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Originally published In Press as doi:10.1074/jbc.M503293200 on June 23, 2005

J. Biol. Chem., Vol. 280, Issue 34, 30055-30062, August 26, 2005
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Protein Kinase A-dependent Phosphorylation of Lutheran/Basal Cell Adhesion Molecule Glycoprotein Regulates Cell Adhesion to Laminin {alpha}5*

Emilie Gauthier{ddagger}§, Cécile Rahuel{ddagger}§, Marie Paule Wautier{ddagger}§, Wassim El Nemer{ddagger}§, Pierre Gane{ddagger}§, Jean Luc Wautier§, Jean Pierre Cartron{ddagger}§, Yves Colin{ddagger}§, and Caroline Le Van Kim{ddagger}§||

From the {ddagger}INSERM U665, Paris, F-75015, §Institut National de la Transfusion Sanguine, Paris, F-75015, France, and University Paris 7, Paris, F-75006, France

Received for publication, March 25, 2005 , and in revised form, June 10, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Lutheran (Lu) blood group and basal cell adhesion molecule (B-CAM) antigens reside on two glycoprotein (gp) isoforms Lu and Lu(v13) that belong to the Ig superfamily and differ only by the size of their cytoplasmic tail. Lu/B-CAM gps have been recognized as laminin {alpha}5 receptors on red blood cells and epithelial cells in multiple tissues. It has been shown that sickle red cells exhibit enhanced adhesion to laminin {alpha}5 when intracellular cAMP is up-regulated by physiological stimuli such as epinephrine and that this signaling pathway is protein kinase A- and Lu/B-CAM-dependent. In this study, we analyzed the relationship between the phosphorylation status of Lu/B-CAM gps and their adhesion function to laminin {alpha}5. We showed that Lu isoform was phosphorylated in sickle red cells as well as in erythroleukemic K562 and epithelial Madin-Darby canine kidney cells and that this phosphorylation is enhanced by different stimuli of the PKA pathway. Lu gp is phosphorylated by glycogen synthase kinase 3 {beta}, casein kinase II, and PKA at serines 596, 598, and 621, respectively. Alanine substitutions of serines 596 and 598 abolished phosphorylation by glycogen synthase kinase 3 {beta} and casein kinase II, respectively, but had no effect on adhesion of K562 cells to laminin under flow conditions. Conversely, mutation of serine 621 prevented phosphorylation by PKA and dramatically reduced cell adhesion. Furthermore, stimulation of K562 cells by epinephrine increased Lu gp phosphorylation by PKA and enhanced adhesion to laminin. It is postulated that modulation of the phosphorylation state of Lu gp might be a critical factor for the sickle red cells adhesiveness to laminin {alpha}5 in sickle cell disease.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Lutheran (Lu)1 blood group and basal cell adhesion molecule (B-CAM) antigens are both carried by two glycoprotein (gp) isoforms, Lu and Lu(v13), that belong to the Ig superfamily and differ only by the size of their cytoplasmic tail (59 versus 19 amino acids). They interact with the erythroid skeleton through spectrin binding of their common cytoplasmic domain (1). In contrast to Lu(v13), the Lu gp isoform contains a specific dileucine motif responsible for its basolateral targeting in epithelial cells (2) as well as potential phosphorylation sites consistent with a receptor signaling function.

The Lu/B-CAM gps represent the unique red cell receptors for laminin {alpha}5 in normal and in sickle red blood cells (3–5). Both Lu and Lu(v13) isoforms bind to soluble and immobilized laminin {alpha}5 (3, 4, 6). Laminins are heterotrimeric proteins composed of {alpha}, {beta}, and {gamma} chains that associate to form at least 15 heterotrimer proteins that are found in all basement membranes (7–9). Cell adhesion to laminin plays a critical role in proliferation, differentiation, and motility and in the progression of malignant tumors through interaction and activation of specific cell surface receptors (10–12). Laminins {alpha}5 ({alpha}5{beta}1{gamma}1 and {alpha}5{beta}2{gamma}1), which contain the {alpha}5 chain, are strong adhesive components for epithelial cells. The major integrin receptors of the laminin {alpha}5 are integrins {alpha}3{beta}1 (13), {alpha}6{beta}1 (14), {alpha}6{beta}4 (15), {alpha}v{beta}3 (16), and {alpha}2{beta}1 (17, 18). Although these integrins are the coreceptors of several laminins, Lu gps bind only to laminin {alpha}5 isoform. Lu/B-CAM gps also act as functional laminin {alpha}5 coreceptors with integrin {alpha}3{beta}1 in renal epithelial and in smooth muscle cells (11, 19, 20).

Mice lacking laminin {alpha}5 chain die during midgestation with multiple morphological abnormalities of several tissues and their compartments (21). In bone marrow, laminin {alpha}5 chain is an adhesive substrate to stem cells and progenitor cells and influences progenitor cell migration in vitro (22). In sickle red blood cells, adhesion to thrombospondin, fibronectin, and laminin in the vasculature may dramatically impact vaso-occlusion events (for review, see Ref. 23). Overexpression of Lu/B-CAM antigens on sickle red cells correlates with an increased adhesion to laminin {alpha}5 (5, 24) and therefore might participate in the reinforced adhesion of sickle red cells to vascular endothelium. It was recently shown that the physiologic stress mediator epinephrine, acting through the {beta}2-adrenergic receptor, increased the adhesion of sickle red blood cells to laminin {alpha}5 via a cAMP and PKA-dependent signaling pathway (25). The authors proposed a classical cascade in which the {beta}2-adrenergic receptor stimulates G{alpha}s proteins that in turn activate adenylyl cyclase, thus elevating the cAMP levels and leading to PKA activation. Lu/B-CAM gps were identified as receptors that mediate the stimulated adhesion of sickle red cells to laminin {alpha}5 under continuous flow conditions (26). This signaling pathway may also participate in vaso-occlusion events. However, the authors failed to reveal any target for phosphorylation by PKA that could explain the Lu/B-CAM-mediated cell adhesion to laminin {alpha}5.

