* Department of Anatomy and Cell Biology, State University of New York, Health Science Center at Syracuse, Syracuse, New
York 13210; 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
Program in Microbial Pathogenesis and Host
Defense, University of California, San Francisco, California 94143
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Abstract |
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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 v
3 inhibits both
phagocytosis and migration mediated by
5
1 on the
same cell, and the
3 cytoplasmic tail is necessary and
sufficient for this regulation of
5
1. Ligation of
5
1
activates the calcium- and calmodulin-dependent protein kinase II (CamKII). This activation is required
for
5
1-mediated phagocytosis and migration. Simultaneous ligation of
v
3 or expression of a chimeric
molecule with a free
3 cytoplasmic tail prevents
5
1-mediated activation of CamKII. Expression of a
constitutively active CamKII restores
5
1 functions
blocked by
v
3-initiated integrin crosstalk. Thus,
v
3
inhibition of
5
1 activation of CamKII is required for
its role in integrin crosstalk. Structure-function analysis
of the
3 cytoplasmic tail demonstrates a requirement for Ser752 in
3-mediated suppression of CamKII activation, while crosstalk is independent of Tyr747 and
Tyr759, implicating Ser752, but not
3 tyrosine phosphorylation in initiation of the
v
3 signal for integrin crosstalk.
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Introduction |
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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 1 (11, 15, 16, 22),
2 (17,
23), or
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 -subunit,
and where it has been examined, the
-subunit cytoplasmic tail has been sufficient for initiation of signaling (3).
We have previously described integrin crosstalk between v
3 and
5
1 in macrophages and in a K562 cell
transfection model of macrophage integrins (2, 3). We
have shown that ligation of
v
3 inhibits
5
1-mediated
phagocytosis, which requires the high affinity state of the
integrin, without affecting
5
1-mediated adhesion, which
is independent of the high affinity state of the integrin.
The cytoplasmic tail of
3 is necessary and sufficient for
this crosstalk.
v
3-mediated inhibition of
5
1 phagocytosis occurs at a step subsequent to
5
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 v
3-to-
5
1 crosstalk. Ligation of
5
1 enhances the activity of the calcium/calmodulin-dependent
protein kinase II (CamKII)1. This increase in CamKII
activity is required for
5
1-dependent migration as well
as
5
1-dependent phagocytosis. Simultaneous ligation of
v
3 inhibits
5
1 activation of CamKII activity, thus
blocking
5
1 migration and phagocytosis. Mutational
analysis of the
3 cytoplasmic tail demonstrates that
Ser752 is required for both
v
3-initiated inhibitory
crosstalk to
5
1 and
v
3 suppression of CamKII activity,
while tyrosine phosphorylation of the
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.
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Materials and Methods |
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Cells and 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 v
3 (K
v
3),
v
3 in which the tyrosine
residue at position 747 or 759 was mutated to phenylalanine (K
v
3Y747F
and K
v
3Y759F, respectively) and Tac
3 (chimera of the extracellular
and transmembrane domain of the IL2 receptor
-chain [Tac subunit] and
the cytoplasmic tail of
3, KTac
3) were derived and maintained as described (2, 3). In addition K562 cells were similarly transfected with cDNA encoding Tac
3 in which the serine residue at position 752 was
replaced with proline (KTac
3S-P), cysteine (KTac
3S-C), alanine (KTac
3S-A), or glutamic acid (KTac
3S-E). Expression of all Tac
3S-X chimeras was equivalent to Tac
3 (3) as determined by flow cytometry as
described (2; see Table I). For construction of Tac
3S-X, the HindIII and
XhoI fragment of pTac
3 encoding the CT of
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 pTac
3 digested with HindIII-XhoI, creating pTac
3S-P, pTac
3S-C, pTac
3S-A, and pTac
3S-E, respectively.
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K562 cells also were transfected with full-length v
3 in which the
serine at position 752 of
3 was mutated to alanine as described for the
mutation of
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
3 CT was replaced with the S-A mutant CT by NdeI-NheI restriction. Transfection, selection, and fluorescent cell sorting for expression levels of
v
3S752A equivalent to wild-type
v
3 was as described previously, resulting in K
v
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-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 -[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:
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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
Kv
3, KTac
3, and KTac
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
-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 3), W6/
32 (anti-human HLA), IC12 (anti-human
v), AP3 (anti-human
3),
P1F6 (anti-human
5), 4E3 (anti-IL2R
, gp55, TAC), and mAb16 (anti-
human
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.
