Functional Mapping of the Cytoplasmic Region of Intercellular Adhesion Molecule-3 Reveals Important Roles for Serine Residues*

(Received for publication, May 5, 1997, and in revised form, July 1, 1997)

Joel S. Hayflick Dagger , Johnny Stine , Raymond Fox , Denise Hoekstra and W. Michael Gallatin

From ICOS Corporation, Bothell, Washington 98021

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Intercellular adhesion molecule-3 (ICAM-3), a ligand for beta 2 integrins, elicits a variety of activation responses in lymphocytes. We describe a functional mapping study that focuses on the 37-residue cytoplasmic region of ICAM-3. Carboxyl-terminal truncations delineated portions involved in T cell antigen receptor costimulation, homotypic aggregation, and cellular spreading. Truncation of the membrane distal 25 residues resulted in loss of T cell antigen receptor costimulation as determined by interleukin 2 secretion. Aggregation and cell spreading were sensitive to truncation of the membrane distal and proximal thirds of the cytoplasmic portion. Phosphoamino acid analysis revealed that ICAM-3 from activated cells contained phosphoserine and phosphopeptide mapping identified Ser489 as a site of phosphorylation in vivo. Mutation of Ser489 or Ser515 to alanine blocked interleukin 2 secretion, aggregation and cell spreading, while mutation of other serine residues affected only a subset of functions. Ser489 was a phosphorylation site in vitro for recombinant protein kinase Ctheta . Finally, treatment of Jurkat cells with chelerythrine chloride, a protein kinase C inhibitor, prevented ICAM-3-triggered spreading. This study delineates separable regions and amino acid residues within the cytoplasmic portion of ICAM-3 that are important for T cell function.


INTRODUCTION

ICAM-31 (CD50) is a member of the Ig superfamily sharing sequence and functional attributes with ICAM-1, -2, -4 (Landsteiner-Weiner blood group glycoprotein), and -5 (telencephalin). ICAM-3 binds to LFA-1 (CD11a/CD18) and the newly described integrin alpha d/CD18 (1). It is constitutively expressed at high levels by most hematopoietic cells, leading to the suggestion that it is involved in early activation steps of an inflammatory response (2-4).

ICAM-3 has been functionally characterized with respect to the five Ig-like extracellular domains. The first amino-terminal domain binds LFA-1 via conserved residues also found in ICAM-1 (5). These conserved sequences of ICAM-3 and -1 may bind to distinct sites of the I domain of LFA-1, suggesting that non-conserved domain 1 residues might contribute to integrin binding (6, 7).

Numerous intracellular signaling events have also been observed to be affected by ICAM-3 engagement. Specifically, activation of intracellular calcium flux and stimulation of tyrosine kinase activity possibly via non-receptor tyrosine kinases p56lck and p59fyn were seen (8, 9). ICAM-3 engagement has also been observed to up-regulate beta 1 and beta 2 integrin function, and to trigger phosphorylation of the cyclin-dependent kinase cdc2 (10-12). Little information, however, is available regarding the molecular mechanisms of these phenomena.

Here we report that ICAM-3 engagement initiates several distinct aspects of lymphocyte function, which involve the 37-amino acid cytoplasmic portion. For these analyses, we developed and characterized an ICAM-3-deficient human T-leukemic Jurkat cell line. Using these cells and gene transfer techniques, a functional map of the cytoplasmic region of ICAM-3 with respect to TCR accessory molecule function, homotypic aggregation, and cell spreading was generated. These data pinpoint serine residues, particularly serine 489, as critical for ICAM-3 function.


MATERIALS AND METHODS

Cells and Cell Culture

Jurkat 77 (J77, a gift from Dr. S. Burakoff, Dana Farber Cancer Research Institute, Boston, MA) and the ICAM-3-deficient J77.50.3 cells were maintained in RPMI complete medium (RPMI supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 1 mM sodium pyruvate) in humidified 5% CO2 at 37 °C.

Monoclonal Antibodies

Murine mAb used in this study were as follows. Anti-ICAM-3 (CD50) mAb ICR1.1 (IgG2a), ICR2.1 (IgG1), ICR9.2 (IgG2a), and anti-ICAM-1 (CD54) mAb 18E3D (IgG1) were generated by the ICOS hybridoma facility. Hybridoma lines secreting anti-TCR (CD3e) OKT3 (IgG2a), anti-CD11a TS1/22 (IgG1), anti-CD18 TS1.18 (IgG1), anti-CD45 4B2 (IgG2a), and anti-MHC class I W6/32 (IgG2a) were obtained from American Type Culture Collection, Rockville, MD. These mAb were purified by protein A column chromatography of mouse ascites fluid. Purified mAb were dialyzed against and stored in PBS. Isotype-matched control mAb used were UPC10 (IgG1) and MOPC 21 (IgG2a) (Sigma). Anti-HA mAb 12CA5 (IgG2a) was from Boehringer Mannheim. Fluorescein isothiocyanate-conjugated sheep anti-mouse IgG F(ab')2 was purchased from Sigma.

Development and Characterization of ICAM-3-deficient Jurkat Cells

A variant of Jurkat 77 cells deficient in the expression of ICAM-3 (J77.50.3) was generated by two rounds of indirect staining and cell sorting using a mixture of ICR1.1 and 9.2 mAb (4). J77.50.3 cells were compared with the parental line for surface expression of numerous membrane proteins by indirect cytofluorometry (FACSCAN, Becton-Dickinson, Mountain View, CA) and found to exhibit similar levels for all except ICAM-3.

