From the Department of Medicine, Microbiology and Immunology, and Jonsson Cancer Center, University of California at Los Angeles School of Medicine, Los Angeles, California 90095
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ABSTRACT |
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The integrin subunit cytoplasmic domains are
important for activation-dependent cell adhesion and
adhesion-dependent signaling events. We report an interaction
between integrin
subunit cytoplasmic domain and Rack1, a Trp-Asp
(WD) repeat protein that has been shown to bind activated protein
kinase C. The Rack1-binding site on integrin
2
subunit resides within a conserved, membrane-proximal region. In the
yeast two-hybrid assay, WD repeats five to seven of Rack1 (Rack1-WD5/7)
interact with integrin
1,
2, and
5 cytoplasmic domain. In eukaryotic cells, Rack1
co-immunoprecipitates with at least two different
integrins,
1 integrins in 293T cells and
2 integrins
in JY lymphoblastoid cells. Whereas Rack1-WD5/7 binds integrins
constitutively, the association of full-length Rack1 to integrins
in vivo requires a treatment with phorbol esters, which
promotes cell spreading and adhesion. These findings suggest that Rack1
may link protein kinase C directly to integrins and participate in the
regulation of integrin functions.
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INTRODUCTION |
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Integrins are heterodimeric adhesion receptors that mediate
attachment of cells to the extracellular matrix and specific cell
counter-receptors (1). Various extracellular stimuli have been shown to
affect the adhesiveness of integrins and regulate attachment of cells
to the extracellular matrix (2). This process known as an
activation-dependent cell adhesion is best illustrated in
leukocytes where the attachment of integrin
LFA11
(
L
2) to intercellular cell adhesion
molecule-1 (ICAM-1) substrates can be promoted by cross-linking T cell
receptors or stimulating cells with phorbol 12-myristate 13-acetate
(PMA) (3). Upon binding to the extracellular matrix, integrins induce
signals required for the reorganization of actin cytoskeleton and the formation of focal adhesion complexes (4-6). The
adhesion-dependent clustering of integrins leads to the
activation of nonreceptor tyrosine kinase focal adhesion kinase and
Ras/mitogen-activated protein kinase pathway, the stimulation of
inositol lipid metabolism, an increase in intracellular
Ca2+ and pH, and the activation of PKC (4, 7, 8).
Each subunit of integrins consists of a large extracellular
ligand-binding domain, a transmembrane domain, and a short cytoplasmic domain that lacks any enzymatic activity. Although the cytoplasmic domains of subunits are variable in size and sequence, the
cytoplasmic domains of
subunits are more conserved in size and
sequence. In particular, three conserved regions, termed cyto-1,
cyto-2, and cyto-3, found in
integrin cytoplasmic domains have been implicated in the recruitment of integrins to the focal adhesion plaques and the regulation of adhesive functions of integrins (9-11).
Although both integrin subunits are required for the ligand binding,
the interaction between intracellular proteins and integrin cytoplasmic
domain can occur in the absence of subunit association. Studies have
shown that chimeric molecules composed of the
integrin cytoplasmic
domains and the extracellular domain of the interleukin-2 receptor can
be directed to the focal adhesion plaques and activate focal adhesion
kinase in the absence of ligand binding (12). Based on these findings,
it has been hypothesized that the integrin
subunit cytoplasmic
domain provides binding sites for proteins involved in the regulation
of integrin functions. Proteins that have been shown to directly
interact with
subunit cytoplasmic domains include known
cytoskeletal proteins
-actinin (13), paxillin (14), talin (11, 15),
and filamin (16); protein kinases focal adhesion kinase (14) and
integrin-linked kinase-1 (ILK-1) (17); and potential regulatory
proteins such as
3-endonexin (18), cytohesin-1 (19), and
integrin cytoplasmic domain-associated protein-1 (Icap-1) (20).
In this study, we report a direct association of receptor for activated
protein kinase C (Rack1) to the integrin subunit cytoplasmic
domain. Rack1 consists of seven repeating units of WD motifs presumed
to be involved in a protein-protein interaction (2, 21). Deletion
studies indicate that the C-terminal three WD motifs (WD-5, WD-6, and
WD-7) of Rack1 interact with the conserved membrane-proximal region of
subunit cytoplasmic domain. Interestingly, interaction of Rack1
with integrins in vivo requires a treatment with PMA, which
promotes cell spreading and integrin-dependent cell
adhesion (3, 22). Our finding suggests a direct linkage between
integrins and PKC through Rack1 and further implicates PKC in
integrin-mediated cell signaling.
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MATERIALS AND METHODS |
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Antibodies and Cell Lines--
The anti-1
integrin antibody producing hybridoma cell line, TS2/16, was generously
provided by Dr. M. Hemler (Dana Farber Cancer Institute, Boston, MA).
