing 1% Nonidet P-40, 1% normal goat serum, 1% gelatin, and 0.01% saponin for 10 min at room temperature. The distribution of class II protein was visualized by staining the cells with the anti-MHC class II {alpha}-chain mAb DA6.147 (1:30 dilution of hybridoma supernatant), and a PE-conjugated goat-anti mouse Ig Ab (1:100) in a buffer of PBS containing 1% normal goat serum, 1% gelatin, and 0.01% saponin. After extensive washing in the above buffer, the cells were mounted using Fluoromount G (Southern Biotechnology Associates, Birmingham, AL). Images were acquired using a Zeiss LSM 410 confocal microscope as described (25).


   Results
Top
 Abstract
 Introduction
 Methods and Materials
 Results
Discussion
 References
 
In vitro phosphorylation of GST-Ii-p35 fusion proteins

Previous studies from our laboratory identified a staurosporine-sensitive serine/threonine kinase as the Ii kinase (20). Ii-p35 is phosphorylated on sites that are flanked by positively charged amino acids, implicating either PKC or PKA in Ii-p35 phosphorylation. In an attempt to identify the Ii kinase, we generated fusion proteins of the cytoplasmic tail of Ii-p33 or Ii-p35 with the amino terminus of GST to examine Ii phosphorylation in vitro. In vitro kinase assays revealed that the GST-Ii-p35 fusion protein served as a substrate for both PKC-mediated (Fig. 1GoA) and PKA-mediated (Fig. 1GoB) phosphorylation. Under these assay conditions, we did not observe any phosphorylation of GST or GST-Ii-p33 fusion proteins by PKC or PKA even at the highest enzyme concentration examined. In addition, we were unable to detect significant phosphorylation of GST Ii-p35 by purified calcium/calmodulin-dependent protein kinase or casein kinase II (data not shown), demonstrating that Ii-p35 phosphorylation was not nonspecific.



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  		FIGURE 1. In vitro phosphorylation of the p35 cytoplasmic domain. GST-Ii-p35 or GST Ii-p33 fusion proteins were incubated with purified PKC (A) or purified PKA (B) in buffers containing [{gamma}-32P]ATP as indicated in the text. The samples were analyzed by reducing SDS-PAGE, the gels were stained to confirm equal loading of GST fusion protein, and the dried gels were subjected to fluorography. C, Phosphorylated Ii-p35 isolated from B-LCL cultured in media containing [32P]orthophosphate or GST-Ii-p35 fusion proteins phosphorylated with [{gamma}-32P]ATP and either PKC or PKA in vitro were isolated from SDS-PAGE gels and digested with trypsin. The tryptic peptides were spotted onto cellulose plates (the origin is indicated by an asterisk) and phosphopeptides were resolved by TLC and autoradiography.

 
Ii-p35 is phosphorylated on the unique cytosolic residues serine 6 and/or serine 8 in vivo (14, 20). To confirm that the GST-Ii-p35 fusion protein was phosphorylated on the same residues as Ii-p35 isolated from APCs, we analyzed tryptic phosphopeptides released from GST-Ii-p35 phosphorylated in vitro with either purified PKC, purified PKA, or from wild-type Ii isolated from B-LCL labeled in vivo with [32P]orthophosphate by TLC. In each case the tryptic peptides released were identical (Fig. 1GoC), indicating that like Ii phosphorylation in vivo, GST-Ii-p35 is phosphorylated on serine 6 and/or 8 by purified PKA or PKC in vitro.

Ii-p35 is phosphorylated in vivo by PKC

In an attempt to identify physiological regulators of Ii phosphorylation, we employed kinase activators and inhibitors in in situ phosphorylation studies using [32P]orthophosphate-labeled B-LCL. Short-term treatment with PMA significantly enhances PKC activity (26), and this treatment resulted in a dramatic increase in Ii phosphorylation (Fig. 2GoA). By contrast, treatment of the cells with the cAMP-activator forskolin did not influence Ii phosphorylation in vivo, strongly suggesting that PKA does not phosphorylate Ii in vivo. The effect of PMA on Ii-p35 phosphorylation was quantitated by densitometry, and in three independent experiments we found that stimulation of PKC activity in professional APCs increased Ii-p35 phosphorylation ~2-fold (Fig. 2GoB). Because ~50% of Ii is constitutively phosphorylated in B-LCL (Fig. 2GoC), a 2-fold increase in Ii-p35 phosphorylation suggests that PMA treatment causes virtually all Ii-p35 to become phosphorylated. To examine this directly, B-LCL were labeled with [35S]methionine for 20 min in the presence of PMA and class II-Ii complexes were immunoprecipitated and analyzed by 2D-PAGE. More than 95% of newly synthesized Ii-p35 was phosphorylated following PMA treatment, as indicated by an almost complete shift in the charge of all Ii-p35 to the position of phosphorylated Ii-p35 as observed in 2D-PAGE gels (Fig. 2GoC). These data demonstrate that incubation of cells in PMA under conditions known to activate PKC activity increases Ii-p35 phosphorylation in vivo.



