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J. Biol. Chem., Vol. 282, Issue 8, 5653-5660, February 23, 2007

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Association of CD38 with Nonmuscle Myosin Heavy Chain IIA and Lck Is Essential for the Internalization and Activation of CD38^*

So-Young Rah¹, Kwang-Hyun Park, Tae-Sik Nam, Sang-Jin Kim, Hyuntae Kim, Mie-Jae Im, and Uh-Hyun Kim²

From the Department of Biochemistry and the Institute of Cardiovascular Research, Chonbuk National University Medical School, Jeonju 561-182, Republic of Korea

Received for publication, October 6, 2006 , and in revised form, November 14, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Activation of CD38 in lymphokine-activated killer (LAK) cells involves interleukin-8 (IL8)-mediated protein kinase G (PKG) activation and results in an increase in the sustained intracellular Ca²⁺ concentration ([Ca²⁺]_i), cADP-ribose, and LAK cell migration. However, direct phosphorylation or activation of CD38 by PKG has not been observed in vitro. In this study, we examined the molecular mechanism of PKG-mediated activation of CD38. Nonmuscle myosin heavy chain IIA (MHCIIA) was identified as a CD38-associated protein upon IL8 stimulation. The IL8-induced association of MHCIIA with CD38 was dependent on PKG-mediated phosphorylation of MHCIIA. Supporting these observations, IL8- or cell-permeable cGMP analog-induced formation of cADP-ribose, increase in [Ca²⁺]_i, and migration of LAK cells were inhibited by treatment with the MHCIIA inhibitor blebbistatin. Binding studies using purified proteins revealed that the association of MHCIIA with CD38 occurred through Lck, a tyrosine kinase. Moreover, these three molecules co-immunoprecipitated upon IL8 stimulation of LAK cells. IL8 treatment of LAK cells resulted in internalization of CD38, which co-localized with MHCIIA and Lck, and blebbistatin blocked internalization of CD38. These findings demonstrate that the association of phospho-MHCIIA with Lck and CD38 is a critical step in the internalization and activation of CD38.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
A type II transmembrane protein, CD38 possesses ADP-ribosyl cyclase and ADP-ribose hydrolase activities (1, 2). These two enzyme activities are involved in the conversion of -NAD⁺ first to cADP-ribose (cADPR)³ and then to ADP-ribose (3–5). The metabolite cADPR is known to increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) by release from intracellular Ca²⁺ stores or by Ca²⁺ influx through plasma membrane Ca²⁺ channels in a variety of cells (6–10). Mounting evidence indicates that cADPR synthesis by ADP-ribosyl cyclases is stimulated through cell-surface heterotrimeric G-protein-coupled receptor signaling. The receptors involved in ADP-ribosyl cyclase activation include the -adrenergic (11, 12), angiotensin II (13), and muscarinic (14) receptors. Activation of ADP-ribosyl cyclase/CD38 by cGMP has been reported (15, 16), and cAMP-dependent activation of the enzyme has also been observed in artery smooth muscle cells (12) and cardiomyocytes (17). However, the molecular mechanism of ADP-ribosyl cyclase/CD38 activation has not been completely elucidated.

The active site of CD38 is located in the extracellular domain, whereas the substrate -NAD⁺ and the targets of the metabolite cADPR are present inside cell (18). This topological paradox of CD38 has been addressed and explained by demonstrating 1) the ligand-induced, vesicle-mediated internalization of CD38, which is followed by an increase in the intracellular cADPR concentration ([cADPR]_i) (19), and 2) the -NAD⁺-transporting function of connexin-43 and the cADPR-transporting function of membrane-bound CD38 or an unidentified cADPR transporter (20). Thus, the exact molecular mechanism of CD38 activation remains to be clarified.

