A Molecular Mechanism of Integrin Crosstalk: alpha vbeta 3 Suppression of Calcium/Calmodulin-dependent Protein Kinase II Regulates alpha 5beta 1 Function

Scott D. Blystone,* Suzanne E. Slater,Dagger Matthew P. Williams,* Michael T. Crow,§ and Eric J. Brownparallel

* Department of Anatomy and Cell Biology, State University of New York, Health Science Center at Syracuse, Syracuse, New York 13210; Dagger  Department of Medicine, Infectious Diseases Division, Washington University School of Medicine, St. Louis, Missouri, 63110; § Vascular Biology Unit, Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224; and parallel  Program in Microbial Pathogenesis and Host Defense, University of California, San Francisco, California 94143

    Abstract
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Many cells express more than one integrin receptor for extracellular matrix, and in vivo these receptors may be simultaneously engaged. Ligation of one integrin may influence the behavior of others on the cell, a phenomenon we have called integrin crosstalk. Ligation of the integrin alpha vbeta 3 inhibits both phagocytosis and migration mediated by alpha 5beta 1 on the same cell, and the beta 3 cytoplasmic tail is necessary and sufficient for this regulation of alpha 5beta 1. Ligation of alpha 5beta 1 activates the calcium- and calmodulin-dependent protein kinase II (CamKII). This activation is required for alpha 5beta 1-mediated phagocytosis and migration. Simultaneous ligation of alpha vbeta 3 or expression of a chimeric molecule with a free beta 3 cytoplasmic tail prevents alpha 5beta 1-mediated activation of CamKII. Expression of a constitutively active CamKII restores alpha 5beta 1 functions blocked by alpha vbeta 3-initiated integrin crosstalk. Thus, alpha vbeta 3 inhibition of alpha 5beta 1 activation of CamKII is required for its role in integrin crosstalk. Structure-function analysis of the beta 3 cytoplasmic tail demonstrates a requirement for Ser752 in beta 3-mediated suppression of CamKII activation, while crosstalk is independent of Tyr747 and Tyr759, implicating Ser752, but not beta 3 tyrosine phosphorylation in initiation of the alpha vbeta 3 signal for integrin crosstalk.

Key words: integrin;  vitronectin;  kinase;  cross- talk;  signaling
    Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

DYNAMIC interaction of cells with the complex protein mixtures found in the extracellular matrix occurs during many biologic and pathologic processes including development, wound healing, hemostasis, metastasis, inflammation, and thrombosis (13, 18). Most cells express multiple integrin receptors capable of interaction with the numerous ligands found in complex tissues. The simultaneous ligation of multiple integrins mandates coordination of the resulting signals. The coordination of integrin signaling into a hierarchy with a net effect on cell behavior has been called integrin crosstalk (2, 3, 17).

Numerous examples of integrin crosstalk have been reported. The common theme of these reports lies in the regulation of the function of one integrin (the target) as a result of coligation of a second integrin (the transducer) on the same cell. Examples of integrin crosstalk have been demonstrated in numerous primary cell types and cell lines including macrophages (2), T cells (17, 23), smooth muscle cells (1), neutrophils (10), monocytes (15), umbilical vein endothelial cells (20), malignant astrocytomas (16), CHO cells (7, 9), K562 cells (2, 3), and embryonic kidney 293 cells (20). Crosstalk may be initiated by transducing integrins belonging to the beta 1 (11, 15, 16, 22), beta 2 (17, 23), or beta 3 (1, 7, 9, 10, 20) family with targets in any of these families as well. Integrin functions affected by crosstalk include phagocytosis (2, 3, 10), soluble ligand binding (7, 15), adhesion (9, 17, 23), migration (1, 17, 20), gene expression (11), and receptor-mediated endocytosis (16).

It is important to note that all reported cases of integrin crosstalk are unidirectional, that is, ligation of the target integrin does not affect the transducer integrin. In many cases, the transducing integrin is much less highly expressed than the target integrin (2, 3). This suggests a hypothesis that the receptor pairs involved in crosstalk are not simply competing for interaction with a signaling molecule, but rather that ligation of the transducing integrin initiates a unidirectional signaling cascade which affects the function of the target integrin. The molecular mechanisms of integrin crosstalk remain undetermined. With a single exception (20), crosstalk signals from the transducing integrin require the cytoplasmic tail of the beta -subunit, and where it has been examined, the beta -subunit cytoplasmic tail has been sufficient for initiation of signaling (3).

We have previously described integrin crosstalk between alpha vbeta 3 and alpha 5beta 1 in macrophages and in a K562 cell transfection model of macrophage integrins (2, 3). We have shown that ligation of alpha vbeta 3 inhibits alpha 5beta 1-mediated phagocytosis, which requires the high affinity state of the integrin, without affecting alpha 5beta 1-mediated adhesion, which is independent of the high affinity state of the integrin. The cytoplasmic tail of beta 3 is necessary and sufficient for this crosstalk. alpha vbeta 3-mediated inhibition of alpha 5beta 1 phagocytosis occurs at a step subsequent to alpha 5beta 1 binding of ligand and is reversed by H7, a pharmacologic inhibitor of serine/ threonine kinases.

