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2 and 7 Leukocyte Integrin Polypeptides1
* Department of Biosciences, Division of Biochemistry, and
Institute of Biotechnology, University of Helsinki, Helsinki, Finland
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Abstract |
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2 leukocyte integrin subunit has previously been shown to become phosphorylated in leukocytes on cytoplasmic serine and functionally important threonine residues. We have now mapped the phosphorylated threonine residues in activated T cells. After phorbol ester stimulation, all three threonine residues (758–760) of the threonine triplet became phosphorylated but only two at a time. CD3 stimulation leads to a strong threonine phosphorylation of the 2 integrin, but differed from phorbol ester activation in that phosphorylation occurred only on threonine 758. The other leukocyte-specific integrin, 7, has also been shown to need the cytoplasmic domain and leukocyte-specific signal transduction elements for integrin activation. Cell activation with phorbol ester, and interestingly, through the TCR-CD3 complex, caused 7 integrin binding to VCAM-1. Additionally, cell activation led to increased phosphorylation of the 7 subunit, and phosphoamino acid analysis revealed that threonine residues became phosphorylated after cell activation. Sequence analysis by manual radiosequencing by Edman degradation established that threonine phosphorylation occurred in the same threonine triplet as in 2 phosphorylation. |
Introduction |
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1, 2, and 7 subfamilies are critical for leukocyte homing (1, 2). 2 and 7 integrins are expressed solely on leukocytes (3, 4). These integrins mediate a number of cell-to-cell interactions by binding to intercellular adhesion molecules, which are expressed on different cells (5). 2 integrins also bind other molecules like E-selectin (6), type I collagen (7), and fibrinogen (8, 9). The integrin 
47 is a cell adhesion receptor mainly expressed on lymphocytes, and mediates their homing to the intestine and associated lymphoid tissue, such as Peyer’s patches (2). 47 is a receptor for the VCAM-1 and the mucosal vascular addressin mucosal addressin cell adhesion molecule-1 on intestinal endothelial cells (10).
The affinity or avidity of integrins for their extracellular ligands is regulated by the activation status of the integrin molecule (5, 11). The different states of activation can be modulated by agents such as phorbol esters (12, 13) or by Abs against the TCR-CD3 complex or other proteins such as CD2 (14, 15). This activation of integrins has been termed inside-out signaling (16, 17). Several integrin families have been shown to undergo activation, among these the 2 integrins in leukocytes (5) and the 1 integrins in hemopoietic cells (18). Also, 47-dependent binding to ligands has been shown to require prior activation (4).
Reversible phosphorylation of proteins is commonly involved in dynamically regulated cellular reactions, and this mechanism is used by cells both in the regulation of adhesion and in integrin-mediated signal transduction (19, 20). However, it is unclear whether or how phosphorylation of integrins themselves affects these activities, and therefore the phosphorylation status of integrins has been extensively studied. The cytoplasmic domains of the integrin subunits contain a number of putative phosphorylation sites. The phosphorylation state of 2 integrins has been reported to change after T cell stimulation by phorbol ester (21, 22, 23, 24) or by CD3 ligation (25, 26). The main phorbol ester-induced phosphorylation site was identified as Ser756 in the 2 cytoplasmic domain (27). The activation also induces threonine phosphorylation in 2, which can be revealed when serine/threonine phosphatases in T cells are inhibited (26). It has been reported previously that two of the three threonine residues (758–760) become phosphorylated after T cell stimulation with phorbol ester (28). Ser745 was recently found to be phosphorylated in the 2 cytoplasmic tail, induced by phorbol ester and strengthened by the use of okadaic acid (OA)3 and CD3 Ab together with phorbol ester (29). The amino acids Thr758–760 and Ser745 have been shown to become phosphorylated by protein kinase C (PKC) (2, 26, 29), and Ser756 by an unknown kinase (29). The importance of the serine phosphorylations of 2 integrin activation is questionable, but the threonine triplet has been shown to be vital for the phorbol ester-induced 2-mediated binding of cells to coated ICAM-1 (27). The threonine triplet has also been shown to be involved in regulating postreceptor events through the 2 integrins, such as cytoskeletal association and cell spreading (30). Threonine-phosphorylated integrins distribute preferentially to the actin cytoskeleton (31). The linkage to the actin cytoskeleton could occur via actin-binding proteins, such as filamin, which interacts directly with 2 integrin (32). Filamin has been shown to interact also with 1 and 7 integrins, and threonine phosphorylation has been speculated to regulate the interaction, and, subsequently, cell migration (33).