Recently, we reported that CD38 in lymphokine-activated killer (LAK) cells is activated by a sequential process involving ligation of the interleukin (IL)-8 receptor, inositol 1,4,5-trisphosphate-induced increase in [Ca²⁺]_i, and activation of protein kinase G (PKG) (16). We also found that PKG does not directly activate or phosphorylate CD38 in vitro. In this study, we investigated the missing link between CD38 and PKG for activation of CD38 in LAK cells. In addition, we also examined whether CD38 is internalized during the activation process of CD38. The results indicate that PKG phosphorylates nonmuscle myosin heavy chain IIA (MHCIIA) upon stimulation of the IL8 receptor and that the association of phosphorylated MHCIIA with CD38 through Lck induces the internalization and activation of CD38.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Reagents and Antibodies—Antibodies were obtained as follows: anti-human CD38 monoclonal antibody (mAb) from BD Biosciences; anti-MHCIIA polyclonal antibody (pAb), anti-FLAG antibody, anti-actin mAb, and anti-FLAG antibody-agarose from Sigma; anti-Lck pAb from Upstate%20Biotechnology">Upstate Biotechnology (Lake Placid, NY); anti-glutathione S-transferase (GST) pAb from Amersham Biosciences AB (Uppsala, Sweden); and horseradish peroxidase-conjugated anti-mouse IgG, anti-goat IgG, and anti-rabbit IgG from Advanced Biochemicals Inc. (Jeonju, Korea). Ficoll-Hypaque and Percoll were obtained from Amersham Biosciences AB. Nylon wool was from Polyscience Inc. (Warrington, PA), and human recombinant IL2 was from Chiron B.V. (Amsterdam, The Netherlands). 8-pCPT-cGMP, (R_p)-8-pCPT-cGMP-S, and blebbistatin were purchased from Calbiochem. Human recombinant IL8, human AB serum, and all other reagents were obtained from Sigma. RPMI 1640 medium and antibiotics were from Invitrogen. Transwell was purchased from Corning Costar Corp. (Cambridge, MA). [³²P]Orthophosphate was from PerkinElmer Life Sciences.

Preparation of LAK Cells—LAK cells were prepared as described previously (21, 22). Briefly, blood obtained from healthy volunteers was layered over Ficoll-Hypaque and centrifuged at 700 x g for 30 min to remove red blood cells. Cell preparations with red blood cells removed were incubated on a nylon-wool column at 37 °C for 1 h in a 5% CO₂ incubator to remove B lymphocytes and macrophages. Nylon-wool nonadherent cells were collected and further separated by Percoll density gradient centrifugation. Four layers of Percoll were used: 37, 44, 52, and 60%. After centrifugation at 700 x g for 20 min, cells from the 52% Percoll layer were collected, washed with serum-free RPMI 1640 medium, and incubated at a density of 2 x 10⁶ cells/ml in culture medium (RPMI 1640 medium supplemented with 10% human AB serum, 0.25 µg/ml amphotericin B, 50 µg/ml gentamycin, 10 units/ml penicillin G, 100 µg/ml streptomycin, 1 mM L-glutamine, 1% nonessential amino acids, and 50 µM 2-mercaptoethanol) containing 3000 IU/ml IL2 in a 5% CO₂ incubator at 37 °C. After incubation for 24 h, the floating cells were removed, and the adherent cells were cultured in culture medium containing 1500 IU/ml IL2. LAK cells induced by IL2 for 10 days were used throughout this study.

Purification of MHCIIA—Jurkat T cells (1-ml packed volume) were lysed in ice-cold lysis buffer containing 20 mM HEPES (pH 7.2), 10% glycerol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1% (v/v) Triton X-100, 150 mM NaCl, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 10 µg/ml aprotinin by a three-round freeze/thaw method. The lysed cells were centrifuged at 20,000 x g for 30 min. The supernatant was collected and loaded onto a small column (250-µl packed volume) of MHCIIA antibody-cross-linked protein G-agarose. Conjugation of the two proteins was achieved with dimethyl pimelimidate (Pierce) using the method recommended by the manufacturer. After the sample was loaded, the column was washed with 5 column volumes of the same lysis buffer, eluted with a small volume (100 µl) of 3 M glycine buffer (pH 2.7), and immediately neutralized with 10 µl of 1 M Tris buffer (pH 8.6). The eluted MHCIIA was stored at -80 °C until used.