The mouse hybridoma cell line TS1/18 producing anti-
2
integrin antibody was obtained from the ATCC (Rockville, MD). The mAb
TS2/16 and TS1/18 recognize the extracellular domains of
1 and
2 integrins, respectively.
Anti-Rack1 antibody was purchased from Transduction Labs. The
anti-
L antibody, directed against the integrin
L cytoplasmic domain, was described previously (20).
Yeast Genetic Screening--
The yeast genetic screening for the
isolation of proteins interacting with the cytoplasmic domain of
2 integrin was carried out essentially as described
previously (20, 23). The C-terminal 46 amino acids (724-769) of the
2 integrin were cloned in frame into LexA coding
sequence to generate a "bait" plasmid, pNlex-
2cyto. pNlex-
2cyto and a JY cDNA library in the pJG45 yeast
expression vector (23) were introduced into a yeast strain containing a chromosomal copy of the Leu2 gene (EGY48 (Mat
trp1 ura3-52
leu2::pLeu2-lexAop6(
UAS leu2)) and an episomal
-galactosidase gene (as a JK103 plasmid) under the control of a
synthetic promoter with LexA-binding sites (23). Yeast transformants
were selected for the formation of blue colonies on
5-bromo-4-chloro-3-indolyl
-D-galactopyranoside indicator plates with leucine. The interacting cDNA clones
were rescued from the selected yeast transformants as described
previously (23).
In Vitro Interaction Assay--
For in vitro GST
interaction assays, the 2 integrin cytoplasmic domain
(amino acids 724-769), a truncation mutant
2(E16) (amino acids 750-769), and the
L integrin subunit
cytoplasmic domain were individually expressed as GST fusion proteins
using the bacterial expression vector pGEX4T1 (Pharmacia Biotech Inc.) (24). Rack1-WD5/7 was synthesized in vitro using a
co-transcription/translation system (Promega) and added to
approximately 2 µg of GST fusion proteins and incubated overnight at
4 °C in NET (25 mM Tris-HCl, pH 7.6, 100 mM
NaCl, 3 mM EDTA) containing 1 mM
dithiothreitol, 1% bovine serum albumin, and 0.1% Triton X-100. The
GST fusion proteins were purified using glutathione-Sepharose beads
(Pharmacia). The beads were washed twice in the binding buffer and
twice in 0.05% Triton X-100 in NET. The bound proteins were eluted by
boiling in SDS sample buffer, subjected to SDS/polyacrylamide gel
electrophoresis, and then transferred to a nitrocellulose membrane
(Bio-Rad). The filter membrane was probed with an anti-Rack1 antibody
(0.06 µg/ml) and then developed using an enhanced chemiluminescence
method (Amersham Corp.).
Eukaryotic Expression and in Vivo Interaction Assay--
A
full-length Rack1 cDNA was obtained from a JY cDNA library
using a polymerase chain reaction and cloned into the eukaryotic expression vectors pcDNA3 (Invitrogen) and pEBG (20). 5-10 µg of
plasmid DNA was transfected into 293T cells by using a calcium phosphate precipitation method (24). 48 h post-transfection, cells
were lysed in TBSM (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 2 mM MgCl2) containing
0.5% Nonidet P-40, leupeptin, aprotinin, and phenylmethylsulfonyl
fluoride. When indicated, the cells were treated with PMA (100 ng/ml,
stock solution 1 mg/ml in Me2SO) for 30 min at 37 °C
prior to lysis. Detergent insoluble materials were removed by
centrifugation at 12,000 × g for 15 min. 500 µg of
the cleared lysates were mixed with an equal volume of TBSM and
incubated with glutathione-Sepharose beads for 3 h at 4 °C. Beads were washed with TBSM + 0.25% Nonidet P-40 once and TBSM alone
three times. Co-precipitation of 1 integrins with the
bound GST-Rack1 was analyzed by immunoblotting with the mAb TS2/16
(anti-
1 integrin). Immunoprecipitation of
1 integrins was carried out by incubating the lysates
with 4 µg/ml of the mAb TS2/16 for 3 h at 4 °C, in the
presence of protein G-agarose beads (Life Technologies, Inc.). JY cell
lysates were prepared as above, and LFA1 was immunoprecipitated using
the mAb TS 1/18 (anti-
2 integrin).
Co-immunoprecipitation of Rack1 with the integrins was analyzed by
immunoblotting using an anti-Rack1 antibody.