In this study, we investigated the phosphorylation status of Lu gps in normal and sickle red cells and in transfected erythroid K562 and epithelial Madin-Darby canine kidney cells (MDCK) cells. We demonstrated that Lu gp but not Lu(v13) isoform is phosphorylated by glycogen synthase kinase 3 {beta} (GSK-3{beta}), casein kinase II (CKII), and PKA, at serines 596, 598, and 621, respectively. The Lu gp phosphorylation is enhanced by forskolin, a major activator of the PKA signaling pathway, in sickle red blood cells and by different stimuli of the PKA-pathway in K562 and MDCK cells. We examined the role of phosphorylation of the Lu gp in regulating the adhesion function to laminin {alpha}5 in K562 cells. We demonstrated that PKA-mediated phosphorylation of the Lu gp at Ser-621 positively regulates the adhesion function of Lu gp under flow conditions.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Primers used in PCR and mutagenesis experiments were from Eurogentec (Seraing, Belgium). The QuikChange site-directed mutagenesis kit was from Stratagene (La Jolla, CA). The pGEX-5X-3 vector, the protein A-Sepharose CL4B beads, and the glutathione-Sepharose 4B beads were purchased from Amersham Biosciences. The Complete protease inhibitor mixture was purchased from Roche Applied Science. Purified human {alpha}5 laminin mixture, epinephrine, butoxamine, and PKA catalytic subunit bovine were purchased from Sigma. CKII, GSK-3{beta}, and br-cAMP were purchased from Calbiochem. Solution of 20% human albumin was from Laboratoire Français du Fractionnement et des Biotechnologies LFB (France).

Antibodies—Monoclonal antibody (mAb) anti-Lub (clone LM342) was a generous gift of Dr R. Fraser (regional Donor Centre, Glasgow, UK). IgGs were purified from cell culture supernatants using the protein A-Sepharose CL-4B method (Amersham Biosciences). Anti-Lu mouse monoclonal antibody, clone F241, was produced in our institute (in collaboration with Dr. D. Blanchard, Etablissement Français du Sang (EFS), Nantes, France), as well as the rabbit polyclonal antibodies, anti-Lu 602 and anti-Lu/B-CAM 603 (INTS, Les Ulis, Courtaboeuf, France).

Lu, Lu(v13), and Lu Mutants Expression Vectors—The PCR-amplified cDNA fragments encoding the C-terminal ends of the Lu gp (residues 569–628, starting from the ATG codon) or Lu(v13) (residues 569–588) were fused with the DNA coding for the GST protein in the pGEX-5X-3 plasmid. Mutants LuSS596–598AA and LuS621A were obtained by in vitro mutagenesis, using the QuikChange site mutagenesis kit, according to the supplier's instructions. Primers used for mutations S621A and SS596–598AA are already described (1). Lub and Lub(v13) cDNAs were subcloned in the pcDNA3 expression vector (Invitrogen) as described (2, 27). Mutations were introduced into Lub-pcDNA3 using the QuikChange site mutagenesis kit. For the SSS596–598-621AAA mutant, Lub SS596–598AA-pcDNA3 mutant was used as template using the same primers as those for mutation S621A. The inserts were sequenced using the ABI-PRISM 310 genetic analyzer (Applied Biosystems).

Cell Culture, Transfection, and Flow Cytometry—Human erythroleukemic K562 cells and MDCK cells were obtained from the American Type Culture Collection. K562 cells were grown in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal calf serum (Dutscher, Brumath, France). MDCK cells were grown in Dulbecco's minimum essential medium (Invitrogen) containing 2 mM GlutaMAX (Invitrogen), 0.1 mM non-essential amino acids, and 1.0 mM sodium pyruvate supplemented with 10% fetal calf serum. Stable K562 and MDCK cells expressing Lu mutants were obtained after transfection of wild-type K562 and MDCK cells with recombinant Lub or Lub(v13) cDNAs cloned in pcDNA3 expression vector using Lipofectin reagent (Invitrogen) as described previously (5, 28). Transfected cells were selected in the presence of 0.8 g/liter Geneticin for K562 cells and 0.6 g/liter Geneticin for MDCK cells. Lub-positive cells were detected by flow cytometry using the anti-Lub mAb LM342. Cells expressing the recombinant proteins were then selected using anti-Lub LM342 followed by anti-mouse IgG-coated beads (Dynabeads-M-450, Dynal A.S., Oslo, Norway). Expression of Lub antigens on transfected cell lines was measured using a FACScan flow cytometer (BD Biosciences) and LM342 mAb as described (27).

In Vitro Phosphorylation Assays—The GST fusion proteins expressed in Escherichia coli BL21 were purified by elution from glutathione-Sepharose 4B beads (150 mM NaCl, 50 mM Tris-HCl, pH 8, 20 mM glutathione) and quantified by absorption at 280 nm. For in vitro phosphorylation assay, 5 µg of GST-Lu fusion protein bound to glutathione-Sepharose 4B beads were mixed with purified kinases (GSK-3{beta}, CKII) in the reaction buffer: 20 mM Tris-HCl, pH 7.4, 20 mM MgCl2, 10 mM dithiothreitol, and 1 µM okadaic acid and incubated with 10 µCi of [{gamma}-32P]ATP (200 µM) 30 min at 30 °C. Reaction products were washed six times with the reaction buffer and then resuspended and boiled 5 min in 1x Laemmli buffer. Samples were subjected to 12.5% SDS-PAGE gel and transferred to nitrocellulose membrane. Bands corresponding to phosphorylated GST-Lu were visualized by autoradiography and were quantified by KODAK ID Image analysis software.