Expression and Purification of GST-fused Lck Domains and FLAG-fused CD38—The construction and preparation of GST-fused Lck domains were performed as described previously (23, 24). Lck domains were transformed in Escherichia coli BL21. Cells were grown to A₅₉₅ = 0.6–0.8, induced with 0.1 mM isopropyl -D-thiogalactopyranoside, and maintained at 25 °C for 5 h. Cells were collected by centrifugation and lysed by sonication in lysis buffer containing 10 mM HEPES (pH 7.5), 150 mM NaCl, 0.5% (v/v) Triton X-100, 10 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. The lysates were cleared by centrifugation at 12,000 x g for 50 min at 4 °C. GST fusion proteins were purified by affinity chromatography on glutathione-Sepharose 4B (Amersham Biosciences AB). FLAG-fused human CD38 was prepared as described, expressed in COS-7 cells, and purified using anti-FLAG antibody-agarose (24). Purified proteins were stored at -80 °C until used.

Immunoprecipitation and Western Blotting—Cells were washed with phosphate-buffered saline (PBS) and then lysed with ice-cold cell lysis buffer containing 20 mM HEPES (pH 7.2), 1% (v/v) Triton X-100, 10% glycerol, 100 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, 1 mM Na₃VO₄, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 10 µg/ml aprotinin. Supernatants were obtained after centrifugation at 20,000 x g for 10 min. For immunoprecipitation, cell lysates (800 µg) precleared with protein G-agarose were incubated with anti-CD38 mAb or anti-MHCIIA pAb overnight at 4 °C and then further incubated with protein G-agarose at 4 °C for 1 h. The immunoprecipitates were washed four times with cell lysis buffer and boiled for 10 min. The immunoprecipitated proteins were subjected to SDS-PAGE on a 8 or 10% gel. The protein G-precleared lysate (10 µg/lane) was also subjected to immunoblotting to verify equal amounts of the proteins used for immunoprecipitation. After transfer to nitrocellulose membranes, the blots were incubated in blocking buffer (10 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.05% Tween 20) containing 5% nonfat dry milk for 2 h at room temperature and then with primary antibodies (CD38, 1:500 dilution; MHCIIA, 1:2000 dilution; actin, 1:5000 dilution; and Lck, 1:2000 dilution) in blocking buffer overnight at 4 °C. The blots were rinsed four times with blocking buffer and incubated with horseradish peroxidase-conjugated anti-mouse IgG (1:5000 dilution), anti-rabbit IgG (1:5000 dilution), or anti-goat IgG (1:5000 dilution) in blocking buffer at room temperature for 1 h. The immunoreactive proteins with the respective secondary antibodies were determined using an enhanced chemiluminescence kit (Amersham Biosciences AB) and exposed to an LAS-1000 ImageReader Lite (Fujifilm, Japan). Protein concentration was determined using a Bio-Rad protein assay kit, and known concentrations of bovine serum albumin (BSA) were used as the standard.

Measurement of [cADPR]_i—The [cADPR]_i was measured using a cyclic enzyme assay as described previously (25). Briefly, cells were treated with 0.5 ml of 0.6 M perchloric acid under sonication. Precipitates were removed by centrifugation at 20,000 x g for 10 min. Perchloric acid was removed by mixing the aqueous sample with a solution containing 3 volumes of 1,1,2-trichlorotrifluoroethane to 1 volume of tri-n-octylamine. After centrifugation for 10 min at 1500 x g, the aqueous layer was collected and neutralized with 20 mM sodium phosphate (pH 8.0). To remove all contaminating nucleotides, the samples were incubated with the following hydrolytic enzymes over-night at 37 °C: 0.44 unit/ml nucleotide pyrophosphatase, 12.5 units/ml alkaline phosphatase, 0.0625 unit/ml NAD glycohydrolase, and 2.5 mM MgCl₂ in 20 mM sodium phosphate buffer (pH 8.0). Enzymes were removed by filtration using a Centricon-3 filter (Amicon). To convert cADPR to -NAD⁺, the samples (0.1 ml/tube) were incubated with 50 µl of cycling reagent containing 0.3 µg/ml Aplysia ADP-ribosyl cyclase, 30 mM nicotinamide, and 100 mM sodium phosphate (pH 8.0) at room temperature for 30 min. The Aplysia ADP-ribosyl cyclase was purified as described (26). The samples were further incubated with the cycling reagent (0.1 ml) containing 2% ethanol, 100 µg/ml alcohol dehydrogenase, 20 µM resazurin, 10 µg/ml diaphorase, 10 µM riboflavin 5'-phosphate, 10 mM nicotinamide, 0.1 mg/ml BSA, and 100 mM sodium phosphate (pH 8.0) at room temperature for 2 h. An increase in the resorufin fluorescence was measured at an excitation of 544 nm and an emission of 590 nm using a SpectraMax Gemini fluorescence plate reader (Molecular Devices Corp.). Various known concentrations of cADPR were also included in the cycling reaction to generate a standard curve.