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  		FIGURE 2. PMA treatment increases Ii-p35 phosphorylation. A, B-LCL were cultured in media containing [32P]orthophosphate for 3 h, and the cells were then either mock treated or treated with 100 nM PMA, 20 µM forskolin/75 µM IBMX, or PMA/forskolin/IBMX for 20 min. The cells were lysed, and Ii was immunoprecipitated from each cell extract and analyzed by SDS-PAGE and autoradiography. The total amount of Ii present in each immunoprecipitate was determined by immunoblotting with the Ii-specific mAb Bu45. B, The PMA-induced increase in Ii phosphorylation was quantitated by phosphorimager analysis (n = 3). The amount of phosphorylation present in the absence of PMA was normalized to 100%. C, B-LCL were treated with 100 nM PMA during a 30-min pulse-labeling with [35S]methionine, the cells were lysed, class II-Ii complexes were isolated by immunoprecipitation, and the immunoprecipitates were analyzed by 2D-PAGE and fluorography. The mobilities of the class II {alpha}-chain, ß-chain, Ii-p33, nonphosphorylated Ii-p35 (arrow), and phosphorylated Ii-p35 (arrow head) are indicated. Note that PMA results in phosphorylation of the entire pool Ii-p35.

 
In addition to the use of kinase activators, specific protein kinase inhibitors are widely used to identify the kinases responsible for substrate phosphorylation in vivo. Pretreatment of B-LCL with BIM, a highly specific inhibitor of PKC (27), significantly reduced Ii phosphorylation in vivo at concentrations that have been reported to specifically inactivate PKC (Fig. 3GoA). As anticipated, anti-Ii immunoblots confirmed that BIM treatment had no effect on total Ii isolated from cells. Similarly, chronic stimulation of PKC activity with PMA leads to a loss of cellular PKC activity due to the degradation of the enzyme (28). Like BIM treatment, inhibition of PKC by chronic stimulation with PMA greatly reduced phosphorylation of Ii-p35 but had no effect on the total amount of Ii isolated from the cells (Fig. 3GoB). Immunoblotting with phospho-specific Abs revealed that phosphorylation of the PKC substrate mitogen-activated protein kinase was also inhibited by ~50% by either drug treatment (data not shown), demonstrating that the inhibition of Ii phosphorylation and mitogen-activated protein kinase phosphorylation occurred at comparable levels with each drug.



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  		FIGURE 3. PKC specific inhibitors prevent Ii-p35 phosphorylation in vivo. A, B-LCL were treated with various concentrations of the PKC-specific inhibitor BIM for 30 min and radiolabeled with [32P]orthophosphate for 2 h in the presence of the indicated concentration of BIM. The cells were lysed, class II molecules were immunoprecipitated from cell extracts, and the immunoprecipitates were analyzed by SDS-PAGE and autoradiography. The total amount of Ii present in each immunoprecipitate was determined by immunoblotting with the Ii-specific mAb Bu45. The extent of phosphorylation in each condition was expressed as a percentage of the phosphorylation observed in the absence of drug treatment. These values were then normalized for total Ii isolated as determined by immunoblotting. B, B-LCL were pretreated with various concentrations of PMA for 24 h and radiolabeled with [32P]orthophosphate for 2 h in the presence of the indicated concentration of PMA. Class II molecules were analyzed as described above. C, B-LCL were treated for 30 min with the PKA-specific inhibitors KT 5720 (20 µM) or PKI (20 µM) and the cells were radiolabeled in media containing [32P]orthophosphate and analyzed as described above.

 
Because the consensus phosphorylation sites for PKA and PKC are very similar and because PKA phosphorylated GST-Ii-p35 in vitro, we evaluated specific inhibitors of PKA for their effect on Ii phosphorylation in B-LCL. Pretreatment of B-LCL with PKI (29, 30) or KT5720 (31) at concentrations well above those reported to inhibit PKA activity had no effect on Ii-p35 phosphorylation (Fig. 3GoC). Together with our data demonstrating that activation of cAMP-levels with forskolin has no effect on Ii phosphorylation (Fig. 2Go), these data strongly suggest that PKA does not phosphorylate Ii in vivo and strongly implicate PKC or a highly related PKC-like kinase as the kinase responsible for Ii-p35 phosphorylation in vivo.

Ii phosphorylation enhances the kinetics of MHC class II-Ii degradation in Ag-processing compartments

To examine the effect of increased Ii-p35 phosphorylation on the transport of class II {alpha}ßI complexes to Ag-processing compartment, we performed pulse-chase analyses. To measure Ii arrival within the endosomal system we assayed for the accumulation of well-characterized Ii degradation intermediates. When APCs are cultured in media containing leupeptin, 20-kDa and 10-kDa leupeptin-induced polypeptide (LIP) fragments of Ii accumulate within Ag-processing compartments (32, 33). Leupeptin-loaded cells were pulse-labeled with [35S]methionine and chased for up to 3 h in the absence or presence of the PKC activator PMA. In the presence of PMA, we routinely observed an increase in LIP generation as compared with cells chased in medium alone (Fig. 4GoA). The average increase in LIP generation in cells treated with PMA was quantitated in six independent experiments, and this analysis revealed that there was a 1.7 ± 0.3-fold increase in Ii-LIP accumulation in cells treated with PMA as compared with mock-treated cells (p < 0.01), strongly suggesting that stimulating PKC activity increases the rate at which class II-Ii complexes are transported to Ag-processing compartments.