Phosphorylation Assays in Transfected Cells—MDCK or K562 cells were washed twice with ice-cold phosphate-buffered saline and then incubated with 200 µCi of orthophosphate 32P (17 mM) for 2 h at 37 °C in the presence of phosphatase inhibitor (1 µM okadaic acid) and Complete protease inhibitor mixture. Cells were treated or not with 5 mM br-cAMP for 30 min, 200 µM forskolin for 20 min, or 200 µM epinephrine for 1 min at 37 °C.

Erythrocyte Phosphorylation Assays—Phospholabeling of red blood cells was performed on normal and sickle red blood cells, as described by Brunati et al. (29). 400 µl of erythrocytes were preincubated in 3.6 ml of buffer A' (150 mM NaCl; 20 mM Tris-HCl, pH 7.5; 10 mM KCl; 1 mM MgCl2; 100 µg/ml streptomycin; 25 µg/ml chloramphenicol) for 4 h at 35 °C to deplete endogenous ATP stores. The cells were then centrifuged 750 x g for 3 min and resuspended in 2.1 ml of buffer A' containing 300 µCi of orthophosphate 32P (25 mM) for 14 h at 35 °C. Cells were then incubated with 200 µM forskolin 20 min at 35 °C and in the presence of 1 µM okadaic acid (phosphatase inhibitor).

Anti-Lu/B-CAM Immunoprecipitation—Cells were lysed for 1 h at 4 °C in Triton lysis buffer A for MDCK and K562 cells (150 mM NaCl, 20 mM Tris-HCl, pH 8, 5 mM EDTA) and Triton lysis buffer A' for red blood cells (150 mM NaCl; 20 mM Tris-HCl, pH 7.5; 10 mM KCl; 1 mM MgCl2), both containing 1% Triton X-100, 0.2% bovine serum albumin, and phosphatase and protease inhibitors. Lysates were centrifuged at 15,000 rpm for 15 min at 4 °C. Aliquots of lysates were mixed with 1x Laemmli buffer and boiled 5 min before loading on 8% SDS-PAGE gels. For Lu gp immunoprecipitation, lysates were submitted to preclearing by incubation with protein A-Sepharose CL4B beads for 2 h at 4 °C. Supernatants were incubated with F241 anti-Lu mAb and protein A-Sepharose CL4B beads overnight at 4 °C. For MDCK and K562 cells, the beads were then washed three times with Triton lysis buffer A, two times with Triton buffer B (500 mM NaCl, 20 mM Tris-HCl, pH 8, 0.5% Triton, 0.2% bovine serum albumin), and once with 50 mM Tris-HCl, pH 8, and for red blood cells, with buffer A' containing 0.1% Triton X-100. Immunocomplexes were resuspended and boiled in 1x Laemmli buffer. Samples were loaded on 8% SDS-PAGE gels and transferred to nitrocellulose membranes. Radioactive proteins were visualized by autoradiography and quantified by KODAK ID Image analysis software.

Electrophoresis and Western Blot Analysis—The SDS-PAGE was performed using 8 and 12.5% polyacrylamide gels according to Laemmli (53). Gels were stained with Coomassie Blue and/or Western blots were performed on nitrocellulose membranes. For immunoblots, membranes were incubated with the rabbit 602 and/or 603 antibodies and detected using the enhanced chemiluminescence (ECL) system according to the manufacturer's instructions (Amersham Biosciences).

Flow Adhesion Assays—K562 cell adhesion to laminin {alpha}5 was measured under physiologic flow conditions using a plate flow chamber as described (30, 31). 3.5 µg of the purified human {alpha}5 laminin mixture (or purified human merosine) were immobilized on clean glass microslides (Camlab, Cambridge, UK) of 3.5 cm2 by incubating overnight at 4 °C. Then, microslides were mounted on a microscope stage and viewed by phase-contrast videomicroscopy. One end of the microslide was attached to Harvard syringe pump, allowing the control of the flow rate through the microslide. The other end of the microslide was attached to a microelectronic valve (Lee Products Ltd., Gerrards Cross, UK), permitting smooth switching between K562 cells suspension and cell-free 0.5% human albumin Hanks' buffer. Following insertion of the microslide into the flow system, 1 ml of K562 cells (3.106/ml) expressing various Lu/B-CAM gps in albumin/Hanks' buffer were flowed across the immobilized laminin for 10 min at shear stress of 0.2 dyne/cm2. Then, adherent cells were washed for 5 min using a flow rate of 0.054, 0.081, 0.108, 0.27, 0.54, 0.81, and 1.08 ml/min, producing shear stresses of 0.2, 0.3, 0.4, 1, 2, 3, and 4 dyne/cm2, successively. After each wash, adherent cells were quantified in six representative areas along the centerline of the microslide by microscopy (x10) using a computerized image analysis system (Optimas 6.1). The counted cells were then averaged and presented as adherent cells per mm2. For cell activation or inhibition experiments, 1 ml of K562 cells (3.106/ml) were preincubated with 20 nM epinephrine for 1 min at 37 °C or 160 µM butoxamine for 20 min at 37 °C, centrifuged, and then flowed across the immobilized laminin {alpha}5.