The 7 subunit shows the closest resemblance to the 2 integrin phosphorylation sites having both a single serine in the position corresponding to the Ser756 as well as a threonine triplet. The similarity between the 2 and 7 sequences in this region is intriguing, because 47, like 2 integrins, is expressed only on leukocytes, and the existence of leukocyte-specific signal transduction elements are hypothesized to be involved in activation of both 2 and 7 integrins (34). The cytoplasmic domain of 7 is important in the regulation of integrin function, because truncation of the 7 subunit cytoplasmic domain resulted in three different activation states of 47: inactive, partially active, and fully active receptors (35).
In this study, we have mapped the phosphorylated threonines in the 2 cytoplasmic domain after T cell activation with OA and phorbol ester. We have also characterized the phosphorylation of 2 after CD3 ligation. T cell activation through the TCR caused phosphorylation both on serine and threonine residues similarly to phorbol esters, but threonine phosphorylation was dominating. Unlike phorbol ester-induced phosphorylation, only one threonine (Thr758) became phosphorylated after CD3 ligation. Interestingly, the main phorbol ester-induced phosphorylation site Ser756 did not become phosphorylated in CD3-activated T cells. Additionally, we have shown that CD3 triggering induced 7 integrin binding to VCAM-1, and we observed 2-like threonine phosphorylation in activated 7 integrins, but mutational analysis showed that the phosphorylated threonine was not needed for adhesion.
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Materials and Methods |
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Phorbol 12,13-dibutyrate (PDBu) was from Sigma-Aldrich (St. Louis, MO), and OA was from Calbiochem-Novabiochem (La Jolla, CA). [32P]Orthophosphate (aqueous solution; 10 mCi/ml; 5000 Ci/mmol) was purchased from the Radiochemical Center (Amersham, U.K.). Recombinant human VCAM-1 was from R&D Systems (Abingdon, U.K.). Sequencing-grade modified trypsin (activity >2; 5 U/mg) was purchased from Promega (Madison, WI).
The mAb R7E4 against the human 2 subunit of leukocyte integrin has been described previously (36). The mAb OKT3, which reacts with CD3, was used in the form of ascites fluid produced by hybridoma cells (clone CRL 8001; American Type Culture Collection (ATCC), Manassas, VA). The anti-7 mAb FIB504 hybridoma was obtained from ATCC. The monoclonal activating Ab against mouse-CD3
and the blocking Ab against mouse 4-chain were purchased from Southern Biotechnology Associates (Birmingham, AL). The monoclonal blocking Ab against mouse 2 (LFA-1) was from R&D Systems, and the monoclonal blocking Ab against human 1 was from Chemicon (Temecula, CA).
PCR mutagenesis of the 7 subunit
The pBluescript constructs encoding the full-length sequence of mouse 7 and 4 were kindly provided by Dr. I. Weissmann (Stanford University, Stanford, CA). The point mutant T782A in mouse 7 cDNA was generated through a two-reaction PCR strategy (37). The mutant and wild-type constructs were subcloned into the mammalian expression vector pEF-BOS (38) using the unique XbaI cloning site. The mutated construct was confirmed by automated DNA sequencing.
Cell lines and cDNA transfection
Buffy coats used for the isolation of T cells were obtained from the Finnish Red Cross Blood Transfusion Service (Helsinki, Finland). T cells were isolated as described previously (28). The murine T cell lymphoma line TK-1 was purchased from ATCC. Cells were grown in RPMI 1640 medium supplemented with 10% FCS, nonessential amino acids, 0.05 mM 2-ME, L-glutamine, and antibiotics.
COS-1 cells were cultured in DMEM supplemented with 10% FCS, L-glutamine, and antibiotics. COS-1 cells were cotransfected with purified 4 and 7 subunit cDNAs using the Fugene 6 transfection reagent according to the manufacturer’s instructions (Roche, Indianapolis, IN). Flow cytometric analysis was used to quantitate cell surface expression of the transfected COS-1 cells.