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  		FIGURE 4. PMA treatment increases transport of newly synthesized class II-Ii complexes to Ag-processing compartments. A, B-LCL were cultured for 2 h in media containing 1 mM leupeptin, washed, pulse-radiolabeled for 30 min with [35S]methionine, and cultured for up to 3 h in complete media the absence or presence of 20 nM PMA. Following cell lysis, class II-associated Ii was isolated using an anti-class II mAb, and the immunoprecipitates were analyzed by SDS-PAGE and autoradiography. The amount of Ii degradation products present in each sample was quantitated by phosphorimager analysis. The amount of Ii-p20 (LIP) and Ii-p10 was expressed as a percentage of total signal present in each sample, thereby accounting for small differences in protein recovery or gel loading. B, B-LCL were pulsed for 30 min in media containing [35S]methionine and chased in complete media containing 20 nM PMA. Following cell lysis, class II molecules were isolated using an anti-class II mAb. SDS-stable class II {alpha}ß dimers were detected by eluting the class II molecules from the immunoprecipitates in SDS-sample buffer at room temperature for 30 min. SDS-stable dimers were quantitated by expressing the amount of {alpha}ß dimer present in each sample as a percentage of total amount of radioactivity present in the sample. C, HeLa cells transiently transfected with Ii-p33 were pulsed-radiolabeled with [35S]methionine (time 0) or chased for 3 h or 4 h in the absence (-) or presence (+) of PMA. Ii molecules were isolated using an anti-Ii mAb and analyzed by SDS PAGE and fluorography. The amount of Ii remaining at each chase point was expressed as a percentage of total material present at time 0 (immediately after pulse-labeling). An anti-Ii immunoblot of each sample confirmed that the steady state amount of Ii in each sample was similar.

 
As an additional indicator of class II traffic to Ag-processing compartments, we measure the kinetics of peptide-loading onto newly synthesized MHC class II molecules. It is well known that class II {alpha}ßI complexes dissociate in SDS at room temperature, whereas after peptide loading class II {alpha}ß-peptide complexes are resistant to denaturation in SDS and migrate as SDS-stable {alpha}ß dimers. Because SDS stability is strictly correlated with Ii proteolysis and dissociation from class II {alpha}ßI complexes (34), this assay indirectly addresses the kinetics of Ii dissociation from class II molecules. B-LCL cells were pulse-labeled with [35S]methionine and chased for up to 3 h in the absence or presence of PMA, class II molecules were isolated by immunoprecipitation, and SDS-stable dimer formation was analyzed by SDS-PAGE under nondenaturing conditions and quantitated by phosphorimager analysis. The addition of PMA during the chase increased the amount of SDS-stable {alpha}ß dimers present at each point (Fig. 4GoB). These data, showing that PMA treatment enhanced the efficiency of class II SDS-stable dimer formation, are in excellent agreement with the hypothesis that PKC-mediated Ii phosphorylation enhances the kinetics of class II trafficking to Ag-processing compartments.

While we have attributed the PMA-induced enhancement of Ii proteolysis to more rapid kinetics of class II trafficking to Ag-processing compartments from the trans-Golgi network, it was theoretically possible that altered Ii proteolysis was not a consequence of trafficking but is instead an indirect consequence of the proteolytic sensitivity of Ii in PMA-treated cells. To address this, we have examined the kinetics of degradation of Ii-p33 in mock-treated or PMA-treated HeLa cells. Because Ii-p33 is not phosphorylated in professional APCs (14, 20), this experiment allows us to address nonspecific effects of PMA on Ii trafficking and proteolysis. Quantitative analysis revealed no significant differences in the kinetics of degradation of Ii-p33 in mock-treated or PMA-treated cells (Fig. 4GoC). Because the lumenal domain of Ii-p33 and Ii-p35 are identical, this data strongly suggests that 1) PMA treatment does not nonspecifically inhibit Ii traffic to lysosomal Ag-processing compartments and 2) that the proteolytic sensitivity of the lumenal domain of Ii is not altered in PMA-treated cells.

A potential problem with the use of specific kinase inhibitors or activators is that it is difficult if not impossible to directly attribute the action of a kinase to a single phosphorylation event. For this reason, we have examined the transport of class II {alpha}ßI complexes to Ag-processing compartments in HeLa cells expressing class II molecules, Ii-33, and either wild-type Ii-p35 or the phosphorylation mutant Ii-p35 (S8A). As we had anticipated, preventing Ii phosphorylation by the use of this mutant inhibited the amount of Ii LIP generated in pulse-chase studies (Fig. 5GoA). Quantitation of multiple experiments revealed that there was a 2-fold reduction in class II traffic to Ag-processing compartments in cells expressing Ii-p35 (S8A) as compared with cells expressing wild-type Ii-p35 (Fig. 5GoB). Importantly, the inhibition of LIP generation obtained by the use of the Ii-p35 phosphorylation mutant was similar to that observed in staurosporine-treated APCs (20), directly demonstrating that Ii phosphorylation regulates the kinetics of class II trafficking to Ag-processing compartments.



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  		FIGURE 5. Mutagenesis of the Ii phosphorylation site inhibits transport of class II-Ii complexes to Ag-processing compartments in HeLa cells. HeLa cells expressing class II {alpha}- and ß-chains, Ii-p33, and either wild-type Ii-p35 (wt) or the phosphorylation mutant Ii-p35 (S8A) were pulse-radiolabeled for 30 min with [35S]methionine and cultured for up to 5 h in complete media containing 1 mM leupeptin. Following cell lysis, class II-associated Ii was isolated using an anti-class II mAb, and the immunoprecipitates were analyzed by SDS-PAGE and autoradiography. The amount of Ii degradation products present in each sample was quantitated by phosphorimager analysis. The amount of Ii-p20 (LIP) and Ii-p10 was expressed as a percentage of the total signal present in each sample. A representative experiment is shown in A, while the percentage of Ii present as Ii-p20 (LIP) and Ii-p10 was quantitated (n = 3) and is shown in B.