In this report, we define a molecular mechanism required for alpha vbeta 3-to-alpha 5beta 1 crosstalk. Ligation of alpha 5beta 1 enhances the activity of the calcium/calmodulin-dependent protein kinase II (CamKII)1. This increase in CamKII activity is required for alpha 5beta 1-dependent migration as well as alpha 5beta 1-dependent phagocytosis. Simultaneous ligation of alpha vbeta 3 inhibits alpha 5beta 1 activation of CamKII activity, thus blocking alpha 5beta 1 migration and phagocytosis. Mutational analysis of the beta 3 cytoplasmic tail demonstrates that Ser752 is required for both alpha vbeta 3-initiated inhibitory crosstalk to alpha 5beta 1 and alpha vbeta 3 suppression of CamKII activity, while tyrosine phosphorylation of the beta 3 cytoplasmic tail has no effect on this activity. These results describe a potential molecular pathway for integrin crosstalk that involves integrin regulation of CamKII activity.

    Materials and Methods
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cells and beta 3 cDNA Mutation

Human peripheral blood monocyte-derived macrophages were prepared as previously described (2). The human erythroleukemic cell line K562 transfected with cDNA encoding alpha vbeta 3 (Kalpha vbeta 3), alpha vbeta 3 in which the tyrosine residue at position 747 or 759 was mutated to phenylalanine (Kalpha vbeta 3Y747F and Kalpha vbeta 3Y759F, respectively) and Tacbeta 3 (chimera of the extracellular and transmembrane domain of the IL2 receptor alpha -chain [Tac subunit] and the cytoplasmic tail of beta 3, KTacbeta 3) were derived and maintained as described (2, 3). In addition K562 cells were similarly transfected with cDNA encoding Tacbeta 3 in which the serine residue at position 752 was replaced with proline (KTacbeta 3S-P), cysteine (KTacbeta 3S-C), alanine (KTacbeta 3S-A), or glutamic acid (KTacbeta 3S-E). Expression of all Tacbeta 3S-X chimeras was equivalent to Tacbeta 3 (3) as determined by flow cytometry as described (2; see Table I). For construction of Tacbeta 3S-X, the HindIII and XhoI fragment of pTacbeta 3 encoding the CT of beta 3 (3) was ligated into HindIII-XhoI-digested pBluescript (Stratagene), creating pBSKSPB3TAIL. This contruct was subjected to PCR using a 5' T7 oligonucleotide (Stratagene) with the 3' oligonucleotide (5'-CCCCCCTCGAGTTAAGTGCCCCGGTACGTGATATTGGTGAAGGT-XXX-CGTGGC-3') where XXX is AGG for S752P, ACA for S752C, GCT for S752A, and TTC for S752E. The resulting products were digested with HindIII-XhoI and ligated into pTacbeta 3 digested with HindIII-XhoI, creating pTacbeta 3S-P, pTacbeta 3S-C, pTacbeta 3S-A, and pTacbeta 3S-E, respectively.

                              
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Table I
Integrin Expression in Transfected K562 Cells

K562 cells also were transfected with full-length alpha vbeta 3 in which the serine at position 752 of beta 3 was mutated to alanine as described for the mutation of beta 3 tyrosine residues (4). In brief, nested PCR was performed on pBLY100 using the overlapping oligonucleotides 5'-GAGGCCACGCCTACCTTCACCAATATCACG-3' and 5'-CTCCGGTGCGGATGGAAGTGGTTATAGTGC-3' encoding the S-A mutation with oligonucleotides in the mutation cassette (4). After the nested PCR reaction, the wild-type beta 3 CT was replaced with the S-A mutant CT by NdeI-NheI restriction. Transfection, selection, and fluorescent cell sorting for expression levels of alpha vbeta 3S752A equivalent to wild-type alpha vbeta 3 was as described previously, resulting in Kalpha vbeta 3S752A (Table I). Modified cDNAs were verified by dideoxy nucleotide sequencing.

Phagocytosis, Adhesion, and Migration

Phagocytosis assays were performed as described (2) by flow cytometry using either FITC-FN- or FITC-mAb16 (anti-alpha 5)-coated 3.0-µm beads. Data are presented as a Phagocytic Index, the number of beads internalized per 100 cells.

Chemotaxis assays were performed in modified Boyden chambers (Neuroprobe) using 14.0-µM polycarbonate filters as described (19). Vitronectin (VN), fibronectin (FN), and BSA were added to basal chambers at 5 µg/ml and mAb at 10 µg/ml were added to apical chambers coincident with cells. Cells in Iscove's Modified Eagle's Medium (IMDM) adjusted to 1 mM Ca2+ and 1 mM Mg2+ with 0.5% human serum albumin and 2.0 mM Mn2+Cl were incubated for 4 h at 37°C in a humidified 5% CO2 atmosphere for migration. Migration was quantitated by counting the number of cells per high power field on the underside of the filter after Giemsa staining.

Adhesion assays were performed as described in FN-coated (10 µg/ml) microtiter wells (2). Data are presented as the percent of added cells adherent after 1 h at 37°C.