Measurement of [Ca²⁺]_i—Cells were washed with Hanks' balanced salt solution (2 mM CaCl₂, 145 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 5 mM D-glucose, and 20 mM HEPES (pH 7.3)) containing 1% BSA and incubated in the same solution containing 1% BSA for 6 h. Starved LAK cells were incubated with 5 µM fluo-3 acetoxymethyl ester (Molecular Probes, Eugene, OR) in Hanks' balanced salt solution containing 1% BSA at 37 °C for 40 min. The cells were washed three times with Hanks' balanced salt solution. Changes in [Ca²⁺]_i in LAK cells were determined at an excitation of 488 nm and an emission of 530 nm using an air-cooled argon laser system (27). The emitted fluorescence at 530 nm was collected using a photomultiplier. One image was scanned every 6 s for 10 min using a Nikon confocal microscope. For the calculation of [Ca²⁺]_i, the method of Tsien et al. d>(F d> is 450 nM for fluo-3 and F is the observed fluorescence level. Each tracing was calibrated for the maximal intensity (F_max) by the addition of ionomycin (8 µM) and for the minimal intensity (F_min) by the addition of EGTA (50 mM) at the end of each measurement.

In Vivo Phosphorylation—LAK cells were washed with phosphate-free medium and then incubated in phosphate-free medium containing 1 mCi of [³²P]orthophosphate at 37 °C for 6 h. The cells were incubated with various agents, washed twice with 10 mM PBS (pH 7.4), and then lysed with ice-cold cell lysis buffer. After centrifugation at 20,000 x g for 10 min, the supernatants were collected for immunoprecipitation. Immunoprecipitation was performed using anti-CD38 mAb as described above.

Determination of Cell Migration—Cell migration was determined as described previously (22). In brief, cells were washed with RPMI 1640 medium, scrapped using a policeman, and washed with RPMI 1640 medium. Transwells with 8-µm pore size polycarbonate filters were used. Lower chambers contained 500 µl of RPMI 1640 medium and 1% BSA. LAK cells (4 x 10⁵) in 100 µl of RPMI 1640 medium containing various agents were placed in the upper chamber and then incubated in a 5% CO₂ incubator at 37 °C for 2 h. After removal of the unattached cells, the filters were removed, fixed with ice-cold 100% methanol, and stained with 15% Wright-Giemsa stain for 7 min. The cells were counted under a phase-contrast microscope.

Confocal Microscopy of Internalized and Co-localized CD38, MHCIIA, and Lck—For localization of CD38 and the cognate proteins, immunofluorescence was performed as described previously (29) with a slight modification. Briefly, LAK cells were incubated with IL8. At the indicated time points, the suspension cells (100 µl) were transferred into 3.7% paraformaldehyde/PBS (900 µl) and incubated at 4 °C for 1 h and then washed three times with ice-cold PBS. The cells were permeabilized with 0.1% Triton X-100 in PBS at 4 °C for 30 min and sequentially incubated with fluorescein isothiocyanate-conjugated anti-CD38 mAb (1:100) and Alexa 633-conjugated anti-Lck (1:1000) and TRITC-conjugated anti-MHCIIA (1:1000) pAbs in the presence of 0.1% BSA at 4 °C for 8 h for each antibody. After being washed three times with PBS, cells were attached to slides precoated with 0.1 mg/ml poly-L-lysine and visualized with a Zeiss confocal microscope.

