(Received for publication, September 17, 1996, and in revised form, November 13, 1996)
From the Dana-Farber Cancer Institute, Harvard
Medical School, Boston, Massachusetts 02115 and the ¶ Division of
Signal Transduction, Beth Israel Hospital and the Departments of Cell
Biology and Medicine, Harvard Medical School,
Boston, Massachusetts 02115
Enzymatic and immunochemical assays show a
phosphatidylinositol 4-kinase in novel and specific complexes with
proteins (CD63 and CD81) of the transmembrane 4 superfamily (TM4SF) and
an integrin (3
1). The size (55 kDa) and
other properties of the phosphatidylinositol 4-kinase (PI 4-K)
(stimulated by nonionic detergent, inhibited by adenosine, inhibited by
monoclonal antibody 4CG5) are consistent with PI 4-K type II. Not only
was PI 4-K associated with
3
1-CD63 complexes in
3-transfected K562 cells, but also it could
be co-purified from CD63 in untransfected K562 cells lacking
3
1. Thus, TM4SF proteins may link PI 4-K
activity to the
3
1 integrin. The
5
1 integrin, which does not associate
with TM4SF proteins, was not associated with PI 4-K. Notably,
3
1-CD63-CD81-PI 4-K complexes are located
in focal complexes at the cell periphery rather than in focal
adhesions. The novel linkage between integrins, transmembrane 4 proteins, and phosphoinositide signaling at the cell periphery may play
a key role in cell motility and provides a signaling pathway distinct
from conventional integrin signaling through focal adhesion kinase.
Cell attachment mediated by transmembrane receptors in the integrin family triggers signal transduction cascades that regulate cell proliferation, apoptosis, morphology, and motility (1, 2). Activation of Rho, a small GTP-binding protein, and focal adhesion kinase (FAK)1 may be central events in signaling cascades initiated by most integrins (3-5). On the other hand, distinct functions for integrins expressed in the same cellular environment are suggestive of additional integrin-specific signaling pathways not yet elucidated (6-9).
Specific association between membrane proteins in the transmembrane-4
superfamily (TM4SF; tetraspan proteins) and certain 1
integrins, including
3
1,
6
1, and
4
1,
was previously demonstrated (10-14). A role for TM4SF proteins in
signaling is suggested by their modulation of intracellular calcium,
tyrosine phosphorylation, and cell proliferation (15-17). However,
there has been little understanding of the mechanisms whereby TM4SF
proteins might signal. Here we found that phosphatidylinositol 4-kinase
is associated with
3
1 integrin and TM4SF
proteins. This supports our hypothesis that integrin-TM4SF complexes
could be a point of convergence for integrin and TM4SF protein
signaling.
Anti-integrin mAbs used were:
anti-3, A3-X8 (18), A3-IVA5 (18), and A3-IIF5 (18);
anti-
5, A5-PUJ2 (19); anti-
6, A6-ELE
(19); anti-
1, A-1A5 (20). Anti-TM4 mAbs used were: anti-CD63, 6H1 (10), and RUU.SP. 2.28 (21); anti-CD81, M38 (22), and
5A6 (23). Anti-vinculin mAb hVIN-1 was from Sigma, and
mAb 8G6 to a 47-kDa cell surface protein named emmprin (24) will be
described.2 The mAb 4C5G specifically
immunoprecipitates type II PI 4-K and inhibits lipid kinase activity
(25).
For
immunoprecipitation, HT1080 cells were lysed in buffer containing 1%
Brij 99, 20 mM Hepes (pH 7.5), 200 mM NaCl, 5 mM MgCl2, 200 µM
Na3VO4, 2 mM NaF, 10 mM
Na4P3O7, 2 mM
phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin,
and immunoprecipitates were prepared as described (11). Prior to
phosphoinositide assay, immunoprecipitates were washed once in HNE
buffer (20 mM Hepes (pH 7.5), 100 mM NaCl, 1 mM EDTA), and assays were performed on the beads as
described (26). Briefly, samples were incubated in 20 mM
Hepes (pH 7.5), 10 mM MgCl2, 50 µM ATP, 200 µM sonicated phosphoinositides
(40% PS, 20% PtdIns, 20% PtdIns 4-phosphate, 20% PtdIns
4,5-diphosphate), and 50 µM [-32P]ATP (8 mCi/nmol) for 10 min at 25 °C. Reactions were stopped with 80 µl
of 1 N HCl, and lipids were extracted with 160 µl of 1:1
(v/v) chloroform:methanol and analyzed by thin layer chromatography (26). Standard PIP, PIP2, and PIP3 are reaction
products generated from an anti-PI 3-K immunoprecipitate obtained using
a polyclonal antibody raised against a glutathione
S-transferase fusion protein containing the N-terminal SH2
domain of the rat p85 subunit of PI 3-K.
To
gain initial clues regarding the functional importance of
integrin-TM4SF complexes, we analyzed their cellular distribution by
immunofluorescence. Previous studies showed that standard fixation and
permeabilization procedures removed substantial amounts of TM4SF
proteins from the cell surface, thereby precluding detailed analysis of
the complex distribution (11). To overcome this problem, we pretreated
cells with chemical cross-linker prior to fixation and
permeabilization. HT1080 cells plated on laminin in serum-free medium
could assemble structures strongly resembling classical integrin focal
adhesions (27, 28) as indicated by staining with anti-integrin
6 (Fig. 1a) or anti-vinculin
(Fig. 1b) mAbs. In addition, integrin
6 (Fig.