CamKII Activity Assay

Transfected K562 cells or monocyte-derived macrophages were stimulated as described in the text, washed once by centrifugation in ice-cold IMDM and suspended in ice-cold homgenization buffer containing Hepes (50 mM), EDTA (4 mM), EGTA (2 mM), sucrose (0.25 M), dithiothreitol (1 mM), phenylmethylsulfonyl fluoride (0.2 mM), Na3VO4 (2.0 mM), NaF (5.0 mM), phenyl-arsine-oxide (10.0 mM), and leupeptin (10 µg/ml), pH 7.5. Suspended cells were sonicated on ice and assayed for CamKII activity against the synthetic substrate autocamtide II (KKALRRQETVDAL) (21). An aliquot of cell extracts was used for protein determination by BCA. Parallel aliquots were assayed for CamKII activity in a 25-µl reaction mixture containing Hepes (50 mM), magnesium acetate (10 mM), Na3VO4 (2.0 mM), NaF (5.0 mM), phenyl-arsine-oxide (10.0 mM), CaCl2 (1 mM), calmodulin (0.1 µM; Sigma Chemical Co.), autocamtide II (20 µM), and gamma -[32P]ATP (0.1 mM, 3,000 cpm/pmol). The reaction was initiated by ATP addition and terminated by addition of trichloroacetic acid to a final concentration of 10%. The reaction mixture was centrifuged through phosphocellulose separation units (Pierce) and washed as described (26). CamKII activation results from phosphorylation events that result in kinase activity which is no longer dependent upon exogenous calcium or calmodulin. CamKII activity in cellular extracts was measured by quantitating the incorporation of radioactive phosphate into a synthetic CamKII substrate (autocamtide-2) in the presence (calcium/calmodulin-independent + calcium/calmodulin-dependent activity) or absence (calcium/calmodulin-independent activity) of calcium and calmodulin. The activation of CamKII (autonomous activity) is expressed as a direct percentage of the total cellular CamKII activity (1) in which:
% CamKII activity=<FR><NU>Autonomous activity </NU><DE>Total activity</DE></FR>×100

where autonomous activity equals the CamKII activity without calcium or calmodulin and total activity equals CamKII activity with calcium or calmodulin.

CamKII expression levels in transfected cell lines was assessed by immunoprecipitation as previously described, followed by Western blot analysis (1).

Infection of K562 Cells with Adenovirus Encoding Constitutively Active CamKII

Kalpha vbeta 3, KTacbeta 3, and KTacbeta 5 were infected with a replication defective adenovirus in which the E1 region was replaced with the CMV early promoter and the cDNA for a constitutively active CamKII (AdCMV.CKIID3) or beta -galactosidase (AdCMV.gal) and viral stocks propagated and titered as described (1). Transfected K562 cells at 5 × 106/ml in IMDM were infected with recombinant adenovirus at a multiplicity of infection of 100 for 1 h followed by the addition of normal growth medium to dilute cells to a concentration of 0.5 × 106/ml. After 4-6 h, cells were harvested for analysis of CamKII activity or functional assay as described in Results. Viability of all infected cell types exceeded 85% at the initiation, and 70% at the conclusion of experimental time courses.

Proteins and Antibodies

FN was purified by gelatin affinity and VN by heparin affinity as previously described (2, 3). Monoclonal antibodies 7G2 (anti-human beta 3), W6/ 32 (anti-human HLA), IC12 (anti-human alpha v), AP3 (anti-human beta 3), P1F6 (anti-human beta 5), 4E3 (anti-IL2Ralpha , gp55, TAC), and mAb16 (anti- human alpha 5) have been previously described and were used in excess at 5.0 µg/ml unless otherwise indicated (2, 3).

Reagents

The kinase inhibitors H7 (50 nM), KN62 (2.5 µM), KN04 (5.0 µM), KT5926 (20 nM), and ML-9 (2 µM) were included in some assays where indicated and all were from LC Laboratories (Woburn, MA). All other reagents were from Sigma Chemical Co. unless otherwise indicated.

Data Presentation

Data are presented as the mean ± SEM from at least three replicates for all studies. Significance was determined by analysis of variance followed by Duncan's comparison testing. A minimum confidence interval of 95% was employed for all studies.

    Results
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

alpha vbeta 3 Crosstalk Regulates alpha 5beta 1-mediated Migration

We have previously described a phenomenon, termed integrin crosstalk, in which ligation of alpha vbeta 3 prevents alpha 5beta 1-mediated phagocytosis in macrophages and in K562 cells expressing transfected alpha vbeta 3. To determine if integrin crosstalk regulated alpha 5beta 1 functions other than phagocytosis, we evaluated the effects of alpha vbeta 3 ligation on the migration of K562 cells on the alpha 5beta 1 ligand FN. K562 cells did not migrate specifically to FN in IMDM containing 1 mM Ca2+ and 1 mM Mg2+. However, addition of 2 mM Mn2+ or the alpha 5beta 1 conformation-stabilizing mAbs 8A2 or A1A5 at 5.0 µg/ml greatly enhanced the FN-specific migration of these cells, consistent with a requirement for high affinity alpha 5beta 1 in migration (data not shown). As shown in Fig. 1 A, the migration of untransfected K562 to FN in the presence of 2 mM Mn2+ was enhanced sixfold over migration to the nonspecific protein casein; this migration was completely inhibited by mAb to alpha 5beta 1 (data not shown). We also examined alpha vbeta 3-mediated migration in K562 expressing this transfected integrin in addition to the endogenous alpha 5beta 1. Kalpha vbeta 3 migrated in response to VN (Fig. 1 A); migration response to VN was inhibited by mAb to alpha v or beta 3 (data not shown). However, migration of Kalpha vbeta 3 to FN was severely impaired compared with untransfected or mock transfected K562 (Fig. 1 A). Migration of Kalpha vbeta 3 to FN was restored by the addition of the ser/thr kinase inhibitor H7 (50 nM), while addition of H7 had no effect on Kalpha vbeta 3 migration to VN (data not shown). Restored migration of Kalpha vbeta 3 to FN in the presence of H7 was completely inhibited by mAb to beta 1 (data not shown). These results completely parallel the previously described alpha vbeta 3-mediated crosstalk which inhibits alpha 5beta 1-mediated phagocytosis (3) and support the hypothesis that the coligation of alpha vbeta 3 by FN regulates alpha 5beta 1-mediated K562 cell migration to FN because this function, like phagocytosis, requires a high affinity form of alpha 5beta 1.


