Phosphorylation of the cytoplasmic domain of the integrin CD18 chain by protein kinase C isoforms in leukocytes.

The CD11/CD18 (beta(2)) integrins are leukocyte-specific adhesion receptors, and their ability to bind ligands on other cells can be activated by extracellular stimuli. During cell activation, the CD18 chain is known to become phosphorylated on serine and functionally important threonine residues located in the intracellular C-terminal tail. Here, we identify catalytic domain fragments of protein kinase C (PKC) delta and PKCbetaI/II as the major protein kinases in leukocyte extracts that phosphorylate a peptide corresponding to the cytoplasmic tail of the integrin CD18 chain. The sites phosphorylated in vitro were identified as Ser-745 and Thr-758. PKCalpha and PKCeta also phosphorylated these residues, and PKCalpha additionally phosphorylated Thr-760. Ser-745, a novel site, was shown to become phosphorylated in T cells in response to phorbol ester stimulation. Ser-756, a residue not phosphorylated by PKC isoforms, also became phosphorylated in T cells after phorbol ester stimulation. When leukocyte extracts were subjected to affinity chromatography on agarose to which residues 751-761 of the CD18 chain phosphorylated at Thr-758 were bound covalently, the only proteins that bound specifically were identified as isoforms of 14-3-3 proteins. Thus, PKC-mediated phosphorylation of CD18 after cell stimulation could lead to the recruitment of 14-3-3 proteins to the activated integrin, which may play a role in regulating its adhesive state or ability to signal.

The CD11/CD18 integrins (␤ 2 integrins) are leukocyte-specific members of the integrin superfamily of heterodimeric cell surface receptors involved in adhesion to the extracellular matrix and to cells. The four different CD11/CD18 integrins (1-2) share a common ␤ chain (CD18) but have different ␣ chains (CD11a-d) and different cell distribution and ligands. CD11/ CD18 integrins are unable to bind their ligands, the intercellular adhesion molecules (ICAMs) 1 in resting cells, but instead need an activating signal for conversion to an adhesive state (2,3). In addition to extracellular activation with divalent cations (4), ligands (5)(6)(7), or certain monoclonal antibodies to the extracellular domains (8 -10), the integrins can also be activated by intracellular signaling, so-called inside-out signaling. This is induced by triggering the T cell receptor (3,11) or other leukocyte surface receptors (11)(12)(13), or by direct activation of PKC by tumor-promoting phorbol esters (14,15). The molecular basis for activation is still poorly understood; however, it is thought to involve changes in avidity via surface redistribution of the integrin (16 -19) and perhaps signaling mediated conformational changes of the integrin extracellular domain (20). Several signaling pathways have been implicated in the activation process, including those that modulate protein kinase C (PKC) (21), phosphoinositide 3-kinase (PI 3-kinase) (22), mitogen-activated protein kinase (23), the small GTP-binding protein Rap1 (21), protein phosphatases (24,25), and the calciumbinding proteins calpain (26) and calmodulin (27). The cytoplasmic domain of the CD18 chain is necessary for the regulation of adhesion (16,28,29), and interestingly, both phorbol esters and CD3 ligation induce CD18 phosphorylation on Ser (30 -32) and Thr (25) residues. Thr phosphorylation is more transient than Ser phosphorylation. The Thr phosphorylation may regulate the adhesive state of the integrin, because mutation of three consecutive threonines in the integrin chain (Thr-758, Thr-759, and Thr-760) reduce integrin binding to ICAM-1 (29) and cytoskeletal association (16) of the integrin molecules. Additionally, phosphorylated integrin molecules preferentially partition with the actin cytoskeleton (33), indicating that phosphorylation of integrins could regulate integrin-cytoskeleton interactions.
In addition to their adhesive properties, the integrins are also involved in activating intracellular signaling pathways and can thus mediate bi-directional signaling across the plasma membrane. In fibroblasts they form focal adhesion complexes with cytoskeletal elements and a wide range of signaling molecules (34). In leukocytes these signaling complexes are believed to be more transient, because leukocytes are involved in relatively short lived interactions with other cells. Several cytoskeletal and signaling molecules have been shown to interact with the CD18 cytoplasmic tail, including the actin-binding proteins talin (33,35), filamin (36), and ␣-actinin (37,38), the adaptor proteins Rack1 (receptor for activated PKC) (39) and cytohesin (40), and the transcription factor Jun activation domain-binding protein (41).
In this study, we present evidence that PKC isoforms are the major protein kinases that phosphorylate the C terminus of the integrin CD18 chain in leukocytes. Ser-745 is identified as a novel phosphorylation site in the integrin cytoplasmic domain. Additionally, we show that a Thr-758-phosphorylated integrin peptide can interact with 14-3-3 proteins in leukocyte lysates and thus potentially initiate signaling complex formation "downstream" of the phosphorylated integrin molecules.