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Results |
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v
3 Crosstalk Regulates
5
1-mediated Migration
We have previously described a phenomenon, termed integrin crosstalk, in which ligation of v
3 prevents
5
1-mediated phagocytosis in macrophages and in K562 cells
expressing transfected
v
3. To determine if integrin
crosstalk regulated
5
1 functions other than phagocytosis,
we evaluated the effects of
v
3 ligation on the migration
of K562 cells on the
5
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
5
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
5
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
5
1 (data not shown). We also examined
v
3-mediated migration in K562 expressing this
transfected integrin in addition to the endogenous
5
1.
K
v
3 migrated in response to VN (Fig. 1 A); migration
response to VN was inhibited by mAb to
v or
3 (data not
shown). However, migration of K
v
3 to FN was severely
impaired compared with untransfected or mock transfected K562 (Fig. 1 A). Migration of K
v
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 K
v
3 migration to VN (data not shown). Restored migration of
K
v
3 to FN in the presence of H7 was completely inhibited by mAb to
1 (data not shown). These results completely parallel the previously described
v
3-mediated
crosstalk which inhibits
5
1-mediated phagocytosis (3)
and support the hypothesis that the coligation of
v
3 by
FN regulates
5
1-mediated K562 cell migration to FN because this function, like phagocytosis, requires a high affinity form of
5
1.
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To demonstrate definitively that v
3 regulation of
5
1-mediated migration was another example of integrin
crosstalk, we examined migration to FN in KTac
3 and
KTac
5, K562 cells expressing chimeric molecules comprised of the extracellular domain of the IL2 receptor and
the cytoplasmic tail domain of the
3 or
5 integrin, respectively. Expression of Tac
3, but not Tac
5, leads to constitutive inhibition of
5
1-mediated phagocytosis in K562
cells (3; see Fig. 7 B). Expression of Tac
3, but not Tac
5
(Fig. 1 A) or Tac lacking a cytoplasmic tail (KTacNT, Fig.
2 A), completely inhibited
5
1-mediated migration to FN.
The constitutive inhibition of migration to FN in KTac
3
was reversed by the addition of 50 nM H7 (Fig. 2 A).
These studies demonstrate that
5
1-mediated migration and
5
1-mediated phagocytosis are similarly regulated by
v
3 or the isolated
3 CT and that this regulation is dependent upon a ser/thr kinase regulated by H7. These data
suggest that both
5
1-mediated migration and
5
1-mediated phagocytosis are regulated by
v
3-initiated crosstalk.
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3 Ser752 Is Required for
3 Crosstalk
We have previously demonstrated that expression of the
isolated 3 cytoplasmic tail is sufficient for initiation of
v
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
3
cytoplasmic tail and analyzed their effects upon
v
3-initiated crosstalk to
5
1-mediated migration.
In a spontaneously occurring Glanzmann's Thrombasthenia mutation, the serine residue at position 752 of the
3 CT is mutated to proline (6). This mutation results in
loss of platelet
3 function and a severe bleeding disorder.
In vitro study has shown that Ser752 of the
3 CT is required for the conformational change associated with elevated affinity of
3 for ligand (8). To test whether Ser752
also is required for integrin crosstalk, we expressed an
v
3 receptor in K562 cells in which Ser752 of
3 was mutated to Ala (Fig. 1 B). While the ligation of wild-type
v
3
blocked
5
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
v
3 on VN (Fig. 1 B), consistent with reports that
this mutation does not affect ligand binding by
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 3 cytoplasmic tail, Tyr747,
has been implicated in activation-dependent
v
3 adhesion to VN (4). In contrast to the S752A mutation, Y747F
had no effect on
v
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 K
v
3Y747F to VN (Fig. 1 B). Mutation of Tyr759 to Phe (Y759F) did not affect either
crosstalk or the migration function of
v
3. These data
demonstrate that the crosstalk signaling and adhesive
functions of
v
3 have distinct and independent sequence
requirements in the
3 cytoplasmic tail.