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Fig. 1.   alpha vbeta 3 crosstalk regulates alpha 5beta 1-mediated migration. Untransfected K562 cells, Kalpha vbeta 3, KTacbeta 3, and KTacbeta 5 were permitted to migrate to vitronectin (VN), fibronectin (Fn) or casein through 14.0-µM pores in a modified Boyden chamber apparatus as described in Materials and Methods (A). For structure-function analysis of integrin crosstalk, untransfected K562 cells and K562 cells expressing transfected wild-type alpha vbeta 3 (Kalpha vbeta 3) or alpha vbeta 3 bearing single amino acid mutations Tyr747-Phe (Kalpha vbeta 3Y747F), Tyr759-Phe (Kalpha vbeta 3Y759F) or Ser752-Ala (Kalpha vbeta 3S752A) were assayed for migration to Fn, VN, or casein as described in Materials and Methods (B). Migration was quantitated manually by counting cells adherent to the basal side of the filter and reported as the number of cells per well. Shown are mean ± SEM of four determinations with no fewer than eight replicates.

To demonstrate definitively that alpha vbeta 3 regulation of alpha 5beta 1-mediated migration was another example of integrin crosstalk, we examined migration to FN in KTacbeta 3 and KTacbeta 5, K562 cells expressing chimeric molecules comprised of the extracellular domain of the IL2 receptor and the cytoplasmic tail domain of the beta 3 or beta 5 integrin, respectively. Expression of Tacbeta 3, but not Tacbeta 5, leads to constitutive inhibition of alpha 5beta 1-mediated phagocytosis in K562 cells (3; see Fig. 7 B). Expression of Tacbeta 3, but not Tacbeta 5 (Fig. 1 A) or Tac lacking a cytoplasmic tail (KTacNT, Fig. 2 A), completely inhibited alpha 5beta 1-mediated migration to FN. The constitutive inhibition of migration to FN in KTacbeta 3 was reversed by the addition of 50 nM H7 (Fig. 2 A). These studies demonstrate that alpha 5beta 1-mediated migration and alpha 5beta 1-mediated phagocytosis are similarly regulated by alpha vbeta 3 or the isolated beta 3 CT and that this regulation is dependent upon a ser/thr kinase regulated by H7. These data suggest that both alpha 5beta 1-mediated migration and alpha 5beta 1-mediated phagocytosis are regulated by alpha vbeta 3-initiated crosstalk.


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Fig. 2.   Role of beta 3 Ser752 in alpha vbeta 3 crosstalk. K562 cells transfected with vector alone (Rc/RSV) or a chimeric molecule composed of the extracellular and transmembrane domains of the IL2 receptor (Tac) and the cytoplasmic tail of integrin subunits; no tail (TacNT), beta 3 (Tacbeta 3), beta 5 (Tacbeta 5), or beta 3 in which Ser752 had been mutated to Pro (Tacbeta 3S-P), Cys (Tacbeta 3S-C), Ala (Tacbeta 3S-A), or Glu (Tacbeta 3S-E) were evaluated for their ability to migrate to FN (A), phagocytose FN opsonized beads (B), and adhere to FN-coated tissue culture plastic (C) as described in Materials and Methods. Parallel samples were treated with 50 nM H7 as indicated. Shown are mean ± SEM of no fewer than three determinations performed in triplicate.

beta 3 Ser752 Is Required for beta 3 Crosstalk

We have previously demonstrated that expression of the isolated beta 3 cytoplasmic tail is sufficient for initiation of alpha vbeta 3 crosstalk (Fig. 1 A and reference 12). To further delineate the required sequence elements of this unique regulatory pathway, we introduced point mutations in the beta 3 cytoplasmic tail and analyzed their effects upon alpha vbeta 3-initiated crosstalk to alpha 5beta 1-mediated migration.

In a spontaneously occurring Glanzmann's Thrombasthenia mutation, the serine residue at position 752 of the beta 3 CT is mutated to proline (6). This mutation results in loss of platelet beta 3 function and a severe bleeding disorder. In vitro study has shown that Ser752 of the beta 3 CT is required for the conformational change associated with elevated affinity of beta 3 for ligand (8). To test whether Ser752 also is required for integrin crosstalk, we expressed an alpha vbeta 3 receptor in K562 cells in which Ser752 of beta 3 was mutated to Ala (Fig. 1 B). While the ligation of wild-type alpha vbeta 3 blocked alpha 5beta 1-mediated migration on FN (Fig. 1 B), the S752A mutant migrated as well as the untransfected cells. In addition, the S752A mutant migrated as well as wild-type alpha vbeta 3 on VN (Fig. 1 B), consistent with reports that this mutation does not affect ligand binding by beta 3 integrins (8). This demonstrates that failure of the S752A mutant to initiate crosstalk did not result from an inability to recognize ligand.

Recently, a tyrosine in the beta 3 cytoplasmic tail, Tyr747, has been implicated in activation-dependent alpha vbeta 3 adhesion to VN (4). In contrast to the S752A mutation, Y747F had no effect on alpha vbeta 3-initiated integrin crosstalk (Fig. 1 B). Consistent with the previous report of a requirement for this tyrosine in firm adhesion, the Y747F mutation did abolish migration of Kalpha vbeta 3Y747F to VN (Fig. 1 B). Mutation of Tyr759 to Phe (Y759F) did not affect either crosstalk or the migration function of alpha vbeta 3. These data demonstrate that the crosstalk signaling and adhesive functions of alpha vbeta 3 have distinct and independent sequence requirements in the beta 3 cytoplasmic tail.