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  		FIG. 1.
Phosphorylation of the Lu gp is stimulated by forskolin in sickle red blood cells. A, amino acid sequence of Lu and Lu(v13) cytoplasmic domains. Black bars indicate consensus motifs potentially recognized by GSK-3{beta}, CKII and PKA. Arrows point to potentially phosphorylated Ser residues (596, 598, and 621). B, Lu but not Lu(v13) is phosphorylated in sickle red blood cells. Normal and sickle red blood cells were radiolabeled with orthophosphate 32P. 32P-radiolabeled intact normal and sickle red blood cells were incubated in the absence (lanes 1, 2, 4, and 5) or in the presence (lanes 3 and 6) of 200 µM forskolin 20 min at 35 °C. Lu/B-CAM gps were immunoprecipitated, and phosphorylation was analyzed by autoradiography and quantified by KODAK ID Image analysis software. Lysates of normal or sickle red blood cells incubated without the F241 anti-Lu/B-CAM mAb overnight (lanes 1 and 4) represent the negative control. Western blots performed with 603 anti-Lu/B-CAM rabbit polyclonal antibody showed equal amounts of Lu gp immunoprecipitated from normal and sickle red blood cells in the presence or absence of forskolin.

 
Statistical Analysis—A Student's t test was used to assess the statistical significance between adhesion of each cell line or adhesion of treated versus untreated cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Phosphorylation of the Lu gp Is Stimulated by Forskolin in Sickle Red Blood Cells—The amino acid sequence of the cytoplasmic domain of Lu gp isoform but not of Lu(v13) contains three consensus serine phosphorylation sites for protein kinases GSK-3{beta}, CKII, and PKA at positions 596, 598, and 621, respectively (Fig. 1A). Consensus sequences for these kinases are (S/T)XXX(S/T), SXX(E/D), RX1–2(S/T)X, respectively (Phosphobase, NetPhos). To determine whether Lu/B-CAM gps are phosphorylated in normal and sickle red blood cells, cells were labeled with orthophosphate 32P, and Lu/B-CAM gps were immunoprecipitated with the F241 monoclonal anti-Lu antibody, which recognizes both Lu and Lu(v13) isoforms. Lu gp isoform (85 kDa), but not Lu(v13), exhibited a weak level of phosphorylation in both normal and sickle red blood cells (Fig. 1B, lanes 2 and 5). These results suggested that Lu gp is phosphorylated in vivo, presumably by either PKA and/or CKII and/or GSK-3{beta} kinases. To define whether the cytoplasmic domain of Lu gp undergoes PKA-dependent phosphorylation in response to stimulation, Lu gp phosphorylation was tested in the presence of 200 µM forskolin, an activator of adenylyl cyclase known to stimulate Lu gp adhesion to laminin {alpha}5 on sickle red blood cells through a cAMP-dependent PKA pathway (25). Stimulation of sickle red blood cells with forskolin resulted in a 2.8-fold increase of Lu phosphorylation (Fig. 1B, lanes 3 and 6), whereas no significant increase in phosphorylation was observed in normal red cells. The different levels of phosphorylation observed in sickle red blood cells before and after treatment with forskolin were significant as similar amounts of Lu gp were immunoprecipitated when tested by Western blot with anti-Lu antibody (Fig. 1B, bottom). These results demonstrated that Lu gp on sickle red blood cells undergoes forskolin-stimulated phosphorylation, presumably by a PKA-dependent pathway.

PKA Phosphorylates Lu gp at Serine 621—To determine whether Lu gp isoform is a substrate of PKA and which serines are involved, phosphorylation experiments were performed in both erythroid K562 and epithelial kidney MDCK cells stably transfected by the pcDNA3-Lu or pcDNA3-Lu(v13) vectors. K562 and MDCK cells were labeled with orthophosphate 32P with or without treatment by br-cAMP, a stable, membrane-permeable cAMP analog that activates PKA. Lu/B-CAM gps were then immunoprecipitated as described under "Experimental Procedures." The Lu gp isoform (85 kDa) (Fig. 2A, lanes 1 and 7) but not Lu(v13) (Fig. 2A, lanes 5 and 11) was phosphorylated in K562 and MDCK transfected cells. As shown in Fig. 2A (lanes 1 and 2 and lanes 7 and 8), phosphorylation of Lu gp was increased following treatment with br-cAMP (2- and 2.1-fold), indicating that Lu gp might be phosphorylated by PKA in these cells. As expected, Lu(v13) was not phosphorylated after stimulation by br-cAMP in both cell lines (Fig. 2A, lanes 5 and 6 and lanes 11 and 12). Treatment with epinephrine or forskolin, two major activators of adenylyl cyclase, also increased phosphorylation of Lu gp in both cell lines (Fig. 2A, lanes 1, 3, and 4 and lanes 7, 9, and 10). To gain more insights into the role of PKA in Lu gp phosphorylation, we generated Lu mutants S621A and SS596–598AA impaired for the PKA and the clustered CKII/GSK-3{beta} consensus phosphorylation sites, respectively (Fig. 1A). As shown in Fig. 2B (lanes 5 and 6), SS596–598AA mutant maintains enhanced phosphorylation after treatment of K562 and MDCK cells with br-cAMP (1.4- and 1.85-fold, respectively), whereas no significant stimulation was observed for the S621A mutant in both cell types (Fig. 2B, lanes 3 and 4). Differences observed in the level of phosphorylation before and after treatment with br-cAMP were significant as similar quantities of Lu gp were immunoprecipitated in each cell line when tested by Western blot with an anti-Lu antibody (Fig. 2, A and B, bottom). These results indicated that PKA phosphorylates Lu gp at Ser-621 in cellular models.