 
Ii phosphorylation does not affect class II egress from the ER

It has been proposed that phosphorylation of Ii-p35 is required for transport of class II {alpha}ßI-p35 complexes molecules out of the ER (14). Because this would indirectly affect class II-Ii trafficking to Ag-processing compartments, we examined class II {alpha}ßI transport out of the ER of HeLa cells expressing class II molecules and either wild-type Ii-p35 or a Ii-p35 phosphorylation mutant Ii-p35 (S8A) in the absence or presence of Ii-p33. The kinetics of traffic out of the ER and through the Golgi apparatus was monitored by following Ii sialylation, a carbohydrate modification that occurs in the late Golgi apparatus. Elimination of the phosphorylation site on Ii-p35 by site-directed mutagenesis only slightly inhibited sialylation of Ii-p35 in HeLa cells expressing only the class II {alpha}- and ß-chains (Fig. 6GoA). However, this mutation did not inhibit transport of Ii-p35 out of the ER in cells expressing the class II {alpha}- and ß-chains together with Ii-p33 (Fig. 6GoB).



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  		FIGURE 6. Phosphorylation in not required for egress of Ii-p35 from the ER. HeLa cells expressing class II {alpha}-chains, ß-chains, and either Ii-p35 (wt) or a the Ii-p35 phosphorylation mutant (S8A) in the absence of Ii-p33 (A) or in the presence of Ii-p33 (B) were metabolically labeled with [35S]methionine for 3 h and chased in nonradioactive media for 2 h. C, The B-LCL JY was mock-treated or treated with 1 µm staurosporine during a 30-min pulse-labeling and 1 h chase. For each set of experiments, MHC class II-Ii complexes were immunoprecipitated using an anti-class II mAb, analyzed by 2D-PAGE (left to right, basic to acidic), and visualized by fluorography. The mobilities of class II {alpha}- and ß-chains and the Ii-p35 high mannose (m) and Ii-p35 sialylated (s) forms are indicated. The high mannose forms of Ii-p35 consist of phosphorylated Ii-p35 (arrowhead) and nonphosphorylated Ii-p35 (arrow). The percent sialylated Ii-p35 was quantitated by phosphorimager analysis and was expressed as a percentage of the total amount of Ii-p35 in the sample.

 
To examine a potential role for Ii-p35 phosphorylation in class II trafficking out of the ER in professional APCs, we performed these studies in B-LCL treated with the serine/threonine kinase inhibitor staurosporine. We have previously shown that this treatment abolishes Ii phosphorylation in vivo without nonspecifically inhibiting protein traffic through the secretory pathway (20). As was observed in HeLa cells expressing class II molecules and the Ii-p35 phosphorylation mutant, staurosporine treatment of B-LCL did not inhibit the ability of Ii-p35 to exit the ER and become sialylated in late Golgi compartments (Fig. 6GoC). Taken together, these data do not support a role for phosphorylation of Ii-p35 in regulating class II transport from the ER and strongly support a role for Ii phosphorylation in a post-Golgi trafficking step.

Expression and phosphorylation of Ii-p35 in dendritic cells

On a single cell basis, dendritic cells are perhaps the most potent of all APC subtypes and are unusual in that class II molecules reside in distinct compartments depending on the maturation state of the cells (35). Interestingly, Ii processing has been proposed to play an important role in regulating class II transport during dendritic cell maturation (36). Because Ii-p35 phosphorylation can greatly influence class II trafficking in B-LCL and transfected heterologous cells, we examined Ii-p35 synthesis and phosphorylation during dendritic cell maturation. FACS analysis confirmed the maturation status of the dendritic cell cultures, as revealed by the dramatic increase in surface expression of the costimulatory molecules CD80 and CD86 (not shown), CD83, and class II molecules (Fig. 7GoA). Furthermore, confocal microscopy indicated that class II molecules were located predominately in intracellular vesicular compartments in immature dendritic cells, whereas in mature dendritic cells class II molecules were located primarily at the plasma membrane (Fig. 7GoB). These results are similar to those observed previously in murine dendritic cells (37) and peripheral blood-derived mature dendritic cells (38) and further highlight the differences between immature and mature dendritic cells.



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  		FIGURE 7. Ii-p35 expression and phosphorylation status do not change during human dendritic cell differentiation. A. Immature and mature dendritic cells were analyzed by flow cytometry for expression of the maturation marker CD83 (left) and HLA-DR (right). Note that maturation leads to dramatic changes in surface expression of each of these proteins. B, The distribution of class II molecules in immature and mature dendritic cells was investigated by confocal immunofluorescence microscopy as described in the text. C, Class II molecules were isolated from PBMC, immature dendritic cells, or mature dendritic cells cultured for 5 h in media containing [35S]methionine. In these experiments, class II-Ii complexes were isolated using the Ii-specific mAb Pin1.1. The immunoprecipitates were analyzed by 2D-PAGE and fluorography.

 
We next examined the synthesis and phosphorylation of Ii isoforms in immature and mature dendritic cells. Due to the charge of phosphate, the ratio of phosphorylated to nonphosphorylated Ii-p35 can readily be estimated by 2D-PAGE analysis. Fig. 7GoC reveals that immature and mature dendritic cells synthesized similar amounts of class II and Ii polypeptides, although the synthesis of the alternatively spliced Ii isoforms Ii-p41 and Ii-p43 was unregulated in dendritic cells as compared with PBMC (Fig. 7GoC). As in B-LCL (Fig. 6GoB) and whole PBMC (Fig. 7GoC), in both immature and mature dendritic cells ~50% of the newly synthesized Ii-p35 was phosphorylated (Fig. 7GoC). These data demonstrate that neither Ii-p35 expression nor phosphorylation significantly change during dendritic cell maturation, and it is therefore unlikely that Ii-p35 phosphorylation regulates the profound class II trafficking differences observed in immature and mature dendritic cells.