Statistical Analysis—Data represent the means ± S.E. of at least three separate experiments. Statistical analysis was performed using Student's t test. A p value <0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Treatment of LAK Cells with IL8 Induces Association of MHCIIA with CD38—Stimulation of the IL8 receptor induces CD38 activation via cGMP/PKG in LAK cells, but CD38 is neither directly phosphorylated nor activated by PKG in vitro (16). To identify the protein(s) located between CD38 and PKG, LAK cells metabolically labeled with [³²P]orthophosphate were treated with IL8 in the absence or presence of a PKG inhibitor, (R_p)-8-pCPT-cGMP-S, and the cell extracts were subjected to immunoprecipitation using anti-CD38 mAb. As shown in Fig. 1A, treatment with IL8 significantly increased the phosphorylation level of an 200-kDa protein compared with the control. The intensity of the phosphoprotein was decreased in the presence of the PKG inhibitor, suggesting that the IL8-induced phosphorylation of the protein is mediated by PKG. The protein band was excised from the gel and subjected to matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis. The results revealed that the protein was MHCIIA (Fig. 1B). To further confirm this observation, immunoprecipitation of MHCIIA by anti-CD38 antibody was examined with and without IL8, the PKG inhibitor, or the cell-permeable cGMP analog 8-pCPT-cGMP. The level of MHCIIA in the immunocomplex was increased upon treatment with IL8 compared with the control (Fig. 1C). The IL8-mediated association of MHCIIA with CD38 was substantially reduced in the presence of the PKG inhibitor. Treatment of LAK cells with the cGMP analog also increased the co-immunoprecipitation of MHCIIA compared with the control. Moreover, pretreatment of LAK cells with the specific MHCIIA inhibitor blebbistatin completely inhibited the IL8-induced co-immunoprecipitation of MHCIIA and CD38 (Fig. 1D). These results suggest that PKG phosphorylates MHCIIA in LAK cells upon IL8 stimulation and that phosphorylated MHCIIA is associated with CD38.

View larger version (66K): [in this window] [in a new window]   FIGURE 1. Association of CD38 with MHCIIA in LAK cells. A, IL8-induced CD38 co-immunoprecipitation of a phosphoprotein is inhibited by PKG inhibitor pretreatment in LAK cells. LAK cells were incubated with 1 mCi of [32P]orthophosphate at 37 °C for 6 h. The labeled cells were treated with 10 pM IL8 or without (Control) for 90 s. LAK cells were preincubated with (Rp)-8-pCPT-cGMP-S (20 µM) for 30 min (Rp-8pCPTcGMP-S + IL8). LAK cell extract was immunoprecipitated with anti-CD38 mAb, and the immunoprecipitates were separated by SDS-PAGE, followed by autoradiography. B, identification of MHCIIA as a protein associated with CD38 by MALDI-TOF analysis. Shown is a MALDI-TOF mass map of peptides obtained after in-gel digestion with trypsin. Matched peptides are shown in boldface and underlined in the protein sequence. C, IL8 induces the association of MHCIIA with CD38 through cGMP/PKG in LAK cells. Cells were treated with 10 pM IL8 or 1 mM 8-pCPT-cGMP for 90 s. Cells pretreated with (Rp)-8-pCPT-cGMP-S (20 µM) for 30 min were incubated with IL8 for 90 s. The cells were extracted with lysis buffer and then subjected to immunoprecipitation using anti-CD38 mAb. The immunoprecipitated proteins were analyzed by immunoblotting with anti-CD38 mAb or anti-MHCIIA pAb. D, IL8-induced association of MHCIIA with CD38 is inhibited by pretreatment of LAK cells with the MHCIIA inhibitor blebbistatin. LAK cells were incubated with blebbistatin (50 µM) for 30 min prior to treatment with IL8 for 90 s. Actin blotting was performed before immunoprecipitation to ensure that equal amounts of samples were subjected to immunoprecipitation. Three or four independent experiments were performed, and similar results were obtained.