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  		FIG. 2.
The Lu gp isoform is phosphorylated by PKA in recombinant erythroid K562 and epithelial renal MDCK cells. 32P-radiolabeled MDCK and K562 cells expressing Lu gp (A, lanes 1–4 and lanes 7–10 and B, lanes 1 and 2), Lu(v13) gp (A, lanes 5 and 6 and lanes 11 and 12), or LuS621A, LuSS596–598AA, and LuSSS596–598-621AAA mutants (B, lanes 3 and 4, lanes 5 and 6, and lanes 7 and 8) were incubated at 37 °C in the absence or presence of 200 µM forskolin for 20 min (A, lanes 3 and 9), 200 µM epinephrine for 1 min (A, lanes 4 and 10), and 5 mM br-cAMP (a specific PKA activator) for 20 min (A, lanes 2 and 8; B, lanes 2, 4, 6, and 8). Lu gp phosphorylation was analyzed by autoradiography after immunoprecipitation and was quantified by KODAK ID Image analysis software. The phosphorylation stimulation factor represents the level of activation of Lu gp phosphorylation in the presence of br-cAMP, forskolin, or epinephrine. Western blots performed with 603 anti-Lu/B-CAM (A) or 602 anti-Lu (B) rabbit polyclonal antibodies showed equal amounts of Lu gp immunoprecipitated from each cell line in the presence or absence of br-cAMP, forskolin, or epinephrine.

 
GSK-3{beta} and CKII Phosphorylate Lu gp at Serines 596 and 598 —We also examined the phosphorylation of Lu gp by CKII and GSK-3{beta} in K562 and MDCK cells. As shown in Fig. 2B, S621A (lane 3) as well as SS596–598AA (lane 5) mutants remained phosphorylated in the absence of additional stimuli, in both cell types. In addition, phosphorylation was abolished for the triple mutant SSS596–598-621AAA (lanes 7 and 8), indicating that CKII and/or GSK-3{beta} and PKA sites were functional and that no additional phosphorylation sites are involved. Altogether, these results indicated that Ser-596 and/or Ser-598 may be used by GSK-3{beta} and/or CKII.

Since GSK-3{beta} and CKII sites are overlapping and neither specific activators nor inhibitors of these enzymes could be used in our cellular models, we also performed phosphorylation reactions using recombinant fusion proteins GST-Lu, GST-Lu(v13), GST-Lu mutants, [{gamma}-32P]ATP, and purified kinases. As shown in Fig. 3A, the cytoplasmic domain of Lu gp (GST-Lu) is clearly a substrate of CKII in contrast to GST-Lu(v13) or GST alone as only GST-Lu is phosphorylated by this enzyme. It is known that GSK-3{beta} requires a primed phosphopeptide for optimal activity with some substrates, i.e. a sequence that carries a phosphate group in the vicinity of the consensus motif (32). As shown in Fig. 3A, phosphorylation of Lu gp by GSK-3{beta} requires the prephosphorylation of Lu gp by CKII since it cannot phosphorylate the cytoplasmic domain of Lu gp when incubated alone with GST-Lu. When serines 596 and 598 were mutated to alanine, no phosphorylation by CKII and GSK-3{beta} was observed, whereas the GST-LuS621A mutant remained phosphorylated (Fig. 3A). Coomassie Blue staining confirmed that equal amounts of GST fusion proteins were used in all assays (Fig. 3B). Together with the experiments performed in cells, these results indicated that the Lu gp isoform is a substrate of CKII and GSK-3{beta} at serines 596 and 598.

Effects of Ser to Ala Substitution on Cell Adhesion to Laminin {alpha}5 under Flow Conditions—We investigated the consequences of phosphorylation of Lu gp on cell adhesion to laminin {alpha}5 under flow conditions. We used erythroid K562 recombinant cells expressing Lu gp (K562-Lu) rather than epithelial MDCK cells as a cellular model since it was previously reported that Lu gp mediates adhesion of sickle red blood cells to laminin {alpha}5 and that epinephrine increased this adhesion (by 1.5–2.5-fold) (25). As control, we also examined adhesion of wild-type K562 (K562-WT) labeled with calcein on the same {alpha}5 laminin mixture coated slide. In this assay, a non-fluorescent form of calcein that diffuses into cells is cleaved by intracellular esterases, producing the highly fluorescent calcein and inhibiting its extrusion ("Experimental Procedures"). K562-Lu and fluorescent K562-WT cells were injected together in the flow at 0.2 dyne/cm2. Very few K562-WT cells adhered to laminin {alpha}5 at shear stresses from 0.2 to 3 dyne/cm2, in contrast to K562-Lu cells (Fig. 4A). Although Lu(v13) gp is not phosphorylated (Fig. 2A), an identical level of adhesion was observed for K562-Lu(v13) as compared with K562-Lu (data not shown). Neither Lu/B-CAM gps nor integrins {alpha}2{beta}1, {alpha}3{beta}1, {alpha}6{beta}1, and {alpha}6{beta}4, which represent laminin {alpha}5 receptors, are expressed endogenously in K562-WT cells (data not shown), indicating that only Lu or Lu(v13) gps expressed in K562 cells are responsible for the observed cell adhesion to laminin {alpha}5. We therefore examined whether the S621A and SS596–598AA mutants of Lu gp, which impair PKA and CKII/GSK-3{beta} phosphorylation sites, respectively, affect adhesion of K562 cells under flow conditions. K562-Lu and K562-LuSS596–598AA cells exhibited the same number of adherent cells at shear stresses of 3 dyne/cm2 (295 versus 308 adherent cells/mm2, p < 0.01), whereas the S621A mutation led to a decrease of adhesion by 200–300-fold (16 adherent cells/mm2) as shown for K562-LuS621A cells (Fig. 4B). To evaluate the effect of this latter mutation on adhesion at physiologic shear stress, K562-LuS621A cells were injected at 0.2 dyne/cm2 and then exposed to increasing shear rates starting from 0.4 dyne/cm2 (Fig. 4C). Approximately 90% of K562-LuS621A mutant cells were detached at 2 dyne/cm2 as compared with 50% of K562-Lu and K562-LuS596–598AA cells. Reduced adhesion of K562-LuS621A cells was not due to lower levels of cell surface Lu proteins since K562-Lu, K562-LuS621A, and K562-LuSS596–598AA cells expressed 67,000, 60,000, and 74,000 Lu recombinant proteins/cell, respectively, as determined by flow cytometry. Thus, adhesion of K562-Lu cells to laminin under flow conditions is correlated with Ser-621 but not Ser-596–598 phosphorylation of the Lu gp isoform.