Antibodies-Two phosphopeptides were synthesized corresponding to residues 740 -751 and 751-761 of the integrin CD18 chain (CKEK-LKpSQWNNDN and CNPLFKpSATTTV, where pS is phosphoserine), with an N-terminal cysteine added for coupling, and conjugated to keyhole limpet hemocyanin as in Ref. 42. The complex was injected into sheep at the Scottish Antibody Production Unit (Carluke, Scotland). The antiserum was passed through agarose to which the relevant phosphorylated peptide had been coupled covalently, and the phosphospecific antibodies were eluted with 0.1 M glycine, pH 2.4, immediately adjusted to pH 8.0 with Tris-HCl, and stored at 4°C. This was carried out by Dr. J. Leitch and C. Clark in our laboratory. The CD18 integrin antibodies R7E4 and R2E7B have been described previously (14,43). The monoclonal antibody OKT3, which reacts with CD3, was used in the form of ascites fluid produced by hybridoma cells (clone CRL 8001, American Type Culture Collection, Manassas, VA). PKC isozymespecific antibodies were from Transduction laboratories (Lexington, KY). The broadly reactive 14-3-3␤ (K-19) antibody was from Santa Cruz Biotechnology (Santa Cruz, CA).
Protein Kinase Assay-Protein kinases were assayed using a peptide corresponding to the integrin CD18 C terminus (RRFEK EKLKS QWNND NPLFK SATTT VMNPK FAES). Recombinant PKC isoforms and activity eluting from column fractions were measured at 30°C in FIG. 1. A kinase activity that phosphorylates the C-terminal integrin peptide is found in leukocyte lysates. A, the CD18 peptide was phosphorylated for 10 min by fractions from the initial MonoQ column as described under "Experimental Procedures." B, the peak fraction from the initial MonoQ column (Q peak) was used to phosphorylate the CD18 C-terminal peptide in the presence or absence of 100 nM Ro 318220 or 1 or 10 M PKC pseudosubstrate inhibitor peptide (PS-pep) for the times indicated. The peptide was subjected to SDS-PAGE and transferred to a PVDF membrane, and the radioactive band was detected by autoradiography.

FIG. 2.
Protocol for the purification of the major CD18 kinase activities in leukocyte lysates. The specific activity and overall yield from Hitrap Q pH 7.5 is shown.
FIG. 3. Purification of the integrin kinase activity. A, fractions from the final gel filtration column were assayed for CD18 C-terminal peptide kinase activity (open circles). The elution position of the standard marker proteins bovine serum albumin (66 kDa) and ovalbumin (44 kDa) are shown. B, aliquots of indicated fractions from A were subjected to SDS-PAGE and stained with Coomassie Blue. The two protein staining bands that coeluted with the activity are marked. C, same as B, except that the fractions were incubated for 30 min with 10 mM magnesium acetate, 0.1 mM [␥-32 P]ATP (10 6 cpm per nmol) in the standard integrin kinase buffer, and the gel was autoradiographed after electrophoresis. The two major 32 P-labeled bands indicated by arrows comigrated with the major protein staining bands in B. D, fractions 2 and 3 (F2 and F3) from the final protamine-agarose column (see "Experimental Procedures"), containing 46 and 54% of the kinase activity, respectively, were subjected to SDS-PAGE and either stained with Coomassie Blue (left-hand panels) or transferred to nitrocellulose membranes and immunoblotted with an antibody raised against the PKC␣ catalytic domain (right-hand panels). The bands identified as PKC␤ and PKC␦ are indicated.
Preparation of Leukocyte Lysates and Purification of Integrin Kinase Activities-Human leukocytes were isolated from buffy coat pools by centrifuging for 20 min at 1500 ϫ g. The leukocyte layer was then subjected to Ficoll gradient centrifugation and washed with phosphatebuffered saline. The cells were lysed in 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM sodium orthovanadate, 10 mM sodium glycerophosphate, 50 mM NaF, 5 mM sodium pyrophosphate, 0.27 M sucrose, 2 M microcystin, 0.1% 2-mercaptoethanol, 1 mM benzamidine, and protease inhibitor mixture. The lysates were frozen in liquid nitrogen and stored in aliquots at Ϫ80°C. The lysates (12 g of protein) were fractionated from 10 to 20% polyethylene glycol 6000 (PEG). After stirring for 3 h at 4°C, the 20% PEG suspension was centrifuged for 30 min at 7000 ϫ g, the supernatant discarded, and the pellet redissolved in 50 ml of buffer A (50 mM Tris-HCl, pH 7.5, 0.1 mM Brij 35). The solution was applied to a 5-ml Hitrap Q column equilibrated in buffer A. After washing with equilibration buffer, the column was developed with a linear salt gradient to 0.7 M NaCl in the same buffer. One major peak of activity was detected eluting at 0.3-0.4 M NaCl, which was pooled, diluted 5-fold in buffer B (50 mM BisTris, pH 6.5, 5% (v/v) glycerol, 0.1% (v/v) 2-mercaptoethanol, 1 mM EGTA, 0.03% (w/v) Brij 35), and applied to a 1-ml Hitrap heparin column equilibrated in buffer B. After washing until no protein could be detected in the eluate, the column was developed with a linear salt gradient to 1 M NaCl. One major peak of activity was detected, eluting at 0.2 M NaCl. This was pooled, diluted in buffer B, and applied to a 1-ml MonoQ column, which was developed with a linear salt gradient to 0.7 M NaCl. The most active fractions, eluting at 0.3 M NaCl were pooled, diluted with buffer C (50 mM Tris-HCl, pH 7.7, 0.1 mM EGTA, 0.1% (v/v) 2-mercaptoethanol, 5% (v/v) glycerol, 0.03% (w/v) Brij 35), and applied to a 1-ml MonoQ column, which was developed with a linear salt gradient to 1 M NaCl. The most active fractions, eluting at 0.3 M NaCl were pooled, concentrated to 25 l with Vivaspin columns, and subjected to gel filtration on Superdex The flow-through from the Hitrap-heparin column was chromatographed successively on 1-ml MonoQ columns equilibrated in buffer B and buffer C, respectively, as described above. A single major activity peak was detected eluting at 0.3 M NaCl in buffer B and 0.35 M in buffer C. The activity was then chromatographed on a 2-ml protamine-agarose column equilibrated in buffer A. The column was washed with 5 ml of buffer A plus 200 mM NaCl and then with buffer A plus 1 M NaCl. Four fractions, each of 6 ml, were collected.