View larger version (41K): [in this window] [in a new window]   FIGURE 2. MHCIIA is involved in CD38 activation. A, the MHCIIA inhibitor blebbistatin inhibits the IL8- or cGMP-induced increase in [cADPR]i in LAK cells. LAK cells were preincubated with blebbistatin (50 µM) for 30 min. After incubation with or without 10 pM IL8 or 1 mM 8-pCPT-cGMP for 90 s, [cADPR]i was determined as described under "Experimental Procedures." The reactions were terminated by the addition of 0.5 ml of 0.6 M perchloric acid at the time points indicated. The means ± S.E. of three independent experiments are shown. *, p < 0.005 (control versus IL8); **, p < 0.005 (IL8 versus IL8 plus blebbistatin); #, p < 0.05 (control versus 8-pCPT-cGMP); ##, p < 0.05 (8-pCPT-cGMP versus blebbistatin). For determination of the cGMP-mediated phosphorylation of MHCIIA, cells were treated with the cell-permeable cGMP analog 8-pCPT-cGMP (1 mM) for 90 s. B, blebbistatin inhibits the IL8- or cGMP analog-induced increase in [Ca2+]i in LAK cells. Three representative Ca2+ traces are shown (n = 10). Arrows indicate the time point of the addition of IL8 (10 pM) or 8-pCPT-cGMP (1 mM). C, IL8- or cGMP analog-induced stimulation of LAK cell migration is blocked by blebbistatin. Cells treated with 50 µM blebbistatin for 30 min were incubated with IL8 (10 pM) for 2 h. Cell migration assay was performed as described under "Experimental Procedures." The means ± S.E. of three independent experiments are shown. *, p < 0.005 (control versus IL8); **, p < 0.005 (IL8 versus IL8 plus blebbistatin); #, p < 0.05 (control versus 8-pCPT-cGMP); ##, p < 0.05 (8-pCPT-cGMP versus blebbistatin).

MHCIIA Is Phosphorylated by PKG in IL8-induced LAK Cells— Next, we examined whether MHCIIA is directly phosphorylated by PKG. Indeed, purified MHCIIA was shown to be a good substrate for PKG by in vitro phosphorylation (Fig. 3A). To determine which amino acid residues of MHCIIA are phosphorylated upon IL8 stimulation, LAK cells were treated with IL8 in the absence and presence of the PKG inhibitor or cGMP analog. Cell extracts were subjected to immunoprecipitation using anti-MHCIIA pAb, and phosphorylated amino acids in MHCIIA were probed with anti-phosphoamino acid antibodies. Treatment with IL8 specifically increased the phosphorylation of the serine residue in MHCIIA, but not the threonine and tyrosine residues (Fig. 3B). Furthermore, the IL8-mediated phosphorylation of the serine residue was substantially reduced in the presence of the PKG inhibitor. The cGMP analog increased the phosphorylation of the serine residue compared with the control.

View larger version (48K): [in this window] [in a new window]   FIGURE 3. MHCIIA is phosphorylated by PKG in IL8-induced LAK cells. A, in vitro phosphorylation of MHCIIA by PKG. Affinity-purified MHCIIA (0.1 µg) was incubated with and without 600 units of PKG in 30 µl of 50 mM Tris-HCl (pH 7.4) containing 20 mM MgCl2, 0.1 mM ATP, 10 µCi of [-32P]ATP, and 10 µM cGMP at 37 °C for 15 min. Reactions were terminated by the addition of SDS-PAGE buffer, followed by boiling in a water bath for 5 min. Proteins were separated by SDS-PAGE (7% gel), followed by autoradiography. WB, Western blot. B, IL8 induces the phosphorylation of MHCIIA through cGMP/PKG in LAK cells. The cells were treated as described for Fig. 1C, extracted with lysis buffer, and then subjected to immunoprecipitation (IP) using anti-MHCIIA pAb. The immunoprecipitated proteins were analyzed by immunoblotting with antiphosphoamino acid antibodies or anti-MHCIIA pAb.