Epinephrine Stimulates Adhesion of K562-Lu but Not K562-Lu(v13) to Laminin {alpha}5 under Flow Conditions—Since Lu gp exhibited enhanced phosphorylation in K562 cells when treated with br-cAMP, forskolin, or epinephrine, we examined the effects of epinephrine on K562 cell adhesion to laminin {alpha}5 as it is the major mediator of the physiologic stress response known to elevate cAMP levels (33). When treated with 20 nM epinephrine during 1 min, adhesion of K562-Lu (177 untreated and 535 treated adherent cells/mm2) but not K562-Lu(v13) cells (1229 untreated and 1026 treated adherent cells/mm2) was increased by 2-fold as compared with untreated cells even at shear stress of 4 dyne/cm2 (Fig. 5A). This result indicated that raising cAMP levels by epinephrine in K562 cells, which enhances phosphorylation of Lu isoform, increased adhesion of Lu gp but not Lu(v13) to laminin {alpha}5. Differences observed in adhesion before and after treatment with epinephrine were significant as K562-Lu and K562-Lu(v13) cells exhibited similar expression levels of recombinant proteins at the cell surface (67,000 and 75,000/cell, respectively).



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  		FIG. 3.
GSK-3{beta} and CKII phosphorylate Lu gp at serines 596 and 598. A, purified recombinant GST-Lu, GST-Lu(v13), GST-LuSS596–598AA, GST-LuS621A, and GST alone as control were phosphorylated in the presence of [{gamma}-32P]ATP by purified CKII or GSK-3{beta} or by both. GST-Lu phosphorylation was analyzed by autoradiography (32P). B, Coomassie Blue staining of the different GST-Lu constructs.

 
Previous studies indicated that the physiologic stress mediator epinephrine acts largely through the {beta}2-adrenergic receptor on sickle red blood cells (33–37). Therefore, we tested whether the {beta}2-selective antagonist butoxamine could inhibit epinephrine-stimulated K562-Lu adhesion to laminin. Fig. 5B shows that butoxamine suppresses the effect of epinephrine as adhesion of K562-Lu (100% of adherent cells/mm2) is no more enhanced in the presence of epinephrine when butoxamine is added (93.65%, p < 0.01). This result was consistent with epinephrine acting primarily via the {beta}2-adrenergic receptor.

Epinephrine Stimulates Adhesion of K562-LuSS596–598AA but Not K562-LuS621A Cells to Laminin {alpha}5—Since mutation of serine 621 abolished enhanced phosphorylation of Lu gp in br-cAMP-stimulated K562 cells (Fig. 2B) and decreased K562-Lu cell adhesion to laminin {alpha}5 under flow conditions (Fig. 4, B and C), we investigated adhesion of K562-LuSS596–598AA and K562-LuS621A cells to laminin {alpha}5 when treated with epinephrine. Adhesion to laminin {alpha}5 of K562-LuSS596–598AA cells (Fig. 6A) was increased by 3-fold following treatment with epinephrine (1000 adherent cells/mm2) at shear stress of 3 dyne/cm2 as compared with untreated cells (308 adherent cells/mm2). It is noteworthy that the level of increase is comparable with that of K562-Lu cells (295 untreated and 839 treated adherent cells/mm2) (Fig. 6). In contrast, this treatment had no effect on adhesion of K562-LuS621A cells (16 untreated and 12 treated adherent cells/mm2) (p < 0.01). This result was confirmed at different shear stresses from 0.4 to 3 dyne/cm2 (Fig. 6B). This indicated that PKA activation by stimulation of transfected K562 cells with epinephrine positively regulates cell adhesion to laminin {alpha}5 and that this activation is mediated specifically by Ser-621 of the Lu gp isoform.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrated that the cytoplasmic domain of the Lu gp, but not of Lu(v13), is weakly phosphorylated in both normal and sickle red blood cells. However, we showed that the phosphorylation of Lu gp was enhanced by forskolin in sickle red blood cells but not in normal red blood cells, implicating a cAMP-dependent PKA phosphorylation mechanism. Phosphorylation of Lu gp was further investigated in recombinant K562 and MDCK cells in which phosphorylation stimulation by epinephrine, forskolin, and br-cAMP was also observed.