Cell adhesion assays
Recombinant soluble human VCAM-1 (0.3 µg/well) was coated on flat-bottom 96-well microtiter plates by overnight incubation at 4°C. The wells were blocked with 3% BSA for 2 h at 37°C. TK-1 cells suspended in binding medium (RPMI 1640, 40 mM HEPES, 0.1% BSA, and 1 mM MgCl2) were stimulated with 200 nM PDBu or anti-CD3 mAb (20 µg/ml or indicated concentrations) and added to each well and allowed to adhere for 30–60 min. In inhibition experiments, TK-1 cells were preincubated for 20 min with OA (1.5 µM). After incubation, unbound cells were removed by gentle washing. The binding was quantitated by counting bound cells under a microscope.
Transfected COS-1 cells suspended in binding medium (DMEM, 10 mM HEPES, 0.1% BSA, and 1 mM MgCl2) were stimulated with 100 nM PDBu and allowed to adhere for 10 min. In inhibition experiments, COS-1 cells were preincubated for 10–15 min with the blocking Ab. After incubation, unbound cells were removed by gentle washing. The binding was quantitated by ELISA.
32P radiolabeling and cell activation
Cell labeling was done as described previously (28), except that TK-1 cells were labeled for 2 h. After labeling, the cells were activated. The cell suspensions were divided into equal aliquots, which were treated with 1.5 µM OA for 25 min at 37°C and/or 200 nM PDBu or 10 µg/ml mouse anti-CD3 mAb for TK-1 cells or 1/200-diluted OKT3 ascites for T cells. Control samples were left untreated. The activation was stopped by adding ice-cold 10 mM EDTA/PBS, and the cells were washed once with ice-cold 2 mM EDTA/PBS. The cells were lysed as described previously (28).
Immunoprecipitation and SDS-PAGE
Cell lysates were precleared with protein G-Sepharose for 45 min at 4°C and incubated with anti-7 mAb on ice overnight or with anti-2 mAb R7E4 for 2.5 h. Immune complexes were captured on protein G-Sepharose. Sepharose beads were washed exclusively with decreasing detergent and salt concentrations. Bound proteins were eluted with SDS and subjected to SDS-PAGE. SDS-PAGE was performed according to Laemmli (39) and blotted onto polyvinylidene difluoride membranes (Millipore, Bedford, MA).
Phosphoamino acid analysis and phosphopeptide mapping
The phosphorylated integrins were localized using a Fuji BAS 1000 PhosphoImager (Tokyo, Japan), and the regions corresponding to integrins were excised. The filter pieces were subjected to hydrolysis in 6 M HCl for 1.5 h at 110°C or subjected to phosphopeptide mapping. The hydrolysate was lyophilized, and standard phosphoamino acids were added. Phosphoamino acids were separated using the two-dimensional HTLE-7000 electrophoresis system (C.B.S. Scientific, Del Mar, CA) on a 20 x 20-cm cellulose plate. The standard phosphoamino acids were visualized by ninhydrin staining, and 32P-labeled amino acids were visualized using the PhosphoImager.
The existence and purity of the 7 integrin submitted for further phosphorylation studies was proved by the liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis. The immune-precipitated 7 protein band was cut out from a silver-stained gel and in-gel digested with trypsin. The peptides produced were first separated by reversed-phase capillary-HPLC, and the eluent was directly injected into a quadrupole/time-of-flight mass spectrometer (Micromass, Manchester, U.K.) equipped with an electrospray ionization source. MS/MS spectra of doubly charged precursor ions were acquired. Database searches were conducted by using Mascot MS/MS ion search (http://www.matrixscience.com). Mouse integrin 7 was identified with the score of 94, and three different peptides with 
7-aa sequence tags were observed. Some trace amounts of IgG were also present in the band, but no other protein with significant score was observed.
For phosphopeptide mapping, the filter pieces were first saturated with polyvinylpyrrolidone 360, and after that, the protein was digested with trypsin at 37°C overnight. The resulting peptides were separated in the first dimension by electrophoresis at pH 8 and in the second dimension by ascending chromatography (40). 32P-Labeled peptides were detected with the PhosphoImager.