Ten micrograms of total RNA isolated from parental J77 and J77.50.3 cells was subjected to blotting, hybridization, and washing as described (13). Labeled probes were generated by random priming of the entire ICAM-3 or glyceraldehyde-3-phosphate dehydrogenase cDNA (14).

ICAM-3 Deletion and Point Mutation Constructs

Coding sequences for HA epitope-tagged ICAM-3 proteins were generated as described (15). To engineer epitope-tagged full-length ICAM-3 construct, three separate PCR fragments that encoded 1) the signal sequence (preceded by a unique HindIII site and Kozak sequence), 2) Ig domains (IgD) I-II of ICAM-3, and 3) a triple (3×) influenza hemagglutinin (HA) epitope tag sequence were synthesized and gel-purified (16). The fragments were combined using PCR in the following order: ICAM-3 signal sequence, HA tag, and ICAM-3 IgD I-II. This product was ligated as a HindIII/ScaI fragment with a ScaI/EcoRI cDNA fragment containing the remainder of the ICAM-3 coding sequence into the HindIII/EcoRI sites of expression vector pMH-neo and all PCR products sequenced (17).

Cytoplasmic region deletions were generated as follows. The region of coding sequence for the extracellular domains described above contained on a HindIII/SacI fragment was combined with SacI/EcoRI PCR fragments encoding cytoplasmic domain truncations and ligated to the HindIII/EcoRI sites of pMH-neo. The PCR fragments were synthesized using the following primers: 1) 5' common anchoring primer CATAATGGTACTTATCAGTGC, and 2) 3' primers D505 (-1/3CT), ATATAGCGGCCGCGGATCCTCACTGCATAGACGTGAG; D493 (-2/3CT), ATATAGCGGCCGCGGATCCTCACCTAACATGGTAACT; and D484 (-CT), ATCACTATGCGGCCGCTCAGTGTCTCCTGAAGACGTACAT. Primer D484 contained a change at codon 483 to increase the membrane anchor region of the maximal cytoplasmic region truncation. Amino acid numbering uses the mature amino terminus for the first residue.

To generate point mutations, the ICAM-3 cDNA was subcloned as a NotI/EcoRI fragment into M13 BM21 replicative form DNA (Boehringer Mannheim) and primers used for mutagenesis by the Kunkel method were designed to make alanine changes at the following codons: serine 487, serine 489, leucine 499, serine 496, serine 503, and serine 515 (18). Leucine 499 was chosen as a control for mutational effect, since it is not a potential phosphorylation site and a conservative change to alanine is expected to maintain similar overall charge. All mutants were sequenced, and each was subcloned as a SacI/EcoRI fragment along with the HindIII/SacI fragment described above into pMH-neo.

Expression and Selection in J77.50.3 Cells

J77.50.3 cells (107) in 0.2 ml of PBS were electroporated in the presence of 100 µg/ml plasmid DNA using a BTX 6000 device (126 V, 1710 microfarads, 72 milliamps, 0.2-cm electrode gap). Drug-resistant cells (G418, 1.25 mg/ml) were screened by indirect fluorescence flow cytometry with ICR1.1, OKT3, or isotype-matched control antibody. Two independent cell lines were chosen for functional studies from each transfection based on similar mean channel fluorescence measurements.

Cell Panning

To enrich for cell lines that expressed higher mean ICAM-3 levels, the drug-resistant lines were panned on mAb-coated plastic. Bacteriologic Petri plates were incubated with 8 ml of ICR2.1 (10 µg/ml) in PBS for 2 h at 37 °C. The plates were rinsed with PBS and cells seeded onto the mAb-coated plastic surface for 8 min at 25 °C. To remove non-adherent cells, the plates were rocked and aspirated. The plates were rinsed with PBS, checked visually to determine the absence of non-adherent cells, and adherent cells removed by trituration. Cells harvested in this manner were expanded and subjected to indirect fluorescence analysis using flow cytometry.

ICAM-3/TCR Costimulation Assay

Plates (96-well, Corning 25860) were coated with OKT3 (0.5 µg/ml, 50 µl/well) in PBS for 16 h at 4 °C. The coating was removed and replaced with PBS alone or mAb at 10 µg/ml in PBS and incubated at 37 °C for 2 h. Wells were rinsed twice with PBS and 2 × 105 cells added in 0.25 ml of RPMI/well. Plates were incubated at 37 °C for 16 h. Conditioned medium from duplicate wells was pooled, diluted serially, and assayed for IL2 concentration by enzyme-linked immunosorbent assay (Biosource International, Camarillo, CA). A dose for OKT3 (25 ng/well) and ICR1.1 (500 ng/well) was chosen for co-stimulation experiments. Assays were repeated a minimum of three times, with similar results observed in each experiment.

Cell Spreading Assay

Dishes (Delta T, 0.5-mm glass; Bioptechs Inc., Butler, PA) were coated with 0.5 ml of mAb in PBS (10 µg/ml). Dishes was incubated at 37 °C for 2 h and rinsed twice with PBS. Cells (2 × 104) were seeded onto coated surfaces for 15 min at 37 °C. Plates were held at 37 °C in the Bioptechs stage insert while being photographed with Ilford Pan F film using a Nikon Diaphot microscope and DIC optics. For PKC inhibitor studies, chelerythrine chloride (in Me2SO) was added to cells at a final concentration of 50 µM and incubated at 37 °C for 10 min prior to seeding into coated dishes.