Together, in vitro and ex vivo phosphorylation assays, as well as site-directed mutagenesis, revealed that GSK-3{beta}, CKII and PKA kinases phosphorylate Lu gp on serines 596, 598, and 621, respectively. We have examined the biological effects of Lu gp phosphorylation in regulating adhesion of K562-Lu cells to laminin {alpha}5. Erythroleukemic K562 cells represent immature erythroid cells and provide a good model for studying phosphorylation of Lu gp and adhesion to laminin {alpha}5 since these non-adherent cells contain all components of the cAMP/PKA-dependent signaling pathway described in epinephrine-stimulated sickle red blood cell adhesion (25, 38, 39). We showed that phosphorylation of LuSS596–598AA, in which the CKII and GSK-3{beta} phosphorylation consensus sites are mutated, but not of the LuS621A mutant, in which the PKA consensus site is disrupted, remained activated by br-cAMP. It is noteworthy that the stimulation factor of Lu gp phosphorylation after cell treatment by epinephrine is weaker than the one after treatment by br-cAMP or forskolin. This can be due to the fact that incubation time of epinephrine is very short (1 min). Furthermore, stimulation of K562-Lu with epinephrine positively regulates cell adhesion to laminin {alpha}5 under flow conditions. This is mediated specifically by Ser-621 phosphorylation since LuSS596–598AA, but not LuS621A mutant, exhibited increased adhesion to laminin {alpha}5.

It was previously shown that adhesion of sickle red blood cells to vasculature can be modulated by signaling events (40). Indeed, in the presence of physiological shear stress, integrin-associated protein (CD47) specifically activates sickle red blood cell adhesion to immobilized thrombospondin via a Gi and tyrosine-kinase-dependent pathway. Moreover, Hines et al. (25) demonstrated that sickle red blood cells from about 46% of patients could be stimulated by epinephrine via a cAMP-dependent activating pathway, leading to an increase of Lu/B-CAM adhesion to laminin {alpha}5. However, these authors failed to demonstrate Lu/B-CAM gps phosphorylation by PKA and suggested that an unidentified protein may act as an intermediate between PKA and Lu/BCAM receptor. In contrast, we have clearly identified the missing link by showing in the present report that Lu gp is phosphorylated by PKA on Ser-621 in sickle red cells and recombinant cell lines and that this phosphorylation regulates K562-Lu cell adhesion to laminin. It is noteworthy that forskolin treatment results in similar stimulation factor of Lu gp phosphorylation (our results) and of adhesion of sickle red blood cells to laminin (25). Interestingly, phosphorylation of LW/ICAM-4 by PKA has been recently shown to be an additional mechanism that activates sickle cell adhesion to endothelium through interaction with the endothelial {alpha}v{beta}3 integrin (41). Our current results, together with those from others (25), support the working hypothesis that the phosphorylation of Lu gp may represent one critical factor that modulates the adhesiveness of sickle red blood cells to laminin in the vasculature of sickle cell patients. Indeed, laminin {alpha}5 is present in endothelial basement membranes and is thus buried beneath endothelial cells that line the vascular wall. However, laminin together with thrombospondin and fibronectin is accessible to circulating blood cells in pathological conditions in which the endothelium is damaged (42). Thus, adhesion of red cells to laminin in the vasculature may dramatically impact the vaso-occlusion events that occur in the sickle cell disease (for review, see Telen (Ref. 23)).



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  		FIG. 4.
Effects of Ser to Ala substitution on cell adhesion to laminin {alpha}5 under flow conditions. A, K562-Lu but not K562-WT cells adhere to laminin {alpha}5 under flow conditions. 3 x 106/ml K562-WT cells labeled with calcein and unlabeled K562-Lu cells were injected together into a microslide coated with laminin {alpha}5. Adherent cells were washed using increasing flow rates producing shear stresses of 0.2–4 dyne/cm2. Photos show adherent cells at shear stress of 0.2, 0.3, 1, and 3 dyne/cm2. Arrows point to the few fluorescent K562-WT cells. Similar results were obtained with K562-Lu(v13) cells (not shown). B and C, S621A substitution decreases K562-Lu cell adhesion. K562-Lu ({circ}), -LuSS596–598AA ({triangleup}), and -LuS621A ({diamond}) cells were allowed to flow over immobilized laminin {alpha}5. Data shown represent adherent cells at the physiologic shear stress of 3 dyne/cm2 (B) and after exposure to increasing shear stress of 0.4–3 dyne/cm2 (C). Each result represents a mean of three experiments. Lu gp and Lu mutant expression levels were similar in all cell lines as measured by flow cytometry.

 
It was previously demonstrated that Lu and Lu(v13) molecules expressed in transfected cells bind to soluble and immobilized laminin {alpha}5 equally well and that these proteins both mediate strong adhesion to immobilized laminin {alpha}5 in flow adhesion assay (5, 43). Accordingly, we showed that both Lu and Lu(v13) gps strongly mediate laminin {alpha}5 binding under flow conditions but that more K562-Lu(v13) cells adhere to laminin as compared with K562-Lu cells (by 1.5–2-fold at shear stresses of 3 and 4 dyne/cm2). Furthermore, we demonstrated that Lu(v13) isoform is not phosphorylated and that stimulation by epinephrine has no effect on K562-Lu(v13) cell adhesion. We suggest that serine 621 phosphorylation may induce conformational changes of Lu gp, thus increasing its affinity for laminin {alpha}5. In contrast, Lu(v13) isoform might be in an activated conformation and constitutively adheres to laminin {alpha}5. Furthermore, Lu(v13) is not the major isoform in normal and sickle red blood cells since its expression is very low as compared with Lu gp isoform (Fig. 1B). Indeed, Lu and Lu(v13) expression was quantified on normal and sickle red cells membranes from 8 and 10 individuals, respectively, by Western blot, and we found that Lu gp expression is 5–10-fold greater than Lu(v13) gp expression.2