To evaluate further the requirement for 3 S752 in integrin crosstalk, additional mutations at that position were
made in the consititutively inhibitory Tac
3 construct. Mutation of Ser752 to Glu, Pro, or Cys as well as Ala abolished the inhibitory activity of Tac
3 on
5
1-dependent
migration (Fig. 2 A) and
5
1-dependent phagocytosis
(Fig. 2 B). Like the wild-type
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
5
1 (Fig. 2 C). The addition of H7 reversed the Tac
3
inhibition of
5
1-mediated migration and phagocytosis
(Fig. 2, A and B).
5
1 and
v
3 Differentially Regulate CamKII
v
3 ligation inhibits the
5
1 high affinity functions of
phagocytosis and migration, without effect upon
5
1-mediated adhesion. Alterations in
5
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
v
3 ligation in smooth muscle cells
was reported (1). Therefore, we evaluated
v
3 regulation of CamKII as a potential mediator of
v
3-initiated crosstalk.
CamKII activity was measured in human monocyte-derived macrophages in the presence and absence of an
5
1-specific phagocytosis target (mAb-16-coated latex
beads) (3). Ligation of macrophage
5
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
v
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,
3 ligation prevented the
5
1-induced rise in CamKII activity.
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To explore further the hypothesis that CamKII mediates v
3 regulation of
5
1, we evaluated the regulation of
CamKII in K
v
3. Binding of mAb-16 beads to K
v
3, and
to vector-transfected K562 (data not shown), resulted in
an increase in CamKII activity (Fig. 3 B) that was not seen
when K
v
3 were incubated with W6/32 beads that bound
to the cells equivalently. As in macrophages, the
5
1-mediated rise in CamKII activity was prevented by ligation of
v
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
5
1-induced increase in
CamKII activity by
v
3 ligation was blocked by KN62,
but not KN04.
Previously we have demonstrated that the cytoplasmic
tail of 3 is both necessary and sufficient for
v
3 inhibitory crosstalk to
5
1 (reference 3 and Fig. 2, A and B). In
the presence of mAb-16 beads, expression of Tac
3, but
not Tac
5, prevented the
5
1-mediated rise in CamKII
activity (Fig. 3 C). These results indicate that
5
1 and
v
3 differentially regulate CamKII activity in macrophages and K562 cells.
To determine if the failure of the Kv
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
v
3 and the
S752A and Y747F mutants. While ligation of
v
3 with the
3-specific mAb 7G2 suppressed mAb-16 bead-induced activation of CamKII, mutation of Ser752 of
3 prevented
the suppression of CamKII activity seen upon
v
3 ligation (Fig. 4 A). However, mutation of Tyr747 or Tyr759
(data not shown) did not affect
v
3 regulation of CamKII.
Thus, Ser752 is required for both
v
3 inhibitory crosstalk
to
5
1 (Fig. 2) and
v
3 regulation of CamKII (Fig. 4).
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To determine the effect of v
3 and mutant
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
v
3 and mutants in transfected K562 cells.
Role of CamKII in v
3 Crosstalk to
5
1
Suppression of the 5
1-dependent increase in CamKII activity by
v
3 ligation or by Tac
3 expression suggested
that CamKII regulation could have a role in
v
3 crosstalk
to
5
1. To determine the role of CamKII in
v
3 crosstalk
to
5
1, K
v
3 cells were incubated with the
5
1 phagocytosis target, mAb-16 beads, or control target, P1F6 (anti-
v
5) beads. Phagocytosis was measured in the presence
and absence of 7G2 to ligate
v
3, the CamKII inhibitor
KN62, or control KN04. As reported previously, phagocytosis via
5
1 was inhibited upon
v
3 ligation with mAb
7G2 (2, 3).
5
1 phagocytosis also was inhibited by KN62
(Fig. 5 A), but not KN04. Combining 7G2 and KN62 resulted in no further decrease in
5
1 phagocytosis. Under
all conditions, there was no significant internalization of
P1F6 beads.
|
To determine the dependence of 5
1 phagocytosis on
CamKII activation, we evaluated phagocytosis in untransfected K562 cells which express
5
1, but not
v
3. The absence of
v
3 in these cells permitted the use of FN-coated
beads as a phagocytosis target for
5
1 rather than the
more selective mAb-16 beads used when
v
3 is present.