Cell Aggregation Assay

Cells were pelleted and resuspended at 8 × 105/ml in complete medium and 0.25 ml distributed to duplicate wells of a 96-well flat bottom plate. mAb ICR1.1 or MOPC 21 control were added to a final concentration of 10 µg/ml. After 1 h of incubation at 37 °C, the percentage of aggregated cells was determined as described previously (19). Quantitative determinations were made by counting free cells in five separate squares of a gridded ocular centered over the well at 125 × magnification. Percent aggregation = {1 - (no. of free cells experimentally treated/no. of free cells control-treated)} × 100.

Labeling of Cellular Proteins

Cells (5 × 107) were starved of methionine and cysteine for 1 h prior to labeling with 100 µCi/ml [35S]methionine and -cysteine (Tran35S-Label; ICN Biochemicals, Irvine, CA) in RPMI, 10% dialyzed fetal bovine serum.

Cells for phosphorylation studies were rinsed in phosphate-free RPMI and labeled with 0.5 mCi of inorganic 32P for 4 h at 37 °C.

Cell Solubilization, Immunoprecipitation, and SDS-PAGE

Labeled cell pellets were suspended in 1 ml of cold lysis buffer (PBS containing 1% Triton X-100, 1 mM PMSF, 1 mM Na3VO4, 1 mM Na2MoO4) and incubated on ice for 20 min with occasional rocking. The insoluble fraction was pelleted by centrifugation in a table top microcentrifuge. Soluble proteins were transferred to a fresh tube and 0.1 ml of Sepharose 4CL beads added (50% slurry equilibrated in lysis buffer without PMSF; Pharmacia Biotech Inc., Uppsula). The tube was rocked for 16 h, after which the beads were briefly spun down. The clarified supernatant was transferred to a fresh tube and antibody added to 10 µg/ml final concentration. Immune complexes were formed by incubation on ice for 1 h and harvested by incubation with protein A beads. The immune complexes were pelleted and washed two times with 1 ml of cold 1% Triton X-100, 1 M NaCl, 1 mM PMSF, 1 mM Na3VO4, 1 mM Na2MoO4, and once with 1 ml of cold lysis buffer. The remaining proteins were eluted by addition of reducing SDS-PAGE loading buffer, boiled for 5 min, and separated by gel electrophoresis.

Immune complexes of labeled proteins were separated by SDS-PAGE, and transferred to polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). Autoradiography of gels containing 32P-labeled proteins was conducted using X-Omat film (Eastman Kodak Corp.) with a single intensifying screen at -70 °C. Gels of 35S-labeled proteins were impregnated with fluor and exposed to film at -70 °C.

Phosphoamino Acid Analysis and Peptide Mapping

Autoradiographs were used to localize 32P-labeled ICAM-3. Bands were excised from the polyvinylidene difluoride sheet and the proteins partially acid hydrolyzed and separated as described in (20). Briefly, the samples were dried in vacuo and resuspended in 6 µl of pH 1.9 buffer containing unlabeled phosphoamino acid standards (Sigma). A portion of each sample, representing equal Cerenkov counts, was spotted on cellulose TLC plates and phosphoamino acids separated by high voltage thin layer electrophoresis (HTLE-7000; CBS Scientific, Del Mar, CA). After ninhydrin staining of the standards, the plates were exposed to film for autoradiography.

Phosphorylation sites were determined by tryptic peptide mapping using in vivo 32P-labeled proteins. Labeled protein bands were incubated with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Worthington Biochemical Corp., Freehold, NJ) for 18 h. The resulting peptide mixture was spotted on a TLC plate and subjected to charge separation and ascending chromatography.

Protein Kinase Assay

Human protein kinase Ctheta was amplified by PCR with a primer that contained a 6-histidine tag at the carboxyl terminus of the protein coding sequence as described (21). The cDNA was subcloned into a BACMID vector (Life Technologies Inc.), and recombinant virus was generated as described by the manufacturer. Infected Sf9 cells were lysed in hypotonic buffer (20 mM Tris, pH 7.5, 250 mM sucrose, 5 mM EDTA, 5 mM EGTA, 100 µg/ml each aprotinin and leupeptin, 1 mM aminoethyl benzenesulfonyl floride) by Dounce homogenization. Active soluble protein kinase was separated from insoluble material by centrifugation (16,000 × g) and 0.04-ml assays performed using 50 µg/ml total protein and 100 µM substrate peptide as described (21). The following substrates were synthesized: ICAM-3 CT, amino acids 482-518 (REHQRSGSYHVREESTYLPLTSMQPTEAMGEEPSRAE), SCR CT (ARSTEQQGMYAESESEELRPGYPEHRSTHTMLPRSVE), SGS (biotin-FREHQRSGSYHVREE), and SGS-P (biotin-FREHQRSGS(PO4)YHVREE).