   Discussion
Top
 Abstract
 Introduction
 Methods and Materials
 Results
 Discussion
References
 
In this paper, we have identified a member of the PKC family of serine/threonine kinases as the kinase responsible for Ii-p35 phosphorylation in a variety of APCs. The functional consequence of Ii phosphorylation is to enhance the kinetics of MHC class II traffic from the trans-Golgi network to the endocytic pathway, as we observed enhanced class II {alpha}ßI transport to prelysosomal Ag-processing compartments under conditions that stimulated Ii phosphorylation and impaired transport to these compartments when Ii phosphorylation is inhibited (20). In addition, we have examined class II transport in transfected heterologous cells expressing class II molecules and Ii-p33 together with either wild-type Ii-p35 or a phosphorylation mutant of Ii-p35. Class II traffic to the endocytic pathway is also inhibited in cells expressing the Ii-p35 phosphorylation mutant, directly demonstrating that phosphorylation of Ii-p35 regulates class II trafficking to Ag-processing compartments.

The use of a variety of pharmacological agents have strongly implicated an isoform of PKC as the prime candidate for the Ii-p35 kinase. Although there are a variety of PKC inhibitors available, many of these have the potential to inhibit other kinases. For example, calphostin C and staurosporine inhibit PKC activity when used at nanomolar concentrations, but have the potential to also inhibit PKA, cGMP-dependent protein kinase, and calcium-calmodulin-dependent protein kinase II when used at higher concentrations. BIM, the most specific PKC inhibitor currently available, acts by competitively inhibiting PKC binding to ATP (27). When used at nanomolar concentrations, BIM has been reported to only inactivate PKC, and we have found that under these conditions BIM treatment of B-LCL specifically inhibits Ii-p35 phosphorylation. The only other kinase potentially effected by BIM is PKA, and we have demonstrated that extremely high concentrations of PKA-specific inhibitors had not effect on Ii-p35 phosphorylation. Therefore, we conclude that a member of the PKC family is responsible for Ii-p35 phosphorylation in vivo.

The PKC family consist of at least 11 known members that have been implicated in the regulation of diverse biological processes (for reviews see Refs. 39 and 40). This family is classified into three groups based upon the enzymes requirements for Ca2+ and diacylglycerol. Members of the "conventional family" require both Ca2+ and diacylglycerol for activity, members of the "novel family" are Ca2+ independent, but diacylglycerol dependent, and members of the "atypical family" require neither Ca2+ nor diacylglycerol for activity. Because PMA is a diacylglycerol analogue, our data showing that brief exposure to PMA increased Ii phosphorylation while chronic PMA treatment reduced Ii phosphorylation strongly suggests that either a conventional or novel PKC family member(s) is responsible for Ii-p35 phosphorylation in vivo.

The 2-fold increase in Ii-p35 phosphorylation in PMA-treated B-LCL correlates almost exactly with the increase in the amount of Ii degradation products generated in endosomal compartments. This is also in excellent agreement with our previous findings that preventing Ii phosphorylation reduced the kinetics of Ii degradation in Ag-processing compartments 2-fold (20). Because the Ii degradation product LIP is only generated in lysosomal Ag-processing compartments, in this and our previous work we have taken the kinetics of LIP generation as an indicator of the kinetics of class II trafficking to Ag-processing compartments. However, it is formally possible that the pharmacological agents employed in these types of experiments could nonspecifically affect cell function. Although it has been reported that phosphorylation of Ii-p35 is required for ER egress (14), we found no evidence for this in cells coexpressing Ii-p35 with class II molecules and Ii-p33 or in B-LCL treated with the serine/threonine kinase inhibitor staurosporine. In addition, PMA-treatment did not affect the kinetics of degradation of a nonphosphorylated isoform of Ii, demonstrating that PMA-treatment did not nonspecifically alter the proteolytic sensitivity of Ii. Therefore, our data is most consistent with a mechanism in which Ii-p35 phosphorylation directly affects class II endosomal transport either by affecting the rate at which class II {alpha}ßI complexes traffic to the endocytic pathway from the trans-Golgi network or by altering the type of endosomal compartment(s) to which the complex is transported. Although distinguishing between these potentially related mechanisms is beyond the scope of this study, it is clear from our data that stimulating Ii phosphorylation enhances the kinetics of class II {alpha}ßI trafficking to the endocytic pathway as well as the loading of peptides onto the resulting class II {alpha}ß dimers.

We have shown that stimulating PKC activity enhances Ii-p35 phosphorylation, whereas inhibiting PKC activity reduces Ii-p35 phosphorylation, and have correlated these changes with enhanced peptide loading onto newly synthesized class II molecules in prelysosomal Ag-processing compartments. In addition, our studies in transfected heterologous cells revealed a role for Ii phosphorylation in traffic to the endocytic pathway in the absence of pharmacological agents. In contrast to our results obtained in human B-LCL, it was reported that short term-PMA treatment inhibited the generation of SDS-stable class II dimers in murine B cells, a result that was attributed to diminished Ii proteolysis (41). However, because murine Ii is not phosphorylated (Ref. 41 and H.A.A., unpublished observation) and mice do not possess the Ii-p35 isoform, species differences alone could account for this apparent discrepancy.