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RESULTS |
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Rack1 Interacts with the Integrin Subunit Cytoplasmic
Domain--
The yeast two-hybrid assays were used to identify cellular
proteins that interact with the cytoplasmic domain of
2
integrin. A bait construct, pNLex-
2cyto, which contains
the entire cytoplasmic domain (amino acids 724-769) of
2 subunit was introduced into EGY48, a yeast strain
harboring Leu2 and
-galactosidase genes under the control of a
synthetic promoter with LexA-binding sites (23). The resulting strain
was used as a host for transformation with a human B cell (JY cells)
cDNA library in the pJG45 yeast expression vector. From ~8 × 105 primary transformants, four overlapping cDNAs
were found to interact with the cytoplasmic domain of
2
integrin but not with the control baits containing unrelated sequences
(data not shown). Sequence analysis revealed that all four clones
encode overlapping regions of the C-terminal portion of the
heterotrimeric G
superfamily protein, Rack1.
WD Domain 5-7 of Rack1 Interacts with the Membrane-proximal Region
of Integrin Subunit Cytoplasmic Domain--
Rack1 is composed of 7 repeating units of WD motif (21). Like G
proteins, Rack1
is expected to form a
-propeller structure, where each WD repeat
composed of four anti-parallel
strands make up a blade of the
propeller (25-27). The four overlapping Rack1 cDNA clones isolated
in yeast two-hybrid screening all contained WD-5 through WD-7,
suggesting these three C-terminal WD repeats are sufficient for the
binding to the
2 integrin cytoplasmic domain (Fig.
1). A deletion of 22 additional amino
acids corresponding to the N-terminal portion of WD-5 (Rack1-
BH1)
completely abolished the interaction. Therefore, we conclude that WD-5
is necessary for the integrin binding. The 22 amino acids deleted in
Rack1-
BH1 includes the fourth
strand and the loop that are
located on the solvent exposed outer surface of the
-propeller (21,
26, 27). Interestingly none of the isolated Rack1 clones contained the
WD motifs N-terminal to WD-4. The full-length Rack1 failed to interact
with the baits containing the
integrin cytoplasmic domains in
yeast, raising a possibility that the interaction between the
full-length Rack1 and the
integrins may be regulated.
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Rack1-WD5/7 Binds Integrin 2 Cytoplasmic Domain in
Vitro--
To confirm the interaction between integrin
subunit and
Rack1 observed in yeast, Rack1-WD5/7 was synthesized in
vitro and incubated with GST fusion proteins containing the
integrin cytoplasmic domains (Fig. 3).
Rack1-WD5/7 associated with a GST fusion protein encoding the complete
cytoplasmic domain of integrin
2 (GST-
2A, amino acids 724-769, Fig. 3, lane 2) but not with GST
fusion proteins encoding truncated forms of integrin
2
cytoplasmic domain (GST-
2B, amino acids 728-769, and
GST-
2(E16), amino acids 750-769, Fig. 3, lanes
1 and 3) or a GST fusion protein encoding the
cytoplasmic domain of integrin
L (Fig. 3, lane
4). The results of in vitro binding assays were
identical to the findings in the yeast two-hybrid interaction
studies.
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Integrins Form an Inducible Complex with Full-length Rack1 in
Vivo--
To demonstrate the interaction of Rack1 and integrins
in vivo, Rack1-WD5/7 or a full-length Rack1 were expressed
as GST fusion proteins in 293T cells. The transfected cells were lysed
in a buffer containing nonionic detergent and incubated with
glutathione-Sepharose beads to purify GST-Rack fusion proteins. The
binding of 1 integrins to the GST-Rack fusion proteins
were analyzed by immunoblotting with the mAb TS2/16
(anti-
1). The
1 integrins were
co-precipitated with GST-Rack1-WD5/7 (Fig.
4A, lane 2) but not
with GST alone used as the controls (Fig. 4A, lane
1). Expression of equivalent amounts of the GST fusion proteins in
293T cells was confirmed by immunoblotting with anti-GST antibody (Fig.
4A, bottom panel).
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DISCUSSION |
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Integrins have been proposed as crucial transmembrane proteins
linking cytoskeleton to the extracellular matrix. Numerous studies have
demonstrated that integrin-dependent cell adhesion is
regulated in response to extracellular stimuli. In platelets, agonist
stimulation induces conformational changes in the extracellular ligand-binding domain of the platelet integrin
IIb
3, leading to an affinity state (7). A
similar affinity modulation has not been clearly demonstrated for other
integrins. Nonetheless, integrin-dependent cell adhesion of
many cell types is enhanced when cell spreading is augmented by a PMA
treatment (3, 22, 30). Both of these responses require integrin
cytoplasmic domains, suggesting that the interaction between integrins
and cellular proteins plays an important role in the regulation of cell
adhesion. To gain insights into cell adhesion regulation, we sought to
identify cellular proteins that directly associate with integrin
subunit cytoplasmic domains. In the present study, we report isolation of Rack1 as an integrin-binding protein. Our evidence for the interaction of Rack1 with integrins is based on (i) the interaction of
the two proteins in a yeast two-hybrid assay, (ii) the binding of
in vitro translated C-terminal portion of Rack1 to
bacterially expressed GST fusion protein containing the
2 integrin cytoplasmic domain, and (iii) the detection
of a complex between Rack1 and integrins in vivo.