View larger version (26K): [in this window] [in a new window]   FIGURE 4. PKG-phosphorylated MHCIIA is associated with CD38 via the Lck SH3 domain. A, PKG-phosphorylated MHCIIA is associated with CD38 via Lck. FLAG-CD38 (2 µg) was incubated with or without GST-Lck (2 µg) and further incubated with phospho-MHCIIA (2 µg) or non-phosphorylated MHCIIA (2 µg) in a 500-µl volume overnight at 4 °C. Phospho-MHCIIA (10 µg) was obtained by incubation with PKG (3000 units) in the presence of 20 mM MgCl2, 0.1 mM ATP, and 10 µM cGMP at 37 °C for 15 min. The proteins were immunoprecipitated (IP) with anti-FLAG antibody-agarose (20 µl of 1:1 suspension/tube). The immunoprecipitated proteins were subjected to immunoblotting using anti-MHCIIA pAb, anti-GST antibody, or anti-FLAG antibody. B, schematic presentation of GST-Lck domain fusion proteins. GST fusion proteins were expressed and purified as described under "Experimental Procedures." C, phosphorylated MHCIIA interacts with the SH3 domain of Lck. PKG-phosphorylated MHCIIA (2 µg) was incubated with FLAG-CD38 (2 µg) and GST-fused Lck domains (2 µg) in a 500-µl volume overnight at 4 °C, and the proteins were immunoprecipitated with anti-FLAG antibody-agarose. The immunoprecipitated proteins were subjected to immunoblotting using anti-MHCIIA pAb, anti-GST antibody, or anti-FLAG antibody. D, IL8-induced association of MHCIIA and Lck with CD38 in LAK cells. The cells were treated as described for Fig. 1C, extracted with lysis buffer, and then subjected to immunoprecipitation using anti-CD38 mAb. The immunoprecipitated proteins were analyzed by immunoblotting with anti-MHCIIA pAb, anti-Lck mAb, or anti-CD38 mAb. Three or four independent experiments were performed, and similar results were obtained.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We demonstrated previously that CD38 in LAK cells is activated through sequential signaling involving the IL8 receptor, inositol 1,4,5-trisphosphate-mediated increase in Ca²⁺, and activation of PKG (16). In this study, we have extended the molecular mechanism of the activation of CD38 in LAK cells. Our results reveal for the first time that MHCIIA is associated with CD38 through Lck and is involved in the internalization and activation of CD38. In addition, the results indicate that PKG phosphorylates the serine residue in MHCIIA upon stimulation of the IL8 receptor and that the phosphorylation of MHCIIA leads to binding to the CD38-Lck complex via the SH3 domain of Lck.

Previous studies demonstrated that CD38 internalization induced by external stimuli such as -NAD⁺ and thiol compounds results in an increase in [cADPR]_i (19, 29). Our results reveal that stimulation of the IL8 receptor (a G-protein-coupled receptor) in LAK cells induces CD38 internalization and increases [cADPR]_i. These findings suggest that CD38 internalization leads to CD38 activation. Our previous data showing that the level of cADPR increases at 30 s after IL8 treatment and reaches a maximal level at 90 s (24) correlate well with the data of CD38 internalization, which occurred as early as 15 s and persisted for up to 30 s (Fig. 5A). However, NAD⁺- or glutathione-induced internalization of CD38 and increase in [Ca²⁺]_i occur relatively slowly and reach maximal levels at 1 h (19). In our study, the CD38 internalization process was mediated by IL8 receptor signaling. Moreover, the intracellular calcium level was increased immediately after IL8 treatment and was sustained. Therefore, we suggest that the discrepancy is due to the different signaling mechanism of CD38 internalization/activation: IL8 receptor signaling-mediated internalization of CD38 versus direct activation of CD38 with the substrate NAD⁺. In addition, although it is unclear whether the internalization of CD38 is sufficient to target the substrate -NAD⁺, it appears, however, that the internalization of CD38 is the critical step for its activation because pretreatment of LAK cells with blebbistatin not only blocked IL8-induced internalization of CD38, but also inhibited IL8-stimulated cADPR production, sustained Ca²⁺ increase, and migration of LAK cells.