RESULTS

Development and Characterization of ICAM-3-deficient Jurkat Cells

Jurkat cells (J77) examined by indirect fluorescence cytometry with antibodies to ICAM-3 routinely display two populations of cells (Fig. 1A). The bulk (97%) of these are ICAM-3-positive, while a small population (3%) is ICAM-3-negative. Enrichment of the ICAM-3-negative population by sequential rounds of cell sorting generated a population displaying >97% ICAM-3-negative cells that we have termed J77.50.3. RNA analysis showed that, in J77.50.3 cells, synthesis of the 2.2-kb ICAM-3 message was below detectable levels (Fig. 1B).


Fig. 1. Comparison of parental Jurkat 77 and J77.50.3 cells. A, J77 and J77.50.3 were stained indirectly for flow cytometry with anti-ICAM-3 ICR1.1, anti-CD3, anti-CD11a, anti-CD18, anti-CD45, IgG1 control, or IgG2a control. B, total RNA blot from parental J77 and J77.50.3 cells for ICAM-3 (upper) versus control glyceraldehyde-3-phosphate dehydrogenase (lower) message. The band detected with the ICAM-3 probe in the J77.50.3 RNA (lane 2) may have represented a low level of ICAM-3 message (less than 0.5% of normalized parental level), and it appeared to have a smaller molecular weight than the band in the parental lane.
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Surface expression of numerous proteins was assessed in J77.50.3 and the parental J77 line. Both populations exhibited similar fluorescence profiles for all mAb studied including CD3e, CD11a, CD18, and CD45 (Fig. 1A), indicating that J77.50.3 cells were similar to J77 except for the ICAM-3 deficiency.

Expression of ICAM-3 in J77.50.3 Cells Restores Function

Optimal T cell activation is thought to require two signals: one from the antigen receptor and the other from one or more of a large number of accessory molecules including ICAM-3 (22). To confirm that ICAM-3/TCR engagement was costimulatory, Jurkat T cells were seeded onto ICR1.1 coimmobilized with increasing concentrations of OKT3. A dose-dependent increase in IL2 production was observed (Fig. 2A). Cells exposed to either immobilized mAb alone showed no induction of IL2 secretion.


Fig. 2. ICR1.1 costimulates with the TCR of J77 but not J77.50.3 cells. A, dose-response graph of IL2 secretion from J77 cells seeded on no mAb, immobilized ICR1.1 mAb alone (500 ng/well), or with an indicated concentration of OKT3. B, J77.50.3 cells are deficient in ICAM-3 mediated costimulation. Cells plated onto ICR1.1 with the suboptimal (low) OKT3 dose (25 ng/well) or a concentrated (high) dose of OKT3 (500 ng/well) alone as described under "Materials and Methods." Control wells were treated with anti-ICAM-1 mAb 18E3D and OKT3. After 16 h, media were removed and IL2 quantitated by enzyme-linked immunosorbent assay. Data are representative results from one experiment that was repeated four times.
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To determined if loss of ICAM-3 expression would impair costimulation, J77.50.3 cells were seeded into mAb-coated wells under conditions that stimulated the parental cells. ICAM-3-deficient J77.50.3 did not secrete IL2 (Fig. 2B). Both cell lines responded to a greater concentration of OKT3 by secreting IL2. Neither cell line responded to a combination of anti-ICAM-1 mAb (18E3D) and OKT3. These data reveal that TCR signaling and the synthetic machinery for IL2 production in J77.50.3 cells was intact. Further, co-engagement of ICAM-1 and CD3 was insufficient to produce IL2.

To evaluate whether ICAM-3 expression in J77.50.3 cells would complement the phenotypic defect, cells were transfected with either a control vector or HA-tagged ICAM-3 (ICAM-3FL). Cells were selected, and several independent lines that maintained stable surface expression were identified (Fig. 3A). Inclusion of the HA tag allowed for validation that the expressed form of ICAM-3 in the transfected cells was from the introduced DNA construct rather than re-expression of the endogenous gene. Indeed, surface staining for either ICAM-3 or HA tag epitopes showed similar levels of fluorescence in the populations (Fig. 3A). While the control-transfected lines lacked the ability to be stimulated by the co-immobilized mAb, J77.50.3 cells expressing ICAM-3FL responded to the costimuli by secreting IL2 into the medium as did the parental J77 cells (Fig. 3B). Therefore, expression of ICAM-3 by J77.50.3 cells restored their ability to respond to ICAM-3/TCR costimulation.


Fig. 3. Surface expression and IL2 secretion from representative transfected J77.50.3 cells. A, flow cytometric analysis of transfected J77.50.3 cells. Top row, anti-ICAM-3 mAb ICR1.1 (filled histogram trace) or IgG2a control (outlined histogram trace) staining of J77 cells (left) and J77.50.3 cells transfected with either vector control (center) or ICAM-3FL (right). Bottom row, anti-HA mAb 12CA5 staining of J77 (left), vector control (middle), or ICAM-3FL (right) transfected J77.50.3 cells. B, IL2 secretion from J77 and J77.50.3 transfectants seeded onto coimmobilized ICR1.1 and OKT3 or OKT3 alone (as in Fig. 2). Data are determinations of means and standard errors from five experiments.
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ICAM-3 Cytoplasmic Region Truncations Have Distinct Functional Consequences

J77.50.3 cells expressing the following HA epitope-tagged cytoplasmic tail truncations were generated to grossly map the cytoplasmic region of ICAM-3: -1/3CT (Gln505 terminus), -2/3CT (Arg493 terminus), and -CT (His484 terminus) (Fig. 4). Characterization of cells expressing each deletion included monitoring surface expression (Fig. 5A and Table I) and immunoprecipitation from lysates of cells that had been metabolically labeled with 35S (Fig. 5B). SDS-PAGE analysis showed that the relative migration of the truncated proteins was of the expected sizes of ~120-140 kDa.