Rack1 is a member of G superfamily, composed exclusively
of seven WD repeats forming an overall
propeller structure (27). WD
repeats represent structural motifs formed by four anti-parallel
strands and have been implicated in protein-protein interaction (25).
Although the precise function of Rack1 is not known, Rack1 binds to PKC
in the presence of Ca2+ and lipid in vitro and
has been proposed as an intracellular receptor for activated protein
kinase C responsible for subcellular localization of PKC (21).
Overlapping cDNA isolates from initial yeast screening indicated
that the WD repeats 5-7 are sufficient for its interaction with
integrins. Rack1-
BH1, lacking a part of WD repeat 5, failed to bind
2 integrin. The region deleted in Rack1-
BH1
corresponds to the outer exposed surface of the
propeller that
would be available to make contacts with other proteins.
The region of the integrin 2 cytoplasmic domain
responsible for the Rack1 binding lies within the membrane-proximal
20-amino acid region (residues 724-743). This region is conserved
among different
subunits, and not surprisingly, Rack1 interacts
with at least two other
subunits,
1 and
5. The presence of three apolar residues immediately
following a Lys residue at the membrane-cytoplasm interface appears to
be critical for the Rack1 binding. Either a 4-amino acid deletion in
this region or a substitution of KALI to KEEE prevented the Rack1
interaction. Several functions, including localization of
1 integrins to the focal adhesion plaques (9) and
affinity regulation of
3 integrins (28), have been
assigned to the membrane-proximal region. It will be of interest to
determine whether these known integrin functions require Rack1.
The most intriguing finding of our study is that the association
between Rack1 and integrins in vivo is inducible and
requires a PMA treatment. Integrin subunits are known to undergo
phosphorylation on serines and threonines upon PMA treatment (29). We,
however, do not favor the possibility that the phosphorylation of
cytoplasmic domain is responsible for the recruitment of Rack1 because
Rack1-WD5/7 binds integrins constitutively both in vitro and
in vivo, and the deletion of major phosphorylation sites on
cytoplasmic domain does not affect this interaction. Based on a
previously reported function of Rack1 as a cytoplasmic receptor of
activated PKC (21), it is conceivable that upon a PMA treatment,
activated PKC binds and phosphorylates Rack1, allowing subsequent
recruitment of Rack1 to the integrin cytoplasmic domain.
Cell stimulation with PMA or receptor activation frequently increases
integrin avidity and promotes cell spreading (22, 30). The functional
consequences of integrin-Rack1 association remains to be seen. In
addition to Rack1, two other proteins, -actinin (31) and
calreticulin (32), have been shown to bind integrin cytoplasmic domain
in an inducible manner.
-Actinin through linking integrins to the
actin cytoskeleton may function to modulate integrin avidity.
Calreticulin, unlike Rack1 or
-actinin, which bind to
subunit
cytoplasmic domains, interacts with
subunits through a high
conserved short KXGFFKR motif (33). The precise function of
calreticulin remains to be seen. However, functional knock-out of
calreticulin by introducing anti-calreticulin antibody into cells or
gene disruption interferes with integrin-dependent cell
adhesion (32, 34). The presence of multiple putative protein-protein
interaction WD domains may allow Rack1 to function as a scaffold
protein to recruit PKC and/or other proteins to the membrane at the
site of membrane-cytoskeletal junction by its ability to bind integrin
cytoplasmic domain. Rack1 may therefore be an integral player in this
membrane-cytoskeletal association and participate in regulation of
cell adhesion.
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ACKNOWLEDGEMENTS |
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We are grateful to R. Brent (Massachusetts General Hospital) for providing the yeast strains and plasmids for the yeast genetic screening. We are also grateful to M. Hemler (Dana-Farber Cancer Institute), T. Springer (Harvard), and K. Shuai (University of California at Los Angeles) for providing reagents and C. Denny for comments on the manuscript.
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FOOTNOTES |
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* This work was supported by the grants from the Margaret E. Early Medical Research Trust, the Searle Scholars Program/The Chicago Community Trust, the James S. McDonnell Foundation, and the Jonsson Cancer Center and by Tumor Immunology Training Grant CA09120-21.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: UCLA School of
Medicine, Div. of Heme-Onc, Factor 11-934, 10833 Le Conte Ave., Los
Angeles, CA 90095. Fax: 310-825-6192; E-mail:
dchang{at}medicine.medsch.ucla.edu.
1 The abbreviations used are: LFA1, leukocyte function antigen-1; GST, glutathione S-transferase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; mAb, monoclonal antibody(ies); GST-Rack1, GST-tagged Rack1; WD, Trp-Asp.
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REFERENCES |
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