Manual radiosequencing of phosphopeptides
The phosphopeptide spots were isolated from the phosphopeptide maps and immobilized on a sequence membrane disc (Millipore). They were subjected to Edman degradation as described (41). Each cycle of degradation consisted of the following: incubation of the disc at 50°C for 10 min with coupling reagent (methanol:water:triethylamine:phenylisothiocyanate; 7:1:1:1, v/v). After the incubation, the reagent was removed. The disc was washed with methanol. Vacuum drying of the disc was followed by trifluoroacetic acid cleavage (50°C for 6 min) of the N-terminal amino acid. The cleaved amino acids were dried and spotted onto a polyvinylidene difluoride membrane. The radioactivities of the spots were visualized with the PhosphoImager and quantified using the Tina 2.09c software (Raytest, Straubenhardt, Germany).
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2 integrin subunit in OA-pretreated and phorbol ester-activated T cells
We have previously shown that, after activation of T cells by OA pretreatment and PDBu, two of the threonine residues in the threonine triplet 758–760 of the 2 cytoplasmic domain become phosphorylated (28). In this study, we wanted to map in detail which threonine residues are phosphorylated in T cells. In resting T cells, the 2 subunit is not phosphorylated and 2 phosphorylation was induced by treatment with OA and PDBu (Ref. 26 ; Fig. 1, A and B). Tryptic phosphopeptide mapping was performed from the 32P-labeled 2 integrin subunits (Fig. 1C). Based on a previously report (28) we identified the spots; the main spot (indicated by y) most likely represents peptides produced by incomplete digestion with trypsin, and the other spot (indicated by ST2) is the peptide with Ser756 and two of the threonine residues 758–760 phosphorylated. The ST2-peptide was isolated from the peptide map, and the phosphopeptide was covalently attached to an aryl amine membrane disc and subjected to Edman degradation. The amount of radioactivity that was released into the liquid phase from the trifluoroacetic acid cleavage reaction was quantified (Fig. 1D). Ser756 became phosphorylated, as observed earlier (27, 29). Additionally, all three threonine residues (758–760) became phosphorylated but only two at a time.
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In the presence of OA, CD3 ligation was shown to induce phosphorylation of the 2 subunit (Fig. 2, A and B), and phosphoamino acid analysis showed that threonine phosphorylation was strongly increased in OKT3-stimulated T cells preincubated with OA (Fig. 2C). About 70% of the total phosphorylation of the 2 subunit occurred on threonine residues. Interestingly, the amount of threonine phosphorylation in PDBu-stimulated T cells treated with OA was only 30% (26). Tryptic phosphopeptide mapping was performed from the phosphorylated 2 subunit (Fig. 2D). In the map produced from OA-pretreated OKT3-activated T cells, two main spots were detected. The spot indicated by y is most likely produced by incomplete digestion with trypsin, because the spot becomes weaker when we prolonged the digestion conditions (data not shown). The phosphoamino acid analysis shows that the phosphorylation in the y-spot occurred mainly on threonine residues and only weakly on serine (not shown). The other spot (indicated by T) is the main phosphopeptide produced by 2 subunit digestion with trypsin. The phosphorylation of the T-spot occurred primarily on threonine residues (data not shown). The T-spot was isolated from a phosphopeptide map and subjected to manual Edman degradation, and the radioactive phosphate release was detected at cycle 3 (Fig. 2E). This result indicates that Thr758 in the 2 cytoplasmic domain is the main target of threonine phosphorylation in OKT3- and OA-stimulated T cells. Note that Ser756, the major site that becomes phosphorylated in response to PDBu, is not phosphorylated after CD3 stimulation.
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47-mediated adhesion to VCAM-1
TK-1 cells were used, because they express high levels of the 47 but do not express very late Ag 4 (41) (42). The 7-mediated adhesion-promoting effect of phorbol esters has been reported previously (4, 43). In contrast to prior reports, we detected some binding to coated VCAM-1 in the absence of activators (Fig. 3, A and B). However, 200 nM PDBu treatment resulted in a 3-fold enhancement in binding to coated human VCAM-1 (Fig. 3A). The increased binding is mediated by 47 as shown by the blocking effect of the anti-7 mAb Fib504 and a blocking Ab against the 4-chain. The Ab against mouse 2 integrin had no effect on adhesion. Moreover, we wanted to examine whether CD3 ligation may also lead to 7 integrin activation. We activated TK-1 cells via the TCR-CD3 complex using different concentrations of anti-CD3 mAb and studied 7-mediated binding to VCAM-1. As seen in Fig. 3B, the anti-CD3 mAb was able to activate TK-1 binding to VCAM-1. The induced adhesion was 47 dependent as shown by the inhibition of the adhesion to background levels with Fib504.