To evaluate further the requirement for beta 3 S752 in integrin crosstalk, additional mutations at that position were made in the consititutively inhibitory Tacbeta 3 construct. Mutation of Ser752 to Glu, Pro, or Cys as well as Ala abolished the inhibitory activity of Tacbeta 3 on alpha 5beta 1-dependent migration (Fig. 2 A) and alpha 5beta 1-dependent phagocytosis (Fig. 2 B). Like the wild-type beta 3 cytoplasmic tail, none of the mutants affected K562 binding to FN-coated surfaces, a function that does not require the high affinity state of alpha 5beta 1 (Fig. 2 C). The addition of H7 reversed the Tacbeta 3 inhibition of alpha 5beta 1-mediated migration and phagocytosis (Fig. 2, A and B).

alpha 5beta 1 and alpha vbeta 3 Differentially Regulate CamKII

alpha vbeta 3 ligation inhibits the alpha 5beta 1 high affinity functions of phagocytosis and migration, without effect upon alpha 5beta 1-mediated adhesion. Alterations in alpha 5beta 1 affinity can be regulated by calcineurin, a calcium/calmodulin-dependent phosphatase and CamKII (calcium/calmodulin-dependent protein kinase II; reference 1). Recently, inhibition of CamKII activity by alpha vbeta 3 ligation in smooth muscle cells was reported (1). Therefore, we evaluated alpha vbeta 3 regulation of CamKII as a potential mediator of alpha vbeta 3-initiated crosstalk.

CamKII activity was measured in human monocyte-derived macrophages in the presence and absence of an alpha 5beta 1-specific phagocytosis target (mAb-16-coated latex beads) (3). Ligation of macrophage alpha 5beta 1 with mAb-16 beads enhanced CamKII activity twofold, while ligation with a control target (W6/32 beads) had no effect (Fig. 3 A). Both basal and stimulated CamKII activities were decreased by the CamKII inhibitor KN62, but not the structurally related, but non-inhibitory KN04. Ligation of alpha vbeta 3 with soluble mAb 7G2 prevented the rise in CamKII activity induced by mAb-16 beads (Fig. 3 A). No additional decrease in CamKII activity was detected when KN62 and 7G2 were combined. Thus, beta 3 ligation prevented the alpha 5beta 1-induced rise in CamKII activity.


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Fig. 3.   Regulation of CamKII activity by alpha 5beta 1 and alpha vbeta 3. Human monocyte-derived macrophages (A) or K562 cells expressing transfected alpha vbeta 3 (Kalpha vbeta 3, B) or integrin beta 5 (KTacbeta 5) or beta 3 (KTacbeta 3) cytoplasmic tail chimeras (C) were assayed for activity of the calcium- and calmodulin-dependent protein kinase II against a synthetic substrate as described in Materials and Methods. Cells were either left unstimulated (no beads) or presented with control phagocytosis targets (W6/32 beads) or with an alpha 5beta 1-specific phagocytosis target (mAb-16 beads) in the presence and absence of KN62 (2.5 µM), KN04 (5.0 µM) or 5.0 µg/ml mAb 7G2 either individually or in combinations as indicated. D shows the inhibition of CamKII activity in Kalpha vbeta 3 after alpha 5beta 1 ligation with mAb-16 beads by soluble mAb 7G2 (5.0 µg/ml), 7G2 Fab' (3.8 µg/ml), and 2.0 mM GRGDSP peptide and the lack of inhibition by mAb P1F6 or 2 mM GRGESP. Regulated CamKII activity is presented as the percent of total activity as described in Materials and Methods. Shown are mean ± SEM for no fewer than three determinations in each group of all panels.

To explore further the hypothesis that CamKII mediates alpha vbeta 3 regulation of alpha 5beta 1, we evaluated the regulation of CamKII in Kalpha vbeta 3. Binding of mAb-16 beads to Kalpha vbeta 3, and to vector-transfected K562 (data not shown), resulted in an increase in CamKII activity (Fig. 3 B) that was not seen when Kalpha vbeta 3 were incubated with W6/32 beads that bound to the cells equivalently. As in macrophages, the alpha 5beta 1-mediated rise in CamKII activity was prevented by ligation of alpha vbeta 3 with soluble mAb 7G2 (Fig. 3 B) and by 7G2 Fab fragments or Arg-Gly-Asp peptide (Fig. 3 D). As seen in macrophages, inhibition of the alpha 5beta 1-induced increase in CamKII activity by alpha vbeta 3 ligation was blocked by KN62, but not KN04.

Previously we have demonstrated that the cytoplasmic tail of beta 3 is both necessary and sufficient for alpha vbeta 3 inhibitory crosstalk to alpha 5beta 1 (reference 3 and Fig. 2, A and B). In the presence of mAb-16 beads, expression of Tacbeta 3, but not Tacbeta 5, prevented the alpha 5beta 1-mediated rise in CamKII activity (Fig. 3 C). These results indicate that alpha 5beta 1 and alpha vbeta 3 differentially regulate CamKII activity in macrophages and K562 cells.