Identification of Proteins by Mass Spectrometry and Phosphoamino Acid Analysis-Proteins of interest were excised from SDS-polyacrylamide gels, digested with trypsin, and identified using a Perspective Biosystems (Framingham, MA) Elite STR matrix-assisted laser desorption-time of flight-mass spectrometer as described previously (46). Phosphoamino acid analysis of integrin CD18 C terminus was also performed as described (47).
Isolation of CD11/CD18 Integrins from Lymphocyte Lysates-Integrins were affinity-purified from human leukocytes using R7E4 columns as described previously (48) and were at least 90% pure as monitored by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining.
Subcellular Fractionation-Fractionation of cells into soluble and cytoskeletal fractions was done after lysis in the presence of Triton X-100 as described (27).
Peptide Affinity Chromatography-The phosphorylated and unphosphorylated forms of the peptides CNPLFKpSATTTV and CLFKSApTT-TVMN corresponding to the sequences surrounding phosphorylated Ser-756 and Thr-758, respectively (where pS and pT indicate phosphoserine and phosphothreonine), were coupled to vinylsulfone-activated agarose via the N-terminal cysteine residue. The cytosol and solubilized cytoskeletal fractions (0.5-1 mg of protein) were then subjected to affinity chromatography on each peptide-agarose column as described (33).
Immunoblotting-For detection of the CD11/CD18 integrin with phospho-specific antibodies, the integrin was first immunoprecipitated from 2.0 to 2.5 mg of lysate protein with 20 g of R7E4 coupled  Fig. 3, B and D, as the catalytic domain of PKC␦ Tryptic peptides from the 42-kDa autophosphorylating bands in Fig. 3, B and D, were analyzed on a Perspective Biosystems Elite STR matrix-assisted laser desorption-time of flight mass spectrometer as described under "Experimental Procedures." The tryptic ions were scanned against the Swiss-Prot and Genpep databases using the SF-FIT program of Protein Prospector. The table summarizes the peptides from the integrin kinase that matched the PKC sequence.
Peptides only were detected in the protamine-agarose eluate.

CD18 Integrin Phosphorylation by PKC
noncovalently to protein G-Sepharose. Bound proteins were eluted with 1% SDS, then subjected to polyacrylamide gel electrophoresis, and transferred to nitrocellulose, and the phosphorylated integrin was immunoblotted and detected using the ECL chemiluminescence detection system (Amersham Biosciences). The phosphospecific antibody that recognizes CD18 phosphorylated at Ser-745 was used at 2 g/ml in the presence of 5 g/ml unphosphorylated peptide conjugated to keyhole limpet hemocyanin. The Ser-756 phosphospecific antibody was used at the same concentration but in the presence of 25 g/ml unphosphorylated peptide conjugated to keyhole limpet hemocyanin. Incubation with both antibodies was carried out overnight in 4°C. The antibody R2E7B which recognizes phosphorylated and unphosphorylated CD18 equally well was used at 1:5000 dilution. The antibody against the PKC␣ catalytic domain, which also reacts with PKC␤ isoforms, was used as a dilution of 1:1000. An antibody that recognizes all 14-3-3 isoforms was used at 1 g/ml.

Identification of the Major Protein Kinases in Human Leukocyte Lysates That Phosphorylate the C Terminus of the Integrin CD18
Chain-To identify the major CD18 kinases, human leukocyte lysates were initially fractionated on MonoQ. All the activity was retained by the column and eluted as one major peak (Fig. 1A), which was pooled and chromatographed on heparin-Sepharose. At this step, 80% of the activity was not retained by the column, whereas 20% was bound. The activity present in both fractions was then further purified as described under "Experimental Procedures." A flow chart of the overall purification protocol is shown in Fig. 2.
The activity that was retained by heparin-Sepharose eluted as a single peak with an apparent molecular mass of 40 kDa at the final gel filtration step (Fig. 3A). SDS-PAGE revealed two major proteins, whose elution position correlated with activity ( Fig. 3B). When the fractions were incubated with Mg[␥-32 P]ATP, both proteins became phosphorylated (Fig. 3C), suggesting that they might be protein kinases capable of autophosphorylation. The bands were excised and subjected to tryptic mass fingerprinting, which revealed that they both corresponded to the ␦-isoform of PKC. All the peptides detected were located in the catalytic domain of PKC␦ (Table I). This observation and the apparent molecular mass of the purified protein (which is much smaller than full-length PKC␦) indicated that it represented an active proteolytic fragment. Such fragments are known to be active in the absence of phospholipids and diacylglycerol, which are required for the activity of full-length PKC␦. This explains why these active fragments were detected, because the assays did not contain the cofactors essential for the activation of PKC isoforms.