Although Ii-p35 represents only 20% of the cellular pool of Ii in human APCs, Ii-p35 can effect the intracellular transport of a substantial portion of the pool of newly synthesized class II molecules. Because Ii exists as a trimer in vivo and there is no evidence for preferential association of Ii isoforms during trimer formation, we estimate that ~50% of all Ii exist with at least one Ii-p35 polypeptide chain.3 In agreement with this, Newcomb and Cresswell found that a significant fraction of all endosomal Ii degradation products in B-LCL are derived from Ii-p35 (42). Therefore, it is apparent that a complete understanding of Ii-p35 biology is essential for a complete understanding of class II function in human APCs.

In this paper, we have demonstrated that ~50% of all Ii-p35 is phosphorylated in transfected HeLa cells, B-LCL, PBMC, and peripheral blood-derived dendritic cells. Immature and mature dendritic cells synthesized similar amounts of Ii-p33 and Ii-p35, and the extent of Ii phosphorylation was similar in each cell type. As in murine dendritic cells (37), confocal immunofluorescence microscopy revealed that most class II molecules were localized in a punctate, perinuclear compartment in immature dendritic cells, whereas maturation resulted in a redistribution of the class II molecules to the plasma membrane. Differences in Ii processing have been proposed to play an important role in regulating the distribution of class II molecules in dendritic cells (36). However, because Ii-p35 expression and Ii phosphorylation were similar in immature and mature dendritic cells, it is unlikely that Ii phosphorylation contributes to the dramatic differences in the subcellular localization of class II molecules observed in these cells.

The intracellular transport of class II molecules has largely been considered a constitutive process, but recent studies have suggested that presentation of Ags by class II molecules can be regulated by cellular signaling (43, 44, 45, 46). This has been best demonstrated in mouse B cells where cross-linking surface Ig or Fc receptors affects presentation of peptide epitopes to T cells (44, 45). In addition, confocal microscopy studies have revealed a rapid reorganization of endosomal compartments following signaling through these cell surface molecules (46). Thus signal transduction events, acting on unidentified targets, can regulate the class II processing and presentation pathway.

Diverse stimuli have been shown to cause a transient increase in cellular PKC activity. Furthermore, it is well documented that PKC stimulation can regulate protein trafficking, presumably by phosphorylating serine/threonine residues near endosomal targeting motifs (47, 48, 49). The results reported in this study provide evidence for a role of PKC in Ii-p35 phosphorylation and endosomal transport of class II molecules. Thus, while Ii-p35 phosphorylation appears to occur constitutively, the possibility remains that signaling pathways activated in the context of specific immunologic events may alter Ii-p35 phosphorylation, thereby affecting the nature of peptide Ags presented by class II molecules.


   Acknowledgments
 
We thank Drs. Alfred Singer and Dinah Singer for critical reading of this manuscript. We also thank Dr. Piergiuseppe DeBerardinis for the use of the GST fusion protein vector, Mr. David Winkler for oligonucleotide synthesis and automated sequence analysis, and Dr. Martin Brown for assistance in using the confocal microscope.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Paul A. Roche, Experimental Immunology Branch, National Institutes of Health, Building 10, Room 4B36, Bethesda, MD 20892. E-mail address: Back

2 Abbreviations used in this paper: Ii, invariant chain; BIM, bisindolylmaleimide I-HCl; ER, endoplasmic reticulum; IBMX, 3-iso butyl-1-methylxanthine; LCL, lymphoblastoid cell line; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; 2D-PAGE, two-dimensional PAGE; LIP, leupeptin-induced polypeptide. Back

3 To simplify our calculations, we will ignore the minor contribution of the alternatively spliced Ii isoforms Ii-p41 and Ii-p43 from our analyses. Quantitative analyses of two-dimensional PAGE (2D-PAGE) gels revealed that Ii-p33 represents 80% of all Ii and Ii-p35 represents 20% of all Ii (H.A.A. and P.A.R., unpublished observations). Assuming that the formation of Ii trimers is a random event, we estimate that 51% of all Ii exists as Ii-p33 homotrimers (0.83), 1% exists as Ii-p35 homotrimers (0.23), and 48% exists as Ii-p33/Ii-p35 heterotrimers ((0.8 x 0.8 x 0.2) x 3) + ((0.8 x 0.2 x 0.2) x 3).) Back

Received for publication May 19, 1999. Accepted for publication August 26, 1999.


   References
Top
 Abstract
 Introduction
 Methods and Materials
 Results
 Discussion
 References
 