Fig. 4. Diagram of ICAM-3 proteins used for these studies. Terminal residues are noted for the truncated proteins -1/3CT, -2/3CT, and -CT. The relative positions of serine and leucine point mutations reported in this study are shown.
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Fig. 5. Surface cytofluorometric and SDS-PAGE analysis of ICAM-3 cytoplasmic tail truncations expressed in J77.50.3 cells. A, ICR1.1 (filled histogram trace) or IgG2a control (outlined histogram trace) staining of cells expressing -1/3CT (left), -2/3CT (center), or -CT (right). B, analysis of ICAM-3FL and truncations from 35S metabolically labeled J77.50.3 cell transfectants by immunoprecipitation and SDS-PAGE. Cell extracts from vector control, ICAM-3FL, -1/3CT, -2/3CT, and -CT (lanes 1-5, respectively) were analyzed.
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Table I. Characterization of different J77.50.3 transfectants


Cell line Mean fluorescence intensitya IL2 secretion (fold stimulation)b Percent aggregationc Percent spread cellsd

Vector 12 0.52 16  ± 4 0
FL 156 5.4 58  ± 3 87  ± 10
 -1/3CT 166 4.8 29  ± 2e 26  ± 1e
 -2/3CT 132 2.2 29  ± 1e 33  ± 3e
 -CT 222 2.2 15  ± 2e 16  ± 1e
Ser487 right-arrow Ala 174 2.0 61  ± 9 77  ± 5
Ser489 right-arrow Ala 153 2.9 12  ± 2e 10  ± 1e
Ser496 right-arrow Ala 157 6.0 58  ± 7 33  ± 3e
Leu499 right-arrow Ala 158 6.0 57  ± 4 74  ± 8
Ser503 right-arrow Ala 137 1.0 66  ± 15 36  ± 2e
Ser515 right-arrow Ala 262 2.1 15  ± 3e 14  ± 3e

a Mean fluorescence intensity for transfectants was determined from live-gated cell populations and expressed in arbitrary units.
b IL2 secreted from cells seeded onto coimmobilized mAb after a 16-h incubation was measured by enzyme-linked immunosorbent assay. Wells were treated with 500 ng/well (ICR1.1 or 18E3D) and 25 ng/well OKT3. Fold stimulation = (IL2 secreted from cells on coimmobilized ICR1.1/OKT3)/(IL2 secreted from cells on coimmobilized 18E3D/OKT3). Results are the means of repeated experiments (n = 3-6) with two cell lines for each truncation/mutation.
c Percent aggregation is based on extrapolation by counting free cells remaining after a 60-min incubation at 37 °C in medium containing ICR1.1 or MOPC21 isotype control (n = 3-5).
d Cell spreading on ICR1.1-coated surfaces is expressed as percent of input cells. After incubation and fixation, 300 cells were counted for each represented cell line. Cells considered spread showed thinned, darkened cytoplasm. Cells considered not spread were refractile and bright (n = 2-5).
e These values were compared with the values from the full-length ICAM-3-expressing cells using one-way analysis of variance (ANOVA) and found to be significantly different. Values for p = <0.05.

The truncations were tested for their ability to trigger J77.50.3 cells to secrete IL2 when costimulated with anti-ICAM-3/TCR mAb. Conditioned media from cells expressing either ICAM-3FL or -1/3CT forms showed 5.4- and 4.8-fold induction, respectively, when normalized for IL2 secretion in negative control-treated wells (Table I). Cells expressing either -2/3CT or -CT forms secreted about 60% less IL2 (2.2-fold each). All of the cell lines tested responded to a more concentrated dose of OKT3 alone by secreting similar levels of IL2 (data not shown).

Immunoregulation of leukocytes has been hypothesized to occur via aggregate formation in which paracrine effects of cytokines (both positive and negative) regulate progression of a cellular immune response (23). J77.50.3 cells expressing ICAM-3FL treated with ICR1.1 mAb responded by forming aggregates (58% aggregated, Table I). This is not due to direct cross-linking of cells by mAb, since Fab fragments of ICR1.1 trigger aggregation as well.2 Expression of -1/3CT resulted in 29% aggregated cells. Truncation to residue Arg493 (-2/3CT) resulted in no further reduction, while cells that expressed -CT aggregated to the same level as vector control cells.

The interface between a T cell and antigen-presenting cell (APC) is an area of intercellular adhesion that is both dynamic and highly intimate (24). T cells spread over a large region of the APC surface during the early phase of contact that coincides with a transient calcium flux (25, 26). Since ICAM-3 is present prior to activation, it is plausible that it would be involved in spreading. J77.50.3 cells expressing ICAM-3FL rapidly flattened and spread on ICR1.1, but remained rounded on the isotype control coating (Fig. 6, A and B, and Table I). Cells expressing either -1/3CT or -2/3CT also spread (Fig. 6, C and D), while cells expressing -CT were incapable of spreading (Fig. 6E).