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47-mediated adhesion to VCAM-1. It was able to inhibit induced adhesion significantly but not totally (Fig. 3C).
Cell stimulation leads to increased phosphorylation of 7 and reveals threonine phosphorylation of 7
The above result obtained with okadaic acid indicated the involvement of serine/threonine phosphorylation in the regulation of 47-dependent functions. The 7 subunit has a serine residue in position Ser779 (corresponding to Ser756 in 2) and a similar triplet of threonines, which have been shown to become phosphorylated in the activated 2 cytoplasmic domain (Fig. 4). TK-1 cells were labeled with 32P and activated with 10 µg/ml anti-CD3 mAb or 200 nM PDBu or left untreated. Some samples were incubated with 1.5 µM OA before the cell activation. The 47 heterodimers were immunoprecipitated and subjected to SDS-PAGE. As shown in Fig. 5, A and B, the 7 subunit is phosphorylated even in unstimulated TK-1 cells. Activation of TK-1 cells with anti-CD3 (Fig. 5A) and PDBu (Fig. 5B) increased the phosphorylation 15–20%. A stronger enhancement of phosphorylation was observed when cells were incubated with OA before activation. OA pretreatment increased the phosphorylation of 7 by 70% in CD3-stimulated TK-1 cells as compared with control and to a similar extent in PDBu-activated cells. In the 4 subunit, we observed a weak and constitutive phosphorylation.
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7 integrin after activation indicated the possibility of qualitative changes in phosphorylation. Thus, we next analyzed phosphorylated amino acids by two-dimensional thin layer electrophoresis (Fig. 5C). In unactivated TK-1 cells, the phosphorylation of 7 occurred primarily on serine. OA alone without activating agent enhanced this serine phosphorylation, but no obvious phosphorylation was observed on threonine residues. Interestingly, threonine phosphorylation was increased in CD3-stimulated, and particularly, in PDBu-stimulated TK-1 cells preincubated with OA.
Identification of threonine phosphorylation sites in the 7 integrin
To determine the threonine phosphorylation site(s) in the 7 integrin, we made tryptic phosphopeptide maps from unactivated (Fig. 6A) and OA-pretreated, PDBu-activated TK-1 cells (Fig. 6B). The map from activated cells had one extra spot as compared with the map from unactivated cells (indicated by X). The phosphoamino acid analysis showed that the phosphorylation of this extra spot occurred mainly on threonine residues and a little on serine (not shown). Thus, the spot was isolated and subjected to manual Edman degradation. The amount of released radioactive phosphate was quantitated (Fig. 6C). The result shows that the fourth amino acid of the tryptic peptide was phosphorylated. This phosphorylated threonine residue is most likely the first threonine (Thr782) of threonine triplet (Thr782–784).
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7 does not affect adhesion in COS-1 cells
We mutated Thr782 to Ala and transfected the wild-type and mutated 47 integrins into COS-1 cells. Both the wild-type and mutated cells bound to a similar extent to coated VCAM-1 (Fig. 7). The binding of nontransfected cells was due to 1 integrins.
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subunits expressed on lymphocytes share a number of common features (Fig. 4), which have been shown to be important in integrin activation and functions. Mutation of the three threonine residues in the sequence 758–760 in the 2 cytoplasmic domain into an alanine triplet caused a dramatic decrease in the leukocyte integrin-mediated adhesion to the ICAM-1 (27). The threonine triplet has also been shown to be involved in regulating postreceptor events through 2 integrins, like cell spreading (30). Interestingly, the other leukocyte-specific integrin class, 7, contains a similar threonine triplet in its cytoplasmic domain. Additionally, the single amino acid Phe766, also found in 7 integrins, is vital for 2 functions. This residue has been hypothesized to be involved in the lymphocyte-specific signal transduction pathway that is required for activation of both 2 and 7 integrins (34).