The activity that was not retained by heparin-Sepharose was purified by chromatography on MonoQ at two different pH values and finally on protamine-agarose. SDS-PAGE of the activity initially eluted at 1 M NaCl (accounting for 46% of the activity) showed five protein-staining bands (Fig. 3D) of which the three most rapidly migrating (apparent molecular masses, of 44, 42, and 30 kDa) became phosphorylated upon incubation with Mg[␥-32 P]ATP (data not shown). The 44-and 42-kDa bands were identified as the catalytic domains of PKC␤ and PKC␤II, respectively, by tryptic mass fingerprinting (Table II), whereas the 30-kDa band appeared to be a mixture of several proteins, none of which was a protein kinase (data not shown). This was confirmed by immunoblotting with an antibody that

CD18 Integrin Phosphorylation by PKC
recognizes the catalytic domains of PKC␣ and PKC␤. More prolonged elution of the protamine-agarose column with 1 M NaCl eluted 54% of the activity. This fraction contained a single protein of apparent molecular mass 42 kDa that was identified as the catalytic domain of PKC␦ by tryptic mass fingerprinting (Table I). This indicated that PKC␦ was only partially retained by heparin-Sepharose.
To establish that PKC isoforms accounted for all the activity eluted from the initial MonoQ column (Fig. 1A), the following additional experiments were performed. First, the activity from this column was shown to be inhibited by 100 nM Ro 318220, a potent inhibitor of conventional PKC isoforms, as well as several other protein kinases (49). Second, the activity was strongly suppressed by a pseudosubstrate peptide, which is believed to be a specific inhibitor of PKC (Fig. 1B).
Identification of Residues in the C Terminus of Integrin CD18 Phosphorylated by PKC Isoforms-The C-terminal peptide was phosphorylated with the purified catalytic domain of PKC␦ and digested with trypsin. The resulting phosphopeptides were then chromatographed on a Vydac C 18 column (Hesperia, CA), which resolved three 32 P-labeled tryptic peptides, termed T1A, T1B, and T2 (Fig. 4A). Each peptide was identified by a combination of Edman sequencing, solid phase sequencing, and mass spectrometry, as described previously (50). Peptides T1A and T2B both corresponded to residues 756 -765 of the CD18 chain (SATTTVMNPK) phosphorylated at Thr-758 (Fig. 4B), whereas peptide T2 corresponded to residues 743-755 (LKSQWNNDNPLFK) phosphorylated at Ser-745 (Fig. 4C).
Phosphorylation of the CD18 C Terminus by Different PKC Isoforms-PKC isoforms have been reported to differ slightly in substrate specificity (51). We therefore examined the ability of seven different PKC isoforms to phosphorylate the C terminus. PKC␣ was the most active of the conventional PKC isoforms, and PKC was the most active novel PKC toward the C terminus of CD18 (Fig. 5A). PKC␤I and PKC␥ phosphorylated the peptide almost exclusively on serine, whereas PKC␣, -␤II, -␦, and -phosphorylated both serine and threonine. PKC⑀ was the only PKC isoform that preferred threonine to serine (Fig.  5B). The sites on the CD18 peptide phosphorylated by PKC were the same as those phosphorylated by PKC␦, namely Ser-745 and Thr-758 (data not shown). PKC␣ phosphorylated the CD18 peptide at a cluster of three tryptic peptides, T1, T2, and T3, as well as another peptide T4 (Fig. 6A). Peptides T1 and T2 corresponded to peptides T1A and T1B in Fig. 3 and were predominantly phosphorylated at Thr-758 (Fig. 6, B and C). Interestingly, peptide T3 consisted of the same peptide phosphorylated predominantly at Thr-760 (Fig. 6D). Peptide T4 corresponded to peptide T2 in Fig. 3 and was phosphorylated at Ser-745 as expected (Fig. 6E).
Phosphorylation of the CD18 Chain in Vivo-It has been shown previously (25, 30 -32) that PDBu in conjunction with FIG. 4. Identification of the phosphorylation sites in the integrin C-terminal peptide phosphorylated by PKC␦. A, the integrin peptide was phosphorylated for 10 min with the catalytic domain of PKC␦ purified from the leukocyte lysates and then subjected to tryptic digestion (see "Experimental Procedures"). The digest was applied to a Vydac C 18 column equilibrated in 0.1% (v/v) trifluoroacetic acid and the column developed with an acetonitrile gradient (straight line). The flow rate was 1 ml/min, and 0.5-ml fractions were collected and analyzed for 32 P radioactivity (open circles) by Cerenkov counting. The major 32 P tryptic phosphopeptides (T1A, T1B, and T2) were identified as described in the text. The phosphopeptides T1B and T2 were sequenced by Edman degradation using an Applied Biosystems 42A protein sequencer (B and C). 32 P radioactivity released after each cycle was measured in a separate experiment by solid phase Edman degradation of the peptides coupled to a Sequelon arylamine membrane as described previously (67). Peptide T1A gave the same result as T1B (not shown). The amino acid sequence is shown using the single letter code for amino acids.

FIG. 5. Phosphorylation of the C-terminal integrin peptide by PKC isoforms.