  1. Cresswell, P.. 1994. Assembly, transport, and function of MHC class II molecules. Annu. Rev. Immunol. 12:259.[Medline]
  2. Watts, C.. 1997. Capture and processing of exogenous antigens for presentation on MHC molecules. Annu. Rev. Immunol. 15:821.[Medline]
  3. Marks, M. S., J. S. Blum, P. Cresswell. 1990. Invariant chain trimers are sequestered in the rough endoplasmic reticulum in the absence of association with HLA class II antigens. J. Cell Biol. 111:839.[Abstract/Free Full Text]
  4. Roche, P. A., M. S. Marks, P. Cresswell. 1991. Formation of a nine-subunit complex by HLA class II glycoproteins and the invariant chain. Nature 354:392.[Medline]
  5. Lamb, C. A., P. Cresswell. 1992. Assembly and transport properties of invariant chain trimers and HLA-DR-invariant chain complexes. J. Immunol. 148:3478.[Abstract]
  6. Anderson, M. S., J. Miller. 1992. Invariant chain can function as a chaperone protein for class II major histocompatibility complex molecules. Proc. Natl. Acad. Sci. USA 89:2282.[Abstract/Free Full Text]
  7. Roche, P. A., P. Cresswell. 1990. Invariant chain association with HLA-DR molecules inhibits immunogenic peptide binding. Nature 345:615.[Medline]
  8. Teyton, L., D. O’Sullivan, P. W. Dickson, V. Lotteau, A. Sette, P. Fink, P. A. Peterson. 1990. Invariant chain distinguishes between the exogenous and endogenous antigen presentation pathways. Nature 348:39.[Medline]
  9. Bakke, O., B. Dobberstein. 1990. MHC class II-associated invariant chain contains a sorting signal for endosomal compartments. Cell 63:707.[Medline]
  10. Lotteau, V., L. Teyton, A. Peleraux, T. Nilsson, L. Karlsson, S. L. Schmid, V. Quaranta, P. A. Peterson. 1990. Intracellular transport of class II MHC molecules directed by invariant chain. Nature 348:600.[Medline]
  11. Roche, P. A.. 1995. HLA-DM: an in vivo facilitator of MHC class II peptide loading. Immunity. 3:259.[Medline]
  12. Strubin, M., E. O. Long, B. Mach. 1986. Two forms of the Ia antigen-associated invariant chain result from alternative initiations at two in-phase AUGs. Cell 47:619.[Medline]
  13. Schutze, M. P., P. A. Peterson, M. R. Jackson. 1994. An N-terminal double-arginine motif maintains type II membrane proteins in the endoplasmic reticulum. EMBO J. 13:1696.[Medline]
  14. Kuwana, T., P. A. Peterson, L. Karlsson. 1998. Exit of major histocompatibility complex class II-invariant chain p35 complexes from the endoplasmic reticulum is modulated by phosphorylation. Proc. Natl. Acad. Sci. USA 95:1056.[Abstract/Free Full Text]
  15. Arunachalam, B., C. A. Lamb, P. Cresswell. 1994. Transport properties of free and MHC class II-associated oligomers containing different isoforms of human invariant chain. Int. Immunol. 6:439.[Abstract/Free Full Text]
  16. Roche, P. A., C. L. Teletski, E. Stang, O. Bakke, E. O. Long. 1993. Cell surface HLA-DR-invariant chain complexes are targeted to endosomes by rapid internalization. Proc. Natl. Acad. Sci. USA 90:8581.[Abstract/Free Full Text]
  17. Warmerdam, P. A., E. O. Long, P. A. Roche. 1996. Isoforms of the invariant chain regulate transport of MHC class II molecules to antigen processing compartments. J. Cell Biol. 133:281.[Abstract/Free Full Text]
  18. Wang, K., P. A. Peterson, L. Karlsson. 1997. Decreased endosomal delivery of major histocompatibility complex class II-invariant chain complexes in dynamin-deficient cells. J. Biol. Chem. 272:17055.[Abstract/Free Full Text]
  19. Saudrais, C., D. Spehner, H. de la Salle, A. Bohbot, J. P. Cazenave, B. Goud, D. Hanau, J. Salamero. 1998. Intracellular pathway for the generation of functional MHC class II peptide complexes in immature human dendritic cells. J. Immunol. 160:2597.[Abstract/Free Full Text]
  20. Anderson, H. A., P. A. Roche. 1998. Phosphorylation regulates the delivery of MHC class II invariant chain complexes to antigen processing compartments. J. Immunol. 160:4850.[Abstract/Free Full Text]
  21. Bono, M. R., J. L. Strominger. 1983. NH2-terminal sequence of the {alpha} and ß chains of human DC-1 antigen isolated from the JY cell line: homology with murine I-A molecules. Immunogenetics 18:453.[Medline]
  22. Blauvelt, A., H. Asada, M. W. Saville, V. Klaus-Kovtun, D. J. Altman, R. Yarchoan, S. I. Katz. 1997. Productive infection of dendritic cells by HIV-1 and their ability to capture virus are mediated through separate pathways. J. Clin. Invest. 100:2043.[Medline]
  23. Geissmann, F., P. Revy, A. Regnault, Y. Lepelletier, M. Dy, N. Brousse, S. Amigorena, O. Hermine, A. Durandy. 1999. TGF-ß1 prevents the noncognate maturation of human dendritic Langerhans cells. J. Immunol. 162:4567.[Abstract/Free Full Text]
  24. Romani, N., D. Reider, M. Heuer, S. Ebner, E. Kampgen, B. Eibl, D. Niederwieser, G. Schuler. 1996. Generation of mature dendritic cells from human blood: an improved method with special regard to clinical applicability. J. Immunol. Methods 196:137.[Medline]
  25. Valdez, A. C., J. P. Cabaniols, M. J. Brown, P. A. Roche. 1999. Syntaxin 11 is associated with SNAP-23 on late endosomes and the trans-Golgi network. J. Cell Sci. 112:845.[Abstract]