View larger version (43K): [in this window] [in a new window]   FIGURE 5. IL8-induced internalization and co-localization of CD38 with MHCIIA and Lck are blocked by blebbistatin. A, IL8-induced internalization and co-localization of CD38 with MHCIIA and Lck. B, pretreatment with blebbistatin blocks the internalization and co-localization of CD38 with MHCIIA and Lck. Serum-starved LAK cells were preincubated in the absence and presence of blebbistatin (50 µM) at 37 °C for 30 min, followed by stimulation with IL8 (10 pM) for the indicated times. The cells were fixed, stained, and examined with a confocal microscope as described "Experimental Procedures." Approximately 60% of the cells showed good capping and comparable staining of the three fluorochromes. C, percentages of cells exhibiting internal localization of CD38 with and without IL8 treatment. D, percentages of cells exhibiting internal localization of CD38 with and without treatment with IL8 plus blebbistatin. n indicates the total number of cells examined under a confocal microscope.

Lck is one of eight members of the Src family of tyrosine kinases, which are activated by T cell stimulation and required for T cell proliferation, IL2 production, and cytotoxicity of LAK cells (39). Lck consists of four domains, viz. the N-terminal, SH2, SH3, and catalytic domains. Our study on the complex formation of CD38 with Lck and MHCIIA has revealed that Lck acts as a linker between CD38 and MHCIIA. Moreover, SH3 domain-containing fusion proteins of Lck are able to interact with purified phosphorylated MHCIIA, but the SH2 domain does not. Several studies have demonstrated that SH3 domainligand interaction is critical for activation of members of the Src kinase family, including Lck (40, 41). However, our data suggest that Lck kinase activity is not involved in CD38 activation and/or internalization because no phosphorylation of tyrosine residues in CD38 or MHCIIA upon IL8 stimulation was observed. It appears that Lck is tightly associated with CD38 in LAK cells, forming a complex, because Lck was immunoprecipitated by anti-CD38 mAb without stimulation of the IL8 receptor and because the level of Lck in the immunoprecipitates was not changed by the receptor stimulation. Because these findings are unexpected, further detailed studies are required.

In conclusion, we have demonstrated that recruitment of phospho-MHCIIA in the CD38-Lck complex is the critical step for the internalization and activation of CD38 and that IL8-activated PKG phosphorylates MHCIIA directly. In addition, the results suggest that Lck acts as an adaptor for the association of CD38 with MHCIIA in IL8/CD38 signaling in LAK cells.

	FOOTNOTES

 
^* This work was supported by Korea Research Foundation Grant KRF-2004-005-E00108 (to U.-H. Kim). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of a BK21 fellowship from the Korea Ministry of Education.

² To whom correspondence should be addressed: Dept. of Biochemistry, Chonbuk National University Medical School, Keumam-dong, Jeonju 561-182, Republic of Korea. Tel.: 82-63-270-3083; Fax: 82-63-274-9833; E-mail: uhkim{at}chonbuk.ac.kr.

³ The abbreviations used are: cADPR, cADP-ribose; [Ca²⁺]_i, intracellular Ca²⁺ concentration; [cADPR]_i, intracellular cADP-ribose concentration; LAK, lymphokine-activated killer; IL, interleukin; PKG, protein kinase G; MHCIIA, nonmuscle myosin heavy chain IIA; mAb, monoclonal antibody; pAb, polyclonal antibody; GST, glutathione S-transferase; 8-pCPT-cGMP, 8-(4-chlorophenylthio)guanosine 3':5'-monophosphate; (R_p)-8-pCPT-cGMP-S, (R_p)-8-(4-chlorophenylthio)guanosine 3':5'-monophosphorothioate; PBS, phosphate-buffered saline; BSA, bovine serum albumin; TRITC, tetramethylrhodamine isothiocyanate; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; SH, Src homology.

	ACKNOWLEDGMENTS

 
We are very grateful to the volunteers for providing blood. We thank Ji-Yeon Shim for excellent technical support and the Korea Basic Science Institute for MALDI-TOF analysis.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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