A, the integrin peptide was phosphorylated for 10 min at 30°C with [␥-32 P]ATP (10 6 cpm per nmol) and the indicated PKC isoforms (each at 3 units/ml). One unit of activity, was that amount which catalyzed the incorporation of 1 nmol of phosphorylate into the standard substrate (histone H3 for PKC␣, -␤I, -␤II, -␥, and a PKC⑀substrate peptide for PKC␦, -⑀, and -) in 1 min. Lipids and calcium ions were included as recommended by the supplier. The phosphorylated peptides were subjected to SDS-PAGE, transferred to PVDF membranes, and subjected to autoradiography. B, the phosphorylated substrates were excised from the PVDF membrane, partially hydrolyzed in 6 N HCl, and phosphoamino acids resolved by thin layer chromatography (see "Experimental Procedures"). PS, phosphoserine; PT, phosphothreonine. the phosphatase inhibitor okadaic acid induces phosphorylation of the integrin CD18 chain in T cells, whereas the CD11 chain is phosphorylated constitutively. We confirmed these findings in the present study and also found that the phosphorylation of CD18 was prevented by 1 M Ro 318220 and 0.5 M of a related compound, Go-6983 (data not shown).
To identify the residues at the C terminus of CD18 whose phosphorylation is induced by PDBu, we raised phospho-specific antibodies capable of recognizing CD18 only when phosphorylated at Ser-745. We also raised phospho-specific antibodies that should recognize CD18 when phosphorylated at Ser-756, because this site has also been reported to become phosphorylated in response to PDBu, as judged by phosphopeptide mapping (44) and mutagenesis (29). We have been unable, thus far, to generate phospho-specific antibodies that recognize CD18 phosphorylated at Thr-758 and Thr-760 and which are sufficiently sensitive to detect the phosphorylation of these residues in cells. However, based on phosphopeptide mapping, it has been reported previously that two of the three threonine residues Thr-758, Thr-759, and Thr-760 become phosphorylated after stimulation with PDBu and okadaic acid (25,44).
The antibody raised against the peptide 740 -751 phosphorylated at Ser-745 was tested for recognition of the phosphopeptide immunogen and the unphosphorylated peptide in the presence of unphosphorylated peptide to block any antibodies present that recognize both the phosphorylated and unphosphorylated forms of the peptide. These experiments demon-strated that, under these conditions, the antibody only recognized the phosphopeptide and not the unphosphorylated peptide (Fig. 7A). Additionally, it only recognized the CD18 protein after phosphorylation by PKC␤ in vitro (Fig. 7B). Recognition by the antibody was prevented by preincubation with the phosphopeptide immunogen but not by the unphosphorylated form of the peptide or the Ser-756 phosphopeptide (Fig.  7B). The specificity of the antibody raised against the peptide comprising residues 751-761 phosphorylated at Ser-756 was established in an analogous manner. However, as protein kinases capable of phosphorylating CD18 at Ser- 756 have not yet been identified, specificity was established using the phosphopeptide immunogen (Fig. 8A). The phospho-specific antibody toward Ser-756 did not recognize the CD18 chain phosphorylated at Ser-745 by PKC␤ (Fig. 7B).
These antibodies were then used to demonstrate that Ser-745 (Fig. 7C) and Ser-756 (Fig. 8B) both become phosphorylated when T cells are exposed to PDBu. No other unspecific bands were seen on the gels (not shown). Phosphorylation of Ser-745 could be detected in the presence of PDBu alone, but the phosphorylation was increased when cells were stimulated with PDBu and okadaic acid in the presence of OKT3, a stimulating antibody raised against the CD3 component of the T cell receptor. Phosphorylation of either site was inhibited by 1 M Ro 318220 (Figs. 7C and 8D). The phosphorylation of Ser-756 could be detected readily after stimulation with high (200 nM) PDBu and OKT3 in the absence of okadaic acid, but OKT3 FIG. 6. Identification of the residues in the C-terminal integrin peptide phosphorylated by PKC␣. A, the integrin peptide was phosphorylated for 60 min at 30°C with PKC␣, subjected to tryptic digestion, and applied to a Vydac C 18 column as in Fig. 3. The major 32 Plabeled tryptic peptides (T1, T2, T3, and T4) were identified as described in the text. B-E, the phosphopeptides were analyzed by Edman and solid phase sequencing, and 32 P radioactivity released after each cycle was measured to identify the sites of phosphorylation. alone in the presence or absence of okadaic acid did not induce Ser-756 phosphorylation (Fig. 8B). Ser-756 phosphorylation can also be induced to similar levels by low (10 nM) PDBu in the presence of the calcium ionophore A12387 (Fig. 8C). Because no PKC isoform tested was able to phosphorylate CD18 at Ser-756 in vitro, this suggested that the phosphorylation of Ser-756 was likely to be catalyzed by another protein kinase activated directly or indirectly by a PKC isoform. To try and identify the signaling pathway in which this putative kinase was located, we examined the effect of inhibitors of other protein kinases on the phosphorylation of Ser-756 in vivo. Interestingly, the phosphorylation of Ser-756 was found to be suppressed by W-7, a calmodulin antagonist (Fig. 8D), but not by PD 98059, an inhibitor of the classical mitogen-activated protein kinase pathway, or by calpeptin, an inhibitor of the calcium-dependent proteinase calpain. Trifluoperazine, another calmodulin antagonist, was also found to suppress phorbol ester-induced Ser-756 phosphorylation (data not shown). Both the phosphorylation of Ser-756 induced by high concentrations of phorbol ester alone and the phosphorylation induced by low levels of phorbol ester in conjunction with the calcium ionophore A23187 were greatly suppressed by W-7 as well as the PKC inhibitor Ro 318220 (Fig.  8E). These observations indicate that calmodulin may be involved in regulating Ser-756 phosphorylation in vivo.