K562 phagocytosis of FN-coated beads via
5
1 was inhibited by the CamKII inhibitor KN62 (Fig. 5 B), but not the
control KN04. Thus, enhanced CamKII activity, initiated
by
5
1 binding of mAb-16 beads, appears to be required
for
5
1 phagocytosis.
These data support the hypothesis that ligation of 5
1
stimulates CamKII activity and that
v
3-mediated suppression of this activity is at least in part responsible for its
inhibition of
5
1-mediated phagocytosis. To demonstrate
that a similar mechanism was responsible for the inhibitory
3 crosstalk to
5
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 KTac
5 (Fig. 5 C). The presence of KN62 did not further attenuate the minimal migration of K
v
3 or KTac
3 cells.
Constitutively Active CamKII Overcomes
v
3-Inhibitory Integrin Crosstalk
To test the hypothesis that v
3 crosstalk to
5
1 was a result of CamKII downregulation by
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
-galactosidase (Fig. 6 A).
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Next, KTac3 and KTac
5 infected with virus encoding
either
-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
5
1-mediated migration in
KTac
3, without any effect on migration in KTac
5 (Fig. 6
B). Thus, expression of active CamKII overcame
v
3-mediated suppression of
5
1 high affinity functions. Unfortunately, safety concerns precluded testing the effect of
the constitutively active CamKII in the phagocytosis assay.
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Discussion |
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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, 5
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
v
3 inhibits the high affinity phagocytic function of
5
1 without effect upon low affinity
5
1-mediated adhesion and that the
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
v
3-mediated suppression of
the high affinity functions of
5
1, and performed structure-function analysis of the
3 cytoplasmic tail to further delineate the required structures for this unique signaling event.
In this study, we show that either v
3 ligation or expression of the isolated
3 cytoplasmic tail exerts an inhibitory effect upon
5
1-mediated migration as well as
phagocytosis. Since both
5
1 migration and phagocytosis
are events that require the high affinity state of the integrin, and since the
v
3-mediated inhibition of
5
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 5
1 function
and a target of integrin crosstalk. First, ligation of
5
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
5
1 is required
for both phagocytosis and migration. In contrast CamKII inhibitors do not affect adhesion which can be effected by
low affinity
5
1. Thus, the requirement for CamKII activation appears to be specific for the high affinity functions
of
5
1.
Coligation of v
3, or exposure of the isolated
3 cytoplasmic tail, prevents
5
1-induced rise in CamKII activity. Since the
3 integrin and the CamKII inhibitor have
the same effect on
5
1 function, the data suggest that suppression of the ability of
5
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
5
1-mediated CamKII activation is required
for the high affinity functions of migration and phagocytosis and that
v
3-activated crosstalk suppresses these functions through inhibition of CamKII activation. Thus,
5
1
and
v
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 5
1 or on suppression by
v
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
5
1
and
v
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
5
1 high
affinity function is myosin light chain kinase (MLCK).
MLCK inhibitors KT5926 and ML9 both reverse
v
3
inhibition of
5
1-mediated phagocytosis and migration
without affecting inhibition of CamKII activation by
v
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 3 cytoplasmic tail in
v
3-mediated crosstalk reveals that Ser752 of
the
3 cytoplasmic tail is required for inhibition of CamKII
and for initiation of integrin crosstalk, while crosstalk is independent of either of the
3 cytoplasmic tail tyrosines.
The requirement for
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
IIb
3 (6, 8). However, detailed analysis has shown
that mutation of Ser752 to Ala does not affect ligand binding by
IIb
3 (8). The failure of the Ser752 to Ala
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
v
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 5
1 for FN. In this in vitro system, CamKII and the
phosphatase calcineurin regulate
5
1 affinity. Because
the
3 suppression of
5
1 phagocytosis occurs subsequent
to
5
1 binding of ligand (3), it is possible that repeated cycling of
5
1 affinity is required for phagocytosis and migration. Binding of ligand-coated beads to high affinity
5
1 would activate CamKII, which would then decrease
integrin affinity. This hypothesis predicts that integrin
crosstalk from
v
3 which blocks CamKII activation would
prevent
5
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 5
1-mediated phagocytosis and migration,
v
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
- and
-chains in association with cytoskeleton,
calreticulin, and signaling molecules, the differences between
v
3 and
5
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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