The phosphorylation of integrin cytoplasmic tails has been proposed as a means of regulating integrin activity. The phosphorylation state of 2 leukocyte integrins has been reported to change in response to cell stimuli (21, 22, 23, 24). The major phosphorylation site has been identified to be Ser756 in the 2 cytoplasmic domain in PDBu-activated T cells (27). A strong threonine phosphorylation of the 2 subunit was observed by using OA (26). The only threonine residue present in 2 cytoplasmic domain is the functionally important threonine triplet. We have now shown that all three threonines, Thr758–760, act as substrates but only two in a given polypeptide. In contrast, if the T cells were stimulated through the TCR, only the first threonine residue in the threonine triplet became phosphorylated. PKC has been recently shown to be the main 2 kinase in leukocytes, and many PKC isoforms are capable of phosphorylating the 2 subunit in vitro (29). The sites phosphorylated by PKC
and PKC were identified as Ser745 and Thr758. PKC additionally phosphorylated Thr760. These results suggest that CD3 stimulation leads to the activation of PKC and/or PKC, but in contrast, PDBu can also activate other PKC isoforms, indicating that different signaling events take place under different activation conditions.
The 2 integrin has been observed to bind to ICAM-1 in response to CD3 cross-linking, without affecting the levels of expression of integrins (14, 15). In this study, we have investigated whether inside-out signaling, initiated by CD3 stimulation, can also regulate the 7 integrin. We showed that brief CD3 stimulation induced 47 integrin-mediated binding to VCAM-1. Previous studies have shown that CD3 stimulation increases the expression levels of the 7 integrin subunit on cell surface (4, 46). However, the effect of CD3 activation on 47 adhesion was so rapid that we suggest that CD3 ligation affects the function of 47 molecules already present on the cell surface.
In contrast to the 2 integrin, the 7 subunit was already phosphorylated in unactivated TK-1 cells. However, the phosphorylation of 7 occurred only on serine residues in unactivated TK-1 cells. This difference may be due to the properties of different types of cells, because TK-1, as a cell line, is continuously dividing. Cell activation with PDBu or CD3 Ab increased the phosphorylation, but only weak phosphorylation was observed on threonine residues. In PDBu- and CD3-stimulated TK-1 cells, the inhibition of phosphatase activity induced significant phosphorylation of threonine in addition to serine. OA alone enhanced serine phosphorylation, but no phosphorylation was observed on threonine residues. This result indicates the presence of a similar continuous threonine phosphorylation/dephosphorylation cycle in 7 subunit as in 2.
The phosphorylation site was determined to be Thr782 corresponding to the first threonine of the threonine triplet in 2. The same site was phosphorylated in the 2 subunit after cell activation. The phosphorylation site could be on a tryptic peptide next to TTT-peptide, because this peptide also has a threonine residue in position four (Fig. 4). However, this alternative threonine residue is found only in mouse 7, and it is unlikely that this is the phosphorylated residue.
The role of threonine phosphorylation in the regulation of integrin activity is still unclear. Phosphorylated 2 integrins have been shown to preferentially associate with the actin cytoskeleton (31), indicating that phosphorylation of integrins could regulate integrin-cytoskeleton interactions. The phosphatase sensitivity of the threonine phosphorylation of leukocyte integrins indicates the possibility of a rapid attachment and detachment of integrins from the cytoskeleton. A possibility is that the cytoskeleton-integrin linkage is mediated by 14-3-3 proteins, which have recently been found to interact specifically with Thr758-phosphorylated 2 integrins (29). The 14-3-3 proteins could possibly initiate a signaling complex formation, further regulating integrin functions.
The threonine phosphorylation has been speculated to have a role also in other integrins. The 3 subunit has a Thr-Ser-Thr sequence corresponding to the threonine triplet in 2. The 3 integrin was found to be phosphorylated mainly on threonine (47). Threonine phosphorylation was speculated to have a negative role on 3 integrin inside-out and outside-in signaling (48), possible by inhibiting the binding of tyrosine-phosphorylated integrins to Shc, a signaling protein (49). However, phosphorylation of 3 integrins has also been speculated to be a major factor in the control of exposure of binding sites for adhesive proteins in the IIb3 integrin complex (50). Mutation of these threonine residues in the integrin 3 cytoplasmic domain has been reported to inhibit cell attachment, cell spreading, and extracellular domain conformation changes (51, 52). Threonine phosphorylation of 1 has not been reported so far, but the VTT sequence in the 1 integrin cytoplasmic domain has been shown to be important for a number of integrin-dependent functions, including signaling, conformational changes in the extracellular domain, cell attachment, and cell spreading (52, 53). However, the functional importance of the phosphorylated Thr782 in 7 remains unclear, because mutation to alanine did not affect adhesion. This finding does not rule out other possible functions of this phosphorylation and needs further study. Another problem may be that COS-1 cells, being monkey cells, do not possess intracellular interactive molecules normally found in T cells.