Fig. 6. Cell spreading assay of J77.50.3 transfectants. Cells expressing ICAM-3FL seeded onto IgG2a control mAb (A) and ICR1.1 (B). Cells expressing -1/3CT (C), -2/3CT (D), and -CT (E) truncations of ICAM-3 seeded onto ICR1.1. Photomicrographs were taken 15 min after seeding at ×400.
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Phosphoamino Acid Composition of ICAM-3

Intracellular protein phosphorylation regulates many signaling cascades that lead to activation or morphological changes. Consequently, we examined the phosphorylation of ICAM-3 under conditions that triggered Jurkat cell activation. ICAM-3FL immunoprecipitated from unstimulated J77.50.3 cells had a basal level of 32P incorporation (Fig. 7A, lane 2). Cells treated with either OKT3 cross-linking or PMA had an increase of 32P uptake onto ICAM-3 (Fig. 7A, lanes 3 and 4). Basal and inducible phosphorylation was also observed with peripheral blood leukocytes.2 Phosphoamino acid analysis revealed that PMA and OKT3 cross-linking elicited phosphorylation of ICAM-3 only on serine residues (Fig. 7B).


Fig. 7. Immunoprecipitation and phosphoamino acid analysis of labeled ICAM-3FL. A, autoradiograph of IgG2a control (lane 1) or anti-ICAM-3 mAb ICR1.1 (lanes 2-4) from unstimulated (lanes 1 and 2), anti-CD3(OKT3)/gam cross-linked (lane 3), or PMA-treated (lane 4) cells after 32P-labeling. Arrow indicates ICAM-3 protein band. B, two-dimensional analysis of ICAM-3 phosphoamino acid composition from PMA (left) or OKT3/gam cross-linked (right) cells. Positions of phosphoserine (pS), phosphothreonine (pT), and phosphotyrosine (pY) are indicated along with the buffer pH used for separations.
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Ser489 Is Phosphorylated in Vivo

Truncation analysis indicated that residues 485-515 were important for costimulation, while aggregation and cell spreading depended on the presence of residues 506-515 and 485-493. To investigate whether these ICAM-3-triggered functions correlated with phosphorylation of specific serine residues in these functionally important regions, phosphorylation site analysis was undertaken by point mutation and peptide mapping. 32P-Labeled ICAM-3FL from PMA-treated cells was subjected to tryptic peptide mapping, which revealed several major phosphopeptides (Fig. 8, A and B). The pattern of separated phosphopeptides was similar whether ICAM-3 was derived from cells stimulated with PMA or CD3 cross-linking (data not shown). The tryptic phosphopeptide map of 32P-labeled ICAM-3 Ser487 right-arrow Ala resulted in the wild type pattern of labeled phosphopeptides (Fig. 8, compare B and C). The Ser489 right-arrow Ala mutation resulted in complete loss of signal for one of the major phosphopeptides (Fig. 8D), indicating that Ser489 is a bona fide phosphorylation site in vivo. Alanine substitution mutants of serine residues 496, 503, and 515 were also subjected to in vivo labeling and endoproteolytic peptide mapping analyses; however, the results were inconclusive (data not shown).


Fig. 8. Phosphopeptide mapping of ICAM-3 FL. A, the cytoplasmic region with the predicted trypsin cleavage sites (arrows) and locations of serines 487 and 489. B-D, tryptic phosphopeptide maps of ICAM-3 FL (B), Ser487 right-arrow Ala (C) and Ser489 right-arrow Ala (D) recovered from 32P-labeled and PMA-stimulated cells. The sample origins are indicated by a dot at the lower left of each panel.
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Mutation of Ser489 Blocks ICAM-3 Function

Phenotypic effects of the alanine point mutants were tested in homotypic aggregation, cell spreading and TCR costimulation assays. Aggregation of Ser489 right-arrow Ala was about 20% of ICAM-3FL levels, similar to the vector control transfectants (12, 58, and 16%, respectively; Table I). Ser489 right-arrow Ala also had a deleterious effect on cell spreading. Conversely, Ser487 right-arrow Ala and Leu499 right-arrow Ala had no detectable inhibition of aggregation or spreading. Induction of IL2 secretion by Ser489 right-arrow Ala in the TCR costimulation assay was also reduced about 50%, compared with Leu499 right-arrow Ala and ICAM-3FL (2.9-, 6.0-, and 5.4-fold, respectively; Table I). The effect of Ser489 right-arrow Ala on costimulation was comparable to the -CT truncation. Cells expressing Ser487 right-arrow Ala were equally impaired when assayed for costimulation but not for aggregation or spreading. Ser496 right-arrow Ala reduced spreading to 33%, while costimulation and aggregation remained intact. Ser503 right-arrow Ala blocked costimulation completely and spreading partially, yet left aggregation intact. Ser515 right-arrow Ala abrogated costimulation, aggregation, and spreading.