  26. Nishizuka, Y.. 1984. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature 308:693.[Medline]
  27. Toullec, D., P. Pianetti, H. Coste, P. Bellevergue, T. Grand-Perret, M. Ajakane, V. Baudet, P. Boissin, E. Boursier, F. Loriolle. 1991. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J. Biol. Chem. 266:15771.[Abstract/Free Full Text]
  28. Larrodera, P., M. E. Cornet, M. T. Diaz-Meco, M. Lopez-Barahona, I. Diaz-Laviada, P. H. Guddal, T. Johansen, J. Moscat. 1990. Phospholipase C-mediated hydrolysis of phosphatidylcholine is an important step in PDGF-stimulated DNA synthesis. Cell 61:1113.[Medline]
  29. Scott, J. D., E. H. Fischer, J. G. Demaille, E. G. Krebs. 1985. Identification of an inhibitory region of the heat-stable protein inhibitor of the cAMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA 82:4379.[Abstract/Free Full Text]
  30. Ward, N. E., C. A. O’Brian. 1993. Inhibition of protein kinase C by N-myristoylated peptide substrate analogs. Biochemistry 32:11903.[Medline]
  31. Kase, H., K. Iwahashi, S. Nakanishi, Y. Matsuda, K. Yamada, M. Takahashi, C. Murakata, A. Sato, M. Kaneko. 1987. K-252 compounds, novel and potent inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases. Biochem. Biophys. Res. Commun. 142:436.[Medline]
  32. Blum, J. S., P. Cresswell. 1988. Role for intracellular proteases in the processing and transport of class II HLA antigens. Proc. Natl. Acad. Sci. USA 85:3975.[Abstract/Free Full Text]
  33. Neefjes, J. J., H. L. Ploegh. 1992. Inhibition of endosomal proteolytic activity by leupeptin blocks surface expression of MHC class II molecules and their conversion to SDS resistance {alpha}ß heterodimers in endosomes. EMBO J. 11:411.[Medline]
  34. Germain, R. N., Jr A. G. Rinker. 1993. Peptide binding inhibits protein aggregation of invariant-chain free class II dimers and promotes surface expression of occupied molecules. Nature 363:725.[Medline]
  35. Mellman, I., S. J. Turley, R. M. Steinman. 1998. Antigen processing for amateurs and professionals. Trends. Cell Biol. 8:231.[Medline]
  36. Pierre, P., I. Mellman. 1998. Developmental regulation of invariant chain proteolysis controls MHC class II trafficking in mouse dendritic cells. Cell 93:1135.[Medline]
  37. Pierre, P., S. J. Turley, E. Gatti, M. Hull, J. Meltzer, A. Mirza, K. Inaba, R. M. Steinman, I. Mellman. 1997. Developmental regulation of MHC class II transport in mouse dendritic cells. Nature 388:787.[Medline]
  38. Engering, A. J., C. D. Richters, D. M. Fluitsma, A. M. van Pelt, E. W. Kamperdijk, E. C. Hoefsmit, J. Pieters. 1998. MHC class II and invariant chain biosynthesis and transport during maturation of human precursor dendritic cells. Int. Immunol. 10:1713.[Abstract/Free Full Text]
  39. Mellor, H., P. J. Parker. 1998. The extended protein kinase C superfamily. Biochem. J. 332:281.
  40. Nishizuka, Y.. 1995. Protein kinase C and lipid signaling for sustained cellular responses. FASEB. J. 9:484.[Abstract]
  41. Barois, N., F. Forquet, J. Davoust. 1997. Selective modulation of the major histocompatibility complex class II antigen presentation pathway following B cell receptor ligation and protein kinase C activation. J. Biol. Chem. 272:3641.[Abstract/Free Full Text]
  42. Newcomb, J. R., P. Cresswell. 1993. Structural analysis of proteolytic products of MHC class II-invariant chain complexes generated in vivo. J. Immunol. 151:4153.[Abstract]
  43. Koppelman, B., J. J. Neefjes, J. E. de Vries, R. de Waal Malefyt. 1997. Interleukin-10 down-regulates MHC class II {alpha}ß peptide complexes at the plasma membrane of monocytes by affecting arrival and recycling. Immunity. 7:861.[Medline]
  44. Wagle, N. M., A. E. Faassen, J. H. Kim, S. K. Pierce. 1999. Regulation of B cell receptor-mediated MHC class II antigen processing by Fc{gamma}RIIB1. J. Immunol. 162:2732.[Abstract/Free Full Text]
  45. Zimmermann, V. S., P. Rovere, J. Trucy, K. Serre, P. Machy, F. Forquet, L. Leserman, J. Davoust. 1999. Engagement of B cell receptor regulates the invariant chain-dependent MHC class II presentation pathway. J. Immunol. 162:2495.[Abstract/Free Full Text]
  46. Siemasko, K., B. J. Eisfelder, E. Williamson, S. Kabak, M. R. Clark. 1998. Signals from the B lymphocyte antigen receptor regulate MHC class II containing late endosomes. J. Immunol. 160:5203.[Abstract/Free Full Text]
  47. Dietrich, J., X. Hou, A. M. Wegener, C. Geisler. 1994. CD3 gamma contains a phosphoserine-dependent di-leucine motif involved in down-regulation of the T cell receptor. EMBO J. 13:2156.[Medline]
  48. Pitcher, C., S. Honing, A. Fingerhut, K. Bowers, M. Marsh. 1999. Cluster of differentiation antigen 4 (CD4) endocytosis and adaptor complex binding require activation of the CD4 endocytosis signal by serine phosphorylation. Mol. Biol. Cell 10:677.[Abstract/Free Full Text]
  49. Guinamard, R., N. Signoret, I. Masamichi, M. Marsh, T. Kurosaki, J. V. Ravetch. 1999. B cell antigen receptor engagement inhibits stromal cell-derived factor (SDF)-1{alpha} chemotaxis and promotes protein kinase C (PKC)-induced internalization of CXCR4. J. Exp. Med. 189:1461.[Abstract/Free Full Text]



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