To determine if the failure of the Kalpha vbeta 3S752A to initiate crosstalk was related to an inability to regulate CamKII, we evaluated CamKII activity after mAb-16 bead binding in K562 cells transfected with wild-type alpha vbeta 3 and the S752A and Y747F mutants. While ligation of alpha vbeta 3 with the beta 3-specific mAb 7G2 suppressed mAb-16 bead-induced activation of CamKII, mutation of Ser752 of beta 3 prevented the suppression of CamKII activity seen upon alpha vbeta 3 ligation (Fig. 4 A). However, mutation of Tyr747 or Tyr759 (data not shown) did not affect alpha vbeta 3 regulation of CamKII. Thus, Ser752 is required for both alpha vbeta 3 inhibitory crosstalk to alpha 5beta 1 (Fig. 2) and alpha vbeta 3 regulation of CamKII (Fig. 4).


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Fig. 4.   Role of beta 3 Ser752 in alpha vbeta 3 regulation of CamKII. Untransfected K562 cells or K562 cells expressing transfected wild-type alpha vbeta 3 (Kalpha vbeta 3) or alpha vbeta 3 in which Tyr747 was mutated to Phe (Kalpha vbeta 3Y747F) or in which Ser752 was mutated to Ala (Kalpha vbeta 3S752A) were assayed for CamKII activity (A) as described in Materials and Methods, after alpha 5beta 1 ligation with mAb-16 beads in the presence or absence of the alpha vbeta 3 ligand 7G2 (5.0 µg/ml). Shown are mean ± SEM of three determinations with no fewer than three replicates. Also as described in Materials and Methods, total cellular CamKII (see arrow) from transfected K562 cell populations used in this study was recovered by immunoprecipitation and revealed by Western blotting (B). For untransfected K562 cells, cell lysates were cleared by immunoprecipitation with anti-CamKII twice (cleared 2×) or once (cleared 1×), before analysis of the final immunoprecipitation to ensure that recovery of protein was complete. Shown is a representative study.

To determine the effect of alpha vbeta 3 and mutant beta 3 on expression of CamKII, immunoprecipitates of CamKII were analyzed by Western blot with CamKII-specific Ab. As shown in Fig. 4 B, cellular expression of CamKII (see arrow) was unchanged by the expression of alpha vbeta 3 and mutants in transfected K562 cells.

Role of CamKII in alpha vbeta 3 Crosstalk to alpha 5beta 1

Suppression of the alpha 5beta 1-dependent increase in CamKII activity by alpha vbeta 3 ligation or by Tacbeta 3 expression suggested that CamKII regulation could have a role in alpha vbeta 3 crosstalk to alpha 5beta 1. To determine the role of CamKII in alpha vbeta 3 crosstalk to alpha 5beta 1, Kalpha vbeta 3 cells were incubated with the alpha 5beta 1 phagocytosis target, mAb-16 beads, or control target, P1F6 (anti-alpha vbeta 5) beads. Phagocytosis was measured in the presence and absence of 7G2 to ligate alpha vbeta 3, the CamKII inhibitor KN62, or control KN04. As reported previously, phagocytosis via alpha 5beta 1 was inhibited upon alpha vbeta 3 ligation with mAb 7G2 (2, 3). alpha 5beta 1 phagocytosis also was inhibited by KN62 (Fig. 5 A), but not KN04. Combining 7G2 and KN62 resulted in no further decrease in alpha 5beta 1 phagocytosis. Under all conditions, there was no significant internalization of P1F6 beads.


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Fig. 5.   Effects of CamKII inhibitors on alpha 5beta 1 function and alpha vbeta 3 crosstalk. Phagocytosis of the alpha 5beta 1-specific target mAb-16 beads, or control target P1F6-beads was evaluated in K562 expressing alpha vbeta 3 in the presence and absence of alpha vbeta 3 ligation by mAb 7G2 (5.0 µg/ml) and/or the CamKII inhibitor KN62 (2.5 µM) or control KN04 (5.0 µM) (A) as described in Materials and Methods. Shown are mean ± SEM of three determinations with four replicates each. alpha 5beta 1-mediated phagocytosis of FN beads was evaluated in untransfected K562 cells in the presence and absence of KN62 (2.5 µM), KN04 (5.0 µM), or the beta 3-specific mAb 7G2 (5.0 µg/ml) (B) as described in Materials and Methods. Shown are the mean ± SEM of two determinations with three replicates each. In C, the migration of untransfected K562 cells, Kalpha vbeta 3, KTacbeta 3, and KTacbeta 5 to FN was assessed in the presence and absence of KN62 (2.5 µM) or KN04 (5.0 µM) as described in Materials and Methods. Shown are mean ± SEM of four determinations with at least three replicates each.

To determine the dependence of alpha 5beta 1 phagocytosis on CamKII activation, we evaluated phagocytosis in untransfected K562 cells which express alpha 5beta 1, but not alpha vbeta 3. The absence of alpha vbeta 3 in these cells permitted the use of FN-coated beads as a phagocytosis target for alpha 5beta 1 rather than the more selective mAb-16 beads used when alpha vbeta 3 is present. K562 phagocytosis of FN-coated beads via alpha 5beta 1 was inhibited by the CamKII inhibitor KN62 (Fig. 5 B), but not the control KN04. Thus, enhanced CamKII activity, initiated by alpha 5beta 1 binding of mAb-16 beads, appears to be required for alpha 5beta 1 phagocytosis.

These data support the hypothesis that ligation of alpha 5beta 1 stimulates CamKII activity and that alpha vbeta 3-mediated suppression of this activity is at least in part responsible for its inhibition of alpha 5beta 1-mediated phagocytosis. To demonstrate that a similar mechanism was responsible for the inhibitory beta 3 crosstalk to alpha 5beta 1 during migration, we evaluated the effects of the CamKII inhibitor KN62 on K562 cell migration in response to FN. KN62, but not the inactive analogue KN04, inhibited the FN-induced migration of mock transfected K562 cells and KTacbeta 5 (Fig. 5 C). The presence of KN62 did not further attenuate the minimal migration of Kalpha vbeta 3 or KTacbeta 3 cells.