In Vitro Phosphorylation of Ser489 by PKCtheta

Numerous protein kinases have been characterized as to the sequence specificity of their substrates. For the family of protein kinase C (PKC) isoforms, a consensus substrate sequence is RXXS/T (27). Examination of the ICAM-3 Ser489 sequence context suggested that it might be a PKC substrate. In addition, TCR cross-linking and PMA treatments, both of which activate multiple PKC isoforms, resulted in the induction of serine phosphorylation of ICAM-3 (Fig. 7). PKCtheta was chosen for in vitro kinase assay since 1) it is found in abundance in cells of the hematopoietic lineage, 2) it can activate the AP-1 element of the IL2 promoter when overexpressed, and 3) it selectively translocates to the T cell/APC contact region in an antigen-dependent manner (28-31). A 37-residue peptide representing the entire cytoplasmic region (amino acids 482-518) was phosphorylated in vitro by recombinant human PKCtheta (Fig. 9A). In contrast, a scrambled ICAM-3 peptide had little detectable incorporation. To address whether Ser489 was a PKCtheta phosphorylation site in vitro, the phosphorylation of shorter substrate peptides (amino acids 481-495) containing Ser489 (SGS) or phospho-Ser489 (SGS-P) was also evaluated. Incorporation of 32P was found only with the SGS peptide and not with the synthetically phosphorylated version, SGS-P (Fig. 9). These data indicate that PKCtheta phosphorylation of the cytoplasmic tail was sequence-specific and that Ser489 was a phosphorylation site in vitro.


Fig. 9. In vitro PKCtheta phosphorylation of ICAM-3 substrate peptides. A, ICAM-3 substrate peptides (100 µM) were incubated with phospholipid micelles and partially purified recombinant PKCtheta as described under "Materials and Methods" prior to termination of the reaction by spotting onto phosphocellulose paper. Mean determinations of PKCtheta activity (black bars) on ICAM-3 substrate peptides ICAM-3 CT (Arg482-Glu518), a scrambled CT sequence (SCR CT), SGS peptide (Phe481-Glu495), and SGS-P peptide (Phe481-Glu495 with phosphoserine 489). Activity from recombinant PKCtheta was compared with a similarly prepared lysate from nonrecombinant virus-infected Sf9 cells (striped bars) after 30 min of incubation at room temperature. B, PKC inhibition of cellular spreading. J77.50.3 cells expressing ICAM-3FL were treated with either 50 µM chelerythrine chloride or vehicle alone (Me2SO) for 10 min prior to seeding into dishes coated with ICR1.1 as described under "Materials and Methods." Magnification, ×800.
[View Larger Version of this Image (40K GIF file)]

Since PKCtheta phosphorylation of Ser489 occurred in vitro and mutation of this amino acid blocked function in vivo, we sought to implicate PKC activity in an ICAM-3-triggered event like cell spreading. Cells expressing ICAM-3FL pretreated with the PKC inhibitor chelerythrine chloride were effectively blocked for spreading, whereas vehicle-treated cells spread as usual (Fig. 9B). This result suggests that PKC activity is required for ICAM-3-dependent cell spreading.


DISCUSSION

We investigated the functional requirements of the cytoplasmic region of ICAM-3 by expression of truncated or point mutated proteins in a variant of the human T leukemic cell line Jurkat. A Jurkat cell line deficient in ICAM-3 expression (J77.50.3) was developed and characterized with regard to surface protein expression, message synthesis, and costimulatory phenotype (Figs. 1 and 2). Expression of surface ICAM-3 restored accessory molecule function as measured by secretion of the T cell activation marker IL2 (Fig. 3). Therefore, the functional deficit in the cell line can be complemented by expression of a single protein, ICAM-3.

The natural occurrence of a subpopulation of ICAM-3-deficient cells in unselected Jurkat cultures is curious. Others have reported that the Jurkat cell line was mixed for the expression of ICAM-3, suggesting that this phenotype is inherent to the cell line itself (32). The ICAM-3 deficiency was stable in continuous culture, and cells with wild type levels of ICAM-3 expression do not arise.

This model cell line was quite useful for investigating the structure/function relationship of the cytoplasmic region of ICAM-3. The 37 residues of the cytoplasmic region are grouped as alternating hydrophilic-hydrophobic-hydrophilic segments, which were used to divide the region for mapping studies (33). The -1/3CT truncation, which lacked five charged residues that gave the distal portion of the native tail its hydrophilic nature, terminated with Gln505 and contained the hydrophobic core residues 498-501 (Fig. 4). The -2/3CT truncation removed the hydrophobic core residues and terminated with Arg493. The -CT truncation terminated with His484 and allowed enough positive charge to achieve membrane stop transfer in the absence of the rest of the cytoplasmic tail.

Functional evaluation of the cytoplasmic region consisted of measuring IL2 secretion following costimulation and anti-ICAM-3-triggered aggregation and cell spreading of stable transfectants expressing truncations or point mutations. IL2 secretion was significantly impaired in cells that expressed truncations of residues 485-505 (-2/3CT and -CT) and partially impaired by truncation of residues 506-518 (-1/3CT). Point mutation analysis showed that particular serine residues (serines 487, 489, 503, and 515), when mutated, would also block triggering of IL2 secretion. These observations suggest that the TCR accessory function of ICAM-3 required the majority of its cytoplasmic region. Since some costimulatory activity remained with -CT truncation compared with the vector control transfectants (2.2 and 0.52, respectively), this suggests that the rest of the protein has a role. Whether this activity is via associated transmembrane proteins or through simple ICAM-3 mAb-mediated enhancement of cell binding to the OKT3-coated surface is unclear. Aggregation and spreading were partially (50-70%) inhibited with deletion of residues 506-518 and an additional 50% with further deletion of amino acids 485-493. Loss of the hydrophobic core residues 494-505 did not result in incrementally greater deficits on aggregation or spreading. In agreement, the point mutant analysis showed that Ser489 right-arrow Ala and Ser515 right-arrow Ala blocked, while Ser496 right-arrow Ala, Leu499 right-arrow Ala, and Ser503 right-arrow Ala had no deleterious effect on aggregation. Effects of the point mutants on spreading were also in agreement with those of the truncations. In particular, mutation of serine residues in the membrane distal or proximal regions either partially or completely inhibited spreading. These results suggest that two distinct and well separated regions (amino acids 506-518 and 485-493) contribute to ICAM-3-triggered aggregation and spreading. Interestingly, mutational effects on costimulation, aggregation, and cell spreading were not absolutely coincidental. This suggests that these three functions are separable and not necessarily interdependent. In general, however, these ICAM-3 triggered functions were effected by mutations within the hydrophilic regions proximal and distal to the membrane-spanning segment.