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  		FIG. 5.
Effects of epinephrine (epi) and butoxamine (butox) on adhesion of K562-Lu and K562-Lu(v13) cells to laminin {alpha}5 under flow. A, effects of epinephrine. Adhesion of K562-Lu ({circ}) and K562-Lu(v13) ({square}) cells to immobilized laminin {alpha}5 was measured using the flow adhesion assay after treatment or not for 1 min at 37 °C with 20 nM epinephrine (• and {blacksquare}). Adherent cells per mm2 are shown after exposure to increasing shear stress of 1–4 dyne/cm2. Each result represents a mean of three experiments. Lu and Lu(v13) expression levels were similar as measured by flow cytometry. B, effects of butoxamine. Inhibition of epinephrine-stimulated adhesion of K562-Lu cells to immobilized laminin {alpha}5 was measured in the flow adhesion assay. K562-Lu cells were injected in the flow following epinephrine-stimulation (20 nM epinephrine for 1 min) or in the presence of the {beta}2-selective antagonist butoxamine (160 µM for 30 min) followed by epinephrine-stimulation (20 nM epinephrine for 1 min). The histogram represents the percentage of adherent K562-Lu cells treated with epinephrine (+ epi), butoxamine and epinephrine (+ butox + epi), and butoxamine alone (+ butox), at shear stress of 1 dyne/cm2, as compared with the 100% of adherent untreated K562-Lu cells. Each result represents a mean of three experiments.

 

Recently, Murphy et al. (44) have demonstrated that cAMP signaling can promote sickle red blood cell adhesion to laminin {alpha}5 via Lu/B-CAM through two signaling pathways, a PKA- or an Epac/Rap1-dependent pathway. Epac is a widely expressed exchange factor for the small GTPases Rap1 and Rap2 and represents a receptor for cAMP (45). Rap1 represents a molecular switch that cycles between an inactive GDP- and active GTP-bound conformation and promotes the activation of integrin adhesion receptor, thus leading to cellular adhesion (45). Although both Epac and Rap1 are endogenously expressed in K562 cells (45, 46), we suggest that mainly the PKA-dependent pathway is required for stimulated K562-Lu cell adhesion since substitution of serine 621 into alanine completely inhibits epinephrine stimulation of K562-Lu cell adhesion to laminin.



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  		FIG. 6.
Epinephrine stimulates adhesion of K562-LuSS596–598AA but not K562-LuS621A cells to laminin {alpha}5. Untreated K562-Lu ({circ}), K562-LuS621A ({diamond}), and K562-LuSS596–598AA ({triangleup}) cells, or cells treated with 20 nM epinephrine for 1 min (•, {diamondsuit}, and {blacktriangleup}), were allowed to flow over immobilized laminin {alpha}5. A, the histogram represents adherent cells per mm2 at the shear stress of 3 dyne/cm2. B, the curves represent the number of adherent cells per mm2 after exposure to increasing shear stress of 0.4–3 dyne/cm2. Each result represents a mean of three experiments.

 
The Lu/B-CAM antigens are also expressed in epithelial tissues (11, 47). This is consistent with the presence of laminin {alpha}5 in these tissues (48). Our current results demonstrated that Lu gp is phosphorylated by GSK-3{beta}, CKII, and PKA in MDCK epithelial cells. It is well known that inside-out regulation of the receptor adhesive properties is frequently promoted by phosphorylation (49–51). For example, adhesion mediated by integrin {alpha}4{beta}1 results in increased T cell migration via a phosphorylation-mediated interaction with paxillin, a signaling adaptor protein (52). Moreover, it has recently been demonstrated that Lu/B-CAM is functionally involved in skin tumor cell binding and migration through the laminin {alpha}5 containing stroma (47). Since laminin also appears to increase tumor cell adhesion and migration, it will be of interest to further analyze the impact of Lu phosphorylation on these biological events in normal and pathological epithelial cells.

In conclusion, our results supported the view that phosphorylation of the intracytoplasmic domain of Lu gp plays a functional role in PKA-dependent adhesion to laminin {alpha}5. Therefore, modulation of the phosphorylation state of Lu gp might be a critical factor for sickle red blood cell adhesiveness to laminin {alpha}5 in sickle cell disease.


   FOOTNOTES
 
* This work was supported by the Institut National de la Transfusion Sanguine (INTS) and INSERM. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Caroline Le Van Kim, Inserm U665, INTS, 6 rue Alexandre Cabanel, 75015 Paris, France. Tel.: 33-1-44-49-30-46; Fax: 33-1-43-06-50-19; E-mail: levankim{at}idf.inserm.fr.

1 The abbreviations used are: Lu, Lutheran; B-CAM, basal cell adhesion molecule; gp, glycoprotein; PKA, protein kinase A; CKII, casein kinase II; GSK-3{beta}, glycogen synthase kinase 3 {beta}; MDCK, Madin-Darby canine kidney; mAb, monoclonal antibody; br-cAMP, 8-bromo-cAMP; WT, wild-type; GST, glutathione S-transferase. Back

2 E. Gauthier, C. Rahuel, M. P. Wautier, W. El Nemer, P. Gane, J. L. Wautier, J. P. Cartron, Y. Colin, and C. Le Van Kim, unpublished data. Back


   ACKNOWLEDGMENTS
 
We are grateful to Emmanuel Collec for technical assistance, to Dr R. Fraser (Regional Donor Centre, Glasgow, UK) for providing anti-Lu (clone LM342) monoclonal antibody, and to Dr D. Blanchard (EFS, Nantes, France) for providing anti-Lu (clone F241) monoclonal antibody.



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W. El Nemer, M.-P. Wautier, C. Rahuel, P. Gane, P. Hermand, F. Galacteros, J.-L. Wautier, J.-P. Cartron, Y. Colin, and C. Le Van Kim
Endothelial Lu/BCAM glycoproteins are novel ligands for red blood cell {alpha}4{beta}1integrin: role in adhesion of sickle red blood cells to endothelial cells
Blood, April 15, 2007; 109(8): 3544 - 3551.
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