Constitutively Active CamKII Overcomes alpha vbeta 3-Inhibitory Integrin Crosstalk

To test the hypothesis that alpha vbeta 3 crosstalk to alpha 5beta 1 was a result of CamKII downregulation by beta 3, K562 cells were infected with an adenovirus-directing expression of a constitutively active form of CamKII (1). Expression of this construct in untransfected K562 cells resulted in an eightfold increase in CamKII activity over a control viral construct encoding beta -galactosidase (Fig. 6 A).


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Fig. 6.   Constitutively active CamKII reverses alpha vbeta 3 inhibitory crosstalk. Untransfected K562 cells (A), or KTacbeta 3 and KTacbeta 5 (B) were infected with an adenovirus encoding either beta -galactosidase (AdCMV.gal) or a constitutively active form of CamKII (AdCMV.CKIID3). CamKII activity against a synthetic substrate was assayed in the presence and absence of calmodulin and the CamKII inhibitor KN62 (2.5 µM) as described in Materials and Methods and reported as the CamKII activity per mg of total cellular protein (A). Migration of KTacbeta 3 and KTacbeta 5 expressing either beta -galactosidase or constitutively active CamKII to FN was assessed as described in Materials and Methods (B). Shown are mean ± SEM of three determinations performed in triplicate.

Next, KTacbeta 3 and KTacbeta 5 infected with virus encoding either beta -galactosidase or constitutively active CamKII were assayed for their ability to migrate in response to FN. Expression of the active kinase specifically overcame the constitutive inhibition of alpha 5beta 1-mediated migration in KTacbeta 3, without any effect on migration in KTacbeta 5 (Fig. 6 B). Thus, expression of active CamKII overcame alpha vbeta 3-mediated suppression of alpha 5beta 1 high affinity functions. Unfortunately, safety concerns precluded testing the effect of the constitutively active CamKII in the phagocytosis assay.

    Discussion
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Integrin crosstalk is an important mechanism for coordinating signals from multiple simultaneously ligated integrins on a single cell for a functional response to extracellular matrix. Although sometimes called "transdominant inhibition," crosstalk may induce, as well as suppress functions of the target integrin, so we believe the more general term, preferable (9). Although the number of examples of integrin crosstalk has rapidly expanded in the past few years, little is known concerning the molecular mechanisms by which one integrin affects the function of another. K562 cells have proved a valuable model for examination of integrin crosstalk because these cells express a single integrin, alpha 5beta 1, permitting a wide variety of genetic experiments exploring the basis of integrin crosstalk. In this system, we have previously shown that ligation of transfected alpha vbeta 3 inhibits the high affinity phagocytic function of alpha 5beta 1 without effect upon low affinity alpha 5beta 1-mediated adhesion and that the beta 3 cytoplasmic tail is both necessary and sufficient for this effect. We now have used this model to explore the biochemical mechanisms involved in crosstalk. Based on a previous report, we examined a potential role for CamKII in alpha vbeta 3-mediated suppression of the high affinity functions of alpha 5beta 1, and performed structure-function analysis of the beta 3 cytoplasmic tail to further delineate the required structures for this unique signaling event.

In this study, we show that either alpha vbeta 3 ligation or expression of the isolated beta 3 cytoplasmic tail exerts an inhibitory effect upon alpha 5beta 1-mediated migration as well as phagocytosis. Since both alpha 5beta 1 migration and phagocytosis are events that require the high affinity state of the integrin, and since the alpha vbeta 3-mediated inhibition of alpha 5beta 1 is reversed by KN62 in both cases, these data suggest that a common signaling mechanism is responsible for these crosstalk events.

Based on the data in this report, we propose the hypothesis that CamKII, a ser/thr kinase with multiple intracellular substrates, is an important regulator of alpha 5beta 1 function and a target of integrin crosstalk. First, ligation of alpha 5beta 1 by specific antibody- or ligand-coated beads enhances the activity of CamKII in both macrophages and K562 cells. Second, activation of CamKII by ligation of alpha 5beta 1 is required for both phagocytosis and migration. In contrast CamKII inhibitors do not affect adhesion which can be effected by low affinity alpha 5beta 1. Thus, the requirement for CamKII activation appears to be specific for the high affinity functions of alpha 5beta 1.

Coligation of alpha vbeta 3, or exposure of the isolated beta 3 cytoplasmic tail, prevents alpha 5beta 1-induced rise in CamKII activity. Since the beta 3 integrin and the CamKII inhibitor have the same effect on alpha 5beta 1 function, the data suggest that suppression of the ability of alpha 5beta 1 to activate CamKII may be an important mechanism of integrin crosstalk. A role for CamKII suppression in integrin crosstalk is supported by the reversal of crosstalk inhibition of migration with constitutively active CamKII. Thus, our data support the hypothesis that alpha 5beta 1-mediated CamKII activation is required for the high affinity functions of migration and phagocytosis and that alpha vbeta 3-activated crosstalk suppresses these functions through inhibition of CamKII activation. Thus, alpha 5beta 1 and alpha vbeta 3 have opposing effects on CamKII activity.