A Thr-758-phosphorylated CD18 Peptide Binds 14-3-3 Proteins from Leukocyte Lysates-To identify potential functions for the phosphorylation of the CD18 C terminus, we investigated whether proteins in T cell lysates were capable of binding to the C terminus when phosphorylated at particular sites. These experiments showed that two proteins of apparent molecular mass 30 and 28 kDa bound specifically to the C-terminal peptide phosphorylated at Thr-758. In contrast, these proteins did not bind to the unphosphorylated C-terminal peptide or to the peptide phosphorylated at Ser-756 (Fig. 9A). The 30and 28-kDa bands were excised and identified by tryptic mass fingerprinting as 14-3-3␣␤ and 14-3-3␦, respectively (Table  III). These results were confirmed by immunoblotting with an antibody that recognizes all 14-3-3 isoforms (Fig. 9B). The presence of 14-3-3 proteins binding to the C-terminal peptide could also be detected in the cytoskeletal fraction of leukocytes, where the major part of the phosphorylated integrins reside (Fig. 9C). DISCUSSION The phosphorylation of integrin cytoplasmic domains has been proposed as a way of regulating integrin activity and/or interaction with cytoplasmic proteins and cell signaling. For example, tyrosine phosphorylation of the integrin ␤ 3 cytoplasmic tail leads to association with Shc (52), an adaptor protein involved in activation of the classical mitogen-activated protein kinase cascade. Tyrosine phosphorylation of the integrin ␤ 3 cytoplasmic tail might also regulate its binding to cytoskeletal elements (53). On the other hand, threonine phosphorylation of ␤ 3 integrins is reported to prevent Shc from binding to the tyrosine-phosphorylated integrin (54).
In contrast to the ␤ 1 and ␤ 3 integrins, the CD18 integrin polypeptide (also called the ␤ 2 integrin) lacks two of the three tyrosines in the conserved NPXY motifs found in the integrin ␤ 1 and ␤ 3 chains. However, the CD18 integrins have been shown to become phosphorylated on serine and threonine residues in cells after stimulation with phorbol ester (30 -32) or T cell receptor engagement (25). The role of these phosphorylation events is not yet understood.
To identify the protein kinases that phosphorylate CD18, we purified and identified the major activities in T cell extracts that phosphorylate a synthetic peptide corresponding to most of the cytoplasmic domain of the integrin CD18 chain. The protein kinases detected were found to be active proteolytic fragments of PKC␤ and PKC␦, which are known to be cleaved intracellularly from the native enzymes by proteases, such as calpain (55,56). The native forms of these and other PKC isoforms were presumably not detected because they are only active in the presence of one or more cofactors, i.e. calcium ions, phospholipids, and diacylglycerol or phorbol esters (57, 58), and it is possible that other PKC isoforms are cleaved proteolyti- FIG. 7. Ser-745 is phosphorylated in human T cells. A, the phosphorylated and unphosphorylated forms of the peptide comprising residues 740 -751 of CD18 (phosphorylated at Ser-745) were conjugated to keyhole limpet hemocyanin and spotted onto nitrocellulose membranes in the amounts indicated. The membranes were then immunoblotted with an antibody raised against the phosphorylated peptide that had been incubated with the unphosphorylated form of the peptide 740 -751 (25 g/ml). B, purified CD18 (1 g/lane) was phosphorylated for 60 min in vitro with purified PKC␤ (CD18ϩK) or without kinase (CD18). Aliquots of the reaction were subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies that recognize CD18 phosphorylated at Ser-745 (anti-pSer745) or Ser-756 (anti-pSer756). The antibodies were first preincubated with or without the indicated peptide antigens (5 g/ml). The peptides comprised residues 740 -751 of CD18 (phosphorylated at Ser-745) and residues 751-761 (phosphorylated at Ser-756) or the unphosphorylated form of the peptide 740 -751. The lowest panel shows immunoblotting with R2E7B, a CD18-antibody that recognizes the phosphorylated and unphosphorylated forms of CD18 equally well. C, human T cells were preincubated for 30 min with or without 1.5 M okadaic acid (OA) or 1 M Ro 318220, activated with 200 nM PDBu (PDBu) or 1:200 dilution of OKT3 (the antibody against the CD3 component of the T cell receptor), and lysed, and the CD11/ CD18 complex immunoprecipitated, subjected to SDS-PAGE, and immunoblotted with the phospho-specific antibody that recognizes CD18 phosphorylated at Ser-745 (anti-pSer745) in the presence of the unphosphorylated peptide (5 g/ml). The immunoblots were stripped and reprobed with a CD18-specific antibody (anti-CD18) to confirm equal loading. cally to a lesser extent than PKC␤ and PKC␦ and thus not detected under our assay conditions. This led us to discover that many of the known PKC isoforms are capable of phosphorylating CD18 in vitro, and indeed two them, PKC␣ and PKC, appeared to be very active toward CD18 (Fig. 4). At present, we cannot say which PKC isoforms are the main kinases phosphorylating CD18 in vivo. The residues on CD18 phosphorylated by PKC␤, PKC␦, and PKC were Ser-745 and Thr-758. Interestingly, however, PKC␣ phosphorylated CD18 on Thr-760 as well as Thr-758. It may also phosphorylate Thr-759 (Fig. 5, B and C), but this has not yet been shown definitively.