Both PDBu and CD3 ligation induce leukocyte integrin phosphorylation also on serine residues (21, 22, 23). The main PDBu-induced serine phosphorylation site in 2 integrin was found to be Ser756, but Ser756 is not essential in the activation of 2 integrins (27). Ser756 has been speculated to be involved in so-called postreceptor events, like cell spreading, that are regulated by different signaling events than integrin activation (45). The corresponding serine is not found in 3, but mutation of the corresponding serine in 1 integrin (Ser785) to methionine has been shown to promote cell spreading and directed migration, but inhibit cell attachment to laminin (54). In contrast, Ser785 mutation to aspartate in 1, which to some degree mimics a constitutively phosphorylated residue, has been shown to reduce localization at focal contacts (55). Interestingly, Ser756 of 2 is not phosphorylated in CD3-stimulated T cells. However, serine phosphorylation is also observed in 2 after CD3 stimulation. It has been shown that Ser745 becomes phosphorylated when T cells are stimulated with PDBu, and that phosphorylation could be increased in the presence of OKT3 plus OA (29). But OKT3 alone in the presence or absence of OA did not induce Ser745 phosphorylation (our unpublished data). In contrast, the stoichiometry of phosphorylation is very low (28), so it is possible that Ser745 phosphorylation escapes detection. The role of Ser756 and Ser745 phosphorylation in 2 is unknown at present.
In conclusion, we have mapped the phosphorylation sites of leukocyte-specific integrins. The phosphorylation of 2 integrin occurs on the first threonine of the threonine triplet in OA-pretreated CD3-stimulated T cells. A similar induced threonine phosphorylation was observed in the 7 integrin. The functional importance of threonine phosphorylation needs further study.
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Acknowledgments |
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4 and 7 cDNAs. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Carl G. Gahmberg, Department of Biosciences, Division of Biochemistry, University of Helsinki, P.O. Box 56 (Viikinkaari 5), FIN-00014 Helsinki, Finland. E-mail address: carl.gahmberg{at}helsinki.fi
3 Abbreviations used in this paper: OA, okadaic acid; PKC, protein kinase C; PDBu, phorbol 12,13-dibutyrate; MS/MS, tandem mass spectrometry.
Received for publication June 21, 2002. Accepted for publication February 12, 2003.
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cytoplasmic domains to regulate "in trans" 1 integrin conformation and function in cell attachment. J. Cell Sci. 112:217.[Abstract]
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1 cytoplasmic domain modulates 1A-integrin-dependent functions. J. Cell Sci. 114:2525.1 integrin localisation. Exp. Cell Res. 222:10.[Medline]This article has been cited by other articles:
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  E. Y. Choi, V. V. Orlova, S. C. Fagerholm, S. M. Nurmi, L. Zhang, C. M. Ballantyne, C. G. Gahmberg, and T. Chavakis Regulation of LFA-1-dependent inflammatory cell recruitment by Cbl-b and 14-3-3 proteins Blood, April 1, 2008; 111(7): 3607 - 3614. [Abstract] [Full Text] [PDF]
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Y. Tanaka, T. Era, S.-i. Nishikawa, and S. Kawamata Forced expression of Nanog in hematopoietic stem cells results in a {gamma}{delta}T-cell disorder Blood, July 1, 2007; 110(1): 107 - 115. [Abstract] [Full Text] [PDF]
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 S. M. Nurmi, M. Autero, A. K. Raunio, C. G. Gahmberg, and S. C. Fagerholm Phosphorylation of the LFA-1 Integrin beta2-Chain on Thr-758 Leads to Adhesion, Rac-1/Cdc42 Activation, and Stimulation of CD69 Expression in Human T Cells J. Biol. Chem., January 12, 2007; 282(2): 968 - 975. [Abstract] [Full Text] [PDF]
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) or presence (
) of 10 µg/ml Fib504, the blocking Ab against 
) or T782A