Neither the upstream events regulating CamKII nor its downstream effector are yet known. Tyrosine kinase inhibitors have no effect either on the high-affinity functions of alpha 5beta 1 or on suppression by alpha vbeta 3, suggesting the possibility that the entire pathway is independent of the well-known effects of integrin ligation on several tyrosine kinases (2, 3). Indeed, the independence of integrin crosstalk from the phosphorylation of Tyr747 further suggests that the signaling involved in the regulation of CamKII may be completely independent of these pathways. A recent report by Wu et al. (25) demonstrates that ligation of alpha 5beta 1 and alpha vbeta 3 have opposite effects on plasma membrane calcium channel activity. Since calcium is an important regulator of CamKII, this voltage gated calcium channel may be important in the differential regulation of CamKII by these two integrins. Based on our preliminary pharmacologic data, one likely effector for CamKII in alpha 5beta 1 high affinity function is myosin light chain kinase (MLCK). MLCK inhibitors KT5926 and ML9 both reverse alpha vbeta 3 inhibition of alpha 5beta 1-mediated phagocytosis and migration without affecting inhibition of CamKII activation by alpha vbeta 3 ligation (Blystone, S.D., and E.J. Brown, unpublished data). MLCK phosphorylation by CamKII is known to inhibit MLCK activity, leading presumably to decreased myosin-induced cell traction (21). This integrin-mediated modulation of myosin function is consistent with the known role for myosin in phagocytosis and migration.

Analysis of structural requirements in the beta 3 cytoplasmic tail in alpha vbeta 3-mediated crosstalk reveals that Ser752 of the beta 3 cytoplasmic tail is required for inhibition of CamKII and for initiation of integrin crosstalk, while crosstalk is independent of either of the beta 3 cytoplasmic tail tyrosines. The requirement for beta 3 Ser752 in crosstalk is unexpected. The importance of Ser752 was suggested by a mutation to proline in a patient with Glanzmann's Thrombasthenia which abolished high affinity binding of fibrinogen by platelet alpha IIbbeta 3 (6, 8). However, detailed analysis has shown that mutation of Ser752 to Ala does not affect ligand binding by alpha IIbbeta 3 (8). The failure of the Ser752 to Ala beta 3 mutation to affect ligand binding is supported by our studies in K562 which demonstrate normal adhesion, normal migration (Fig. 2, B and C), and normal generation of the ligand-induced binding site (LIBS) recognized by the antibody LIBS-1 in response to RGD peptide in this mutant (data not shown). In contrast, integrin crosstalk is entirely abolished by the S752A mutation, as it is by mutation to Pro (the original Glanzmann's mutation), to Glu (to mimic a potential phosphorylation), and to Cys (as a conservative mutation). Thus, it appears that Ser is absolutely required at this position. While this suggests the possibility of Ser phosphorylation in integrin crosstalk, we have been unable to demonstrate such phosphorylation so far. In contrast, Tyr747, which is absolutely required for stimulated adhesion and for alpha vbeta 3-mediated migration (Fig. 1) in K562 cells, is not involved in integrin crosstalk. Thus, these two amino acids, closely spaced in the relatively short cytoplasmic domain of one chain of an integrin, mediate two entirely distinct signaling cascades.

In a recent report, Bouvard et al. (5) showed that increased CamKII levels resulted in a decrease in the affinity of alpha 5beta 1 for FN. In this in vitro system, CamKII and the phosphatase calcineurin regulate alpha 5beta 1 affinity. Because the beta 3 suppression of alpha 5beta 1 phagocytosis occurs subsequent to alpha 5beta 1 binding of ligand (3), it is possible that repeated cycling of alpha 5beta 1 affinity is required for phagocytosis and migration. Binding of ligand-coated beads to high affinity alpha 5beta 1 would activate CamKII, which would then decrease integrin affinity. This hypothesis predicts that integrin crosstalk from alpha vbeta 3 which blocks CamKII activation would prevent alpha 5beta 1 movement to the low affinity state. This is entirely consistent with reports of receptor activation rather than inactivation by integrin crosstalk (14, 24) which measured ligand binding rather than functions that require affinity modulation.

Finally, these data demonstrate that, while increased CamKII activity is required for alpha 5beta 1-mediated phagocytosis and migration, alpha vbeta 3 can perform these same functions independent of any increase in CamKII. This is a startling example of the diversity of signaling and function among the integrins. It suggests that there may be fundamental differences within this family of closely related receptors in how they mediate even their most basic functions. While many studies have emphasized common features of integrin alpha - and beta -chains in association with cytoskeleton, calreticulin, and signaling molecules, the differences between alpha vbeta 3 and alpha 5beta 1 in requirements for phagocytosis and migration suggest that there will be profound differences among integrins even as they perform similar functions.

    Footnotes

Address correspondence to Dr. Scott D. Blystone, Anatomy and Cell Biology, SUNY Health Science Center at Syracuse, 750 East Adams Street, Syracuse, NY 13210. Tel.: (315) 464-8512. Fax: (315) 464-8535. E-mail: blystons{at}vax.cs.hscsyr.edu

Received for publication 30 April 1998 and in revised form 8 March 1999.

   S.D. Blystone is an investigator of the Arthritis Foundation. This work was supported by grants AI24674 and GM38330 from the National Institutes of Health to E.J. Brown. During these studies, S.D. Blystone was a recipient of NRSA AI08990-02 and a grant from the Lucille P. Markey Foundation.

The authors wish to thank all contributors of cDNAs and antibodies used in these studies and the members of the Brown laboratory for suggestions and advice on the performance of these studies.

    Abbreviations used in this paper

CamKII, calcium/calmodulin-dependent protein kinase II; FN, fibronectin; IMDM, Iscove's Modified Eagle's Medium; LIBS, ligand-induced binding site; MLCK, myosin light chain kinase; VN, vitronectin.

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