The finding that the major serine residue phosphorylated by PKC isoforms in vitro was Ser-745 was somewhat surprising, because the major site in the C-terminal domain that becomes phosphorylated in response to phorbol esters is Ser-756 (29), which is not phosphorylated by any PKC isoform in vitro.
However, the PDBu-induced phosphorylation of CD18 at Ser-756 is prevented by inhibitors of PKC (Fig. 8), suggesting that Ser-756 is phosphorylated by a protein kinase that is activated by a PKC isoform. Alternatively, a PKC isoform may inhibit a protein phosphatase that dephosphorylates Ser-756 in cells. Interestingly, we found that the phosphorylation of Ser-756 induced by low concentrations of PDBu only occurred in the presence of the calcium ionophore A23187, a phenomenon reported previously (59) for total CD18 phosphorylation. Moreover, the phosphorylation of Ser-756 induced by low concentrations of PDBu and A23187, or high concentrations of PDBu in the absence of A23187, was suppressed by the calmodulin antagonist W-7. This raises the possibility that a PKC-activated Ser-756 kinase might also be dependent on calcium ions and calmodulin. The phosphorylation of Ser-756 does not seem to be important in adhesion, because its mutation to alanine FIG. 8. Ser-756 phosphorylation is induced by low concentrations of PDBu in the presence of calcium ionophore and inhibited by the calmodulin antagonist W-7. A, the phosphorylated and unphosphorylated forms of the peptide comprising residues 751-761 of CD18 (phosphorylated at Ser-756) were conjugated to keyhole limpet hemocyanin and spotted onto nitrocellulose membranes in the amounts indicated. The membranes were then immunoblotted with an antibody raised against the phosphorylated peptide that had been incubated without additions (none), with the unphosphorylated form of the peptide 751-761, or with the phosphorylated form of the same peptide (each at 100 g/ml). The figure shows that the antibody only became phospho-specific after incubation with the unphosphorylated peptide. All subsequent experiments with this antibody were therefore performed in the presence of excess unphosphopeptide (25 g/ml). B, the experiment was carried out as in Fig. 7B  has no effect on phorbol ester-induced binding of CD18 to ICAM-1, a major ligand of the integrin (29). However, we have shown previously that calmodulin antagonists are strong suppressors of PDBu-induced T cell aggregation (27). Because a calmodulin antagonist also reduces Ser-756 phosphorylation, it is possible that Ser-756 phosphorylation plays a role in these events.
Based on phosphopeptide mapping, it has been reported previously (44) that two of the three threonine residues Thr-758, Thr-759, and Thr-760 become phosphorylated when T cells are stimulated with PDBu, although the protein phosphatase inhibitor okadaic acid also had to be added to the cells. The cytoplasmic domain also becomes phosphorylated on a threonine residue(s) when the T cell receptor is activated (25). In the present study, we have shown that Ser-745 becomes phosphorylated, albeit somewhat weakly, when T cells are stimulated with PDBu alone, and that phosphorylation could be increased in the presence of OKT3, an antibody that binds to the CD3 component of the T cell receptor, plus okadaic acid. Because PKC isoforms are the major protein kinase activities in leukocyte lysates responsible for the phosphorylation Ser-745, and the phosphorylation of Ser-745 is inhibited by Ro 318220 in vivo, it would appear that a PKC isoform mediates the phosphorylation of CD18 at Ser-745 in cells. However, since Ro 318220 and Go-6983, which both inhibit CD18 phosphorylation, are not exclusively specific PKC inhibitors, it cannot be completely excluded that another unknown phorbol ester-activated kinase is mediating the phosphorylation of CD18 at Ser-745. It is also possible that Ser-745 becomes phosphorylated more strongly in response to other signals or combinations of signals that have yet to be identified.
The mutation of Ser-745 to Ala, like the mutation of Ser-756 to Ala, has no effect on phorbol ester-induced binding of CD11/ CD18 to ICAM-1 (29), indicating that these phosphorylation events may play a different role. Ser-745 is not conserved in the ␤ 1 or ␤ 3 integrins, and it may thus play a role in CD11/CD18specific signaling events. The stoichiometry of CD18 integrin phosphorylation in vivo after phorbol ester treatment in the presence of okadaic acid has been determined as 0.92 mol per mol of protein (44). Because Ser-756, Ser-745, and Thr-758/ Thr-759/Thr-760 phosphorylation take place under these conditions, each of these sites is clearly phosphorylated to only a low stoichiometry. However, this does not exclude them playing important roles physiologically if, by analogy with receptor tyrosine phosphorylation, their function is to recruit other signaling molecules to the plasma membrane.