Since many protein functions are regulated by phosphorylation status, the phosphoamino acid composition of ICAM-3 was determined under basal and stimulated conditions. In vivo labeling of ICAM-3, via the intracellular ATP pool, resulted in phosphoserine content only (Fig. 7). In addition, ICAM-3 from activated blood-derived leukocytes (including neutrophils) and leukocytic cell lines, failed to yield immunoreactivity with anti-phosphotyrosine mAb.2 In agreement with our results, Lozano et al. (32) have reported inducible serine phosphorylation of ICAM-3. In contrast, Skubitz et al. (34) reported inducible phosphorylation predominantly on tyrosine. This discrepancy may be attributed to differences in cell isolation procedures or the manner in which the cells were labeled.

We determined that Ser489 is a major phosphorylation site in vivo by tryptic peptide mapping of serine to alanine point mutants and that recombinant PKCtheta phosphorylated this site in vitro (Figs. 8 and 9). The requirement for Ser489 in ICAM-3 function was demonstrated by mutation of this site, which abrogated all ICAM-3-triggered events tested (Table I). Ser489 is closely apposed to the cytoplasmic face of the plasma membrane, which could facilitate its phosphorylation by PKC upon recruitment to the lipid microenviroment of the membrane by events like TCR stimulation or phorbol ester treatment. In fact, only under conditions of TCR stimulation or PMA treatment have increased levels of ICAM-3 phosphorylation been found by us. Since phosphorylation is a dynamic event, in which rapid dephosphorylation can occur, specialized conditions may be required for the isolation of hyper-phosphorylated ICAM-3 from aggregated or spread cells. Alternatively, ICAM-3-triggered aggregation and spreading require Ser489 for structural integrity independent of phosphorylation state. In this case, an alanine mutant blocks function by altering important structural features. Whether other serine residues (496, 503, and 515) function, at least in part, by their phosphorylation status remains to be determined.

The studies described here delineate portions of the cytoplasmic region of ICAM-3 that are important for T cell biology. Since the regions implicated in TCR costimulation partially encompass regions linked to aggregation and cell spreading, our data suggest that these functions might share underlying mechanisms, perhaps reorganization of the cytoskeleton. Links between cytoskeleton dynamics and activation have been observed in T cells during the process of APC/T adhesion and contact (35-38). At initial contact, engagement of TCR by MHC-antigen complexes activates PKC which leads to phosphorylation of ICAM-3, similar to that observed using anti-CD3 mAb. T cell spreading over the APC surface, perhaps driven by actin polymerization, provides threshold levels of TCR triggering by antigen-MHC and allows other co-stimulatory molecular interactions to occur, such as CD28-B7. For sustained T cell signaling, actin-based cytoskeletal changes are required and, as the present work suggests, could be induced by engagement of ICAM-3 (37). Preliminary studies suggested that ICAM-3 engagement induced actin polymerization.2 Later, dephosphorylation of ICAM-3 could contribute to cell dissociation and rounding, as observed with Ser489 mutation, contributing to clonal expansion. Further biochemical studies are required to delineate the mechanisms by which T cell behavior is regulated by the functionally important amino acids of the ICAM-3 cytoplasmic region identified here.


FOOTNOTES

*   This work was supported by ICOS Corp. and Abbott Laboratories.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom reprint requests and correspondence should be addressed: ICOS Corp., 22021-20th Ave. SE, Bothell, WA 98021. Tel.: 425-485-1900 (ext. 2213); Fax: 425-486-0300; E-mail: jhayflick{at}icos.com.
1   The abbreviations used are: ICAM, intercellular adhesion molecule; LFA-1, lymphocyte function antigen-1; IL2, interleukin 2; TCR, T cell receptor; mAb, monoclonal antibody; PBS, phosphate-buffered saline; PKC, protein kinase C; APC, antigen-presenting cell; PCR, polymerase chain reaction; HA, hemagglutinin; MHC, major histocompatibility complex; PMSF, phenylmethylsulfonyl fluoride; PMA, phorbol 12-myristate 13-acetate.
2   J. Hayflick, unpublished observations.

ACKNOWLEDGEMENTS

We thank Dr. Steven J. Burakoff for the Jurkat 77 cell line, Drs. Meri Hoekstra and Don Staunton and Louise Band for critical reading of the manuscript, and Dr. Owen Lockerbie for advice during the project. We thank Christi Wood and Dina Leviten for expert DNA sequencing and synthesis, Tony DiMaggio and Adam Kashishian for advice with the phosphorylation studies, David Hynds for peptide synthesis, and Rick Jasman and Anne Jensen for expert monoclonal antibody production and characterization.


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