PKC has been implicated previously in the regulation of integrin function. PKC␣ associates with ␤ 1 integrins and regulates their internalization (60), whereas PKC⑀ has been implicated in the regulation of integrin-dependent cell spreading (61,62). Interestingly, the PKC isoforms ␣, ␤I, ␤II, and ␦, but not PKC, activate integrin-mediated adhesion to ICAM-1 in a model system (21). Moreover, the receptor for activated PKC (Rack1), a PKC␤-interacting protein that is believed to regulate its localization and substrate specificity (63,64), interacts with the membrane-proximal part of the integrin CD18 cytoplasmic tail in phorbol ester-activated leukocytes (39). An attractive hypothesis is that the binding of Rack1 to the integrin cytoplasmic tail could recruit active PKC␤ to the integrin and allow it to phosphorylate Ser-745 and Thr-758.
To investigate the functions of the C-terminal phosphorylation on CD18, we initially studied whether proteins present in the cytoplasm of T cells bound to the C-terminal peptide when it was phosphorylated at particular sites. This led us to find that the C terminus of CD18 binds specifically to 14-3-3 proteins when it is phosphorylated at Thr-758. The 14-3-3 proteins were recruited both from the soluble and, importantly, from the cytoskeletal fractions of leukocytes, where most of the phosphorylated integrins have been shown to reside (33). In contrast, the 14-3-3s did not bind to the unphosphorylated C-terminal peptide or the peptide phosphorylated at Ser-756. One additional protein of 16 kDa bound to the C-terminal peptide (Fig.  9A), but this was independent of phosphorylation. In coprecipitation studies, we failed to detect 14-3-3 binding to CD18 in activated cells, but this could be due to the low stoichiometry of threonine phosphorylation under these circumstances. Alternatively, the phosphorylation of Ser-756, Thr-759, and/or Thr-760 may interfere with binding of 14-3-3s to the Thr-758phosphorylated integrins. Further work is clearly needed to evaluate whether 14-3-3 binding to CD18 occurs in vivo.
14-3-3s are adaptor proteins that bind to phosphoserine-and phosphothreonine-containing motifs (65) and are known to be involved in regulating a number of signaling molecules (66). They have been shown to dimerize and could thus recruit other signaling proteins to form complexes. The optimal consensus sequence for 14-3-3 binding is RXXpS/pTXP (65), which does not conform to the sequence surrounding Thr-758. However, other phosphorylated sequences have been shown to interact with 14-3-3 proteins (67, 68). Indeed, recently, 14-3-3␤ was identified in a yeast two-hybrid screen with ␤ 1 integrins, but FIG. 9. 14-3-3 proteins bind to a Thr-758-phosphorylated integrin C-terminal peptide but not to a Ser-756-phosphorylated peptide. Human leukocyte lysates (A and B) or cytoplasmic and cytoskeletal fractions (C) were subjected to affinity chromatography on agarose to which the peptides indicated below had been attached covalently (see "Experimental Procedures"). The protein bound to each column was eluted with SDS, subjected to SDS-PAGE, and either stained with Coomassie Blue (A) or transferred to nitrocellulose and immunoblotted with an antibody that recognizes all 14-3-3 isoforms (B and C). A, lane 1, leukocyte lysate; lane 2, eluate from a control protein G-Sepharose column; lane 3, eluate from agarose to which the unphosphorylated peptide 751-761 had been bound; lane 4, eluate from agarose to which the peptide 751-761 phosphorylated at Ser-756 had been bound; lane 5, eluate from agarose to which the unphosphorylated peptide 753-763 had been bound; lane 6, eluate from agarose to which the peptide 753-763 phosphorylated at Thr-758 had been bound. B, fractions 3-6 from A were electrophoresed and immunoblotted with an anti-14-3-3 antibody. C, same as B, except that cytoplasmic (lanes 1 and 2) and cytoskeletal (lanes 3 and 4) fractions were subjected to affinity chromatography on agarose to which the unphosphorylated form of the peptide 753-763 (lanes 1 and 3) or the same peptide phosphorylated at Thr-758 (lanes 2 and 4) had been bound. the interaction was not thought to be phosphorylationdependent (69).
The TTT motif (Thr-758, Thr-759, and Thr-760) appears to play a pivotal role in integrin regulation. Mutation of these threonines singly or in combination decreases binding to ICAM-1 in response to PDBu (29). In addition, an activating mutation (L732R) that induces phorbol ester responsiveness of CD11a/CD18 integrins in K562 cells that normally do not respond to phorbol esters is abolished by the mutation of Thr-758 to Ala (70). The mutation of the TTT motif also causes defects in post-receptor signaling events, whereby the integrin receptor interacts with the actin cytoskeleton and induces cell spreading (16), and phosphorylated integrins have been shown to associate with the actin cytoskeleton preferentially (33). It is therefore tempting to speculate that threonine phosphorylation of CD18 recruits 14-3-3 proteins to the plasma membranecytoskeleton connection and that the 14-3-3s in turn recruit further proteins to regulate cell spreading and/or integrin signaling.  Fig. 9 binding to the Thr 758-phosphorylated C terminus of CD18 as 14-3-3 ␣/␤ and /␦ Tryptic peptides from the bands marked 14-3-3 in Fig. 9A were analyzed by mass fingerprinting. The table summarizes the peptides from the bands that matched the 14-3-3 sequences.