1a) and vinculin (Fig. 1b) were distributed in
complexes throughout the cell periphery in a pattern strongly
resembling plasma membrane "focal complexes" that were recently
described (29). In comparison, two TM4SF proteins (CD63 and CD81) that
associate with the
6 integrin (10, 11) were detected in
focal complexes but excluded from focal adhesions (Fig. 1, c
and d). The
3
1 integrin (Fig.
1e) also showed peripheral focal complex-type staining but
no focal adhesion staining, whereas another prominent membrane protein,
emmprin (24), showed a uniform punctate distribution and was not
present in either focal adhesions or focal complexes (Fig.
1f). Notably, in
3-transfected RD cells (18)
plated on laminin-1, fibronectin, or a 40-kDa fragment of fibronectin,
both
3
1 integrin and TM4SF proteins were
again detected in focal complexes and excluded from the focal adhesions
(data not shown). Inability of TM4SF proteins to cluster into focal
adhesions even when an appropriate integrin is present (e.g.
6
1 in Fig. 1a) suggests that
function of the integrin-TM4SF complexes may be specifically relevant
to focal complexes rather than focal adhesions. In subsequent
experiments we focused on the
3
1-CD63-CD81 complex because it is far
more abundant in HT1080 cells than
6
1-CD63-CD81.
Integrin-TM4SF Protein Association with Phosphatidylinositol 4-Kinase
Lamellipodial and filopodial focal complexes may trigger
signal(s) leading to the reorganization of actin cytoskeleton and focal
adhesion assembly (29, 30). Because phosphoinositides may be potent
effectors of actin polymerization (31-33), we investigated whether
phosphoinositide kinase activity could be co-purified with the
3
1-TM4SF complex. Integrin and TM4SF
immunoprecipitates prepared from HT1080 cells were assayed for
phosphoinositide kinase activity. The reaction products co-migrated on
TLC plates with standard PIP but not PIP2 or
PIP3 (Fig. 2A). Incorporation of 32P into PIP was observed with
3,
1, CD63, and CD81 immunoprecipitates (Fig.
2A, lanes a and c-e) but not with
5 or negative control P3 immunoprecipitates (Fig.
2A, lanes b and f). This result is consistent with previous results showing that even when
5
1 is abundantly expressed
(e.g. on HT1080 and K562 cells), it is not associated with
TM4SF proteins (10-12). Notably, an immunoprecipitate of NAG2, another
TM4SF protein associated with
3
1
integrin,3 did not exhibit associated
phosphatidylinositol kinase activity (data not shown). This provides
additional evidence for the specificity of interaction between
3
1-CD63-CD81 and phosphatidylinositol kinase.
Specific association of PI 4-K with
3
1-TM4SF complexes. A, integrins or TM4SF proteins
were immunopurified, and phosphoinositide kinase activity in the
immunoprecipitates was assayed as described under "Materials and
Methods." Arrows indicate TLC migration positions of the
phosphoinositide markers, derived from a PI 3-K immunoprecipitation. B, [32P]PtdIns phosphate produced by
CD63-associated phosphoinositide kinase activity (as in A)
was eluted from a TLC plate, deacylated, and analyzed by HPLC as
described (26). A tritiated PtdIns 4-phosphate (DuPont NEN) standard
was deacylated and included with the 32P-labeled product in
the HPLC run. Migration positions of PtdIns 4-phosphate standard and
PtdIns 3-phosphate are indicated. C, integrins or CD63 were immunopurified
and probed by Western blotting with a rabbit polyclonal antibody raised
against recombinant PI 4-K
, a 97-kDa protein that shares similar
enzymatic characteristics with type II PI 4-K (46). D,
integrins or CD63 were immunopurified from K562 or
3-transfected K562 (18) cells, and the presence of PI
4-K activity in the immunoprecipitates was tested as in A.
The lanes marked PI 3-K show PIP, PIP2, and
PIP3 standards generated by a PI 3-K reaction.
Although TLC analysis separated PIP from PIP2 and
PIP3, it did not discriminate between different PtdIns
phosphate species, e.g. PtdIns 3-phosphate and PtdIns
4-phosphate (and PtdIns 5-phosphate, if such a product exists). To
determine the position of phosphorylation, the
[32P]PtdIns phosphate product generated by the CD63
immunoprecipitate was extracted from the TLC plate, deacylated, and
analyzed by HPLC. This deacylated product co-migrated identically with
authentic deacylated 3H-labeled phosphatidylinositol
4-phosphate but apart from standard phosphatidylinositol 3-phosphate
(Fig. 2B). Thus, there is PI 4-K activity in the
3
1-TM4SF complex.
To determine the type of PI 4-K associated with the
3
1-TM4 complexes, lipid kinase reactions
on
3 and CD63 immunoprecipitates were carried out in the
presence of Triton X-100 (0.3%), adenosine (200 nM) or mAb
4C5G (5 µg/ml). Previous data showed that activity of PI 4-K type II
can be stimulated by nonionic detergent and inhibited by adenosine and
the 4C5G mAb, whereas all three reagents have little or no effect on PI
4-K type III (25, 34). Adding adenosine and 4C5G mAb to the reactions
decreased the activity of the enzyme by 70-80%, whereas Triton X-100
had a stimulatory effect (15-20-fold) (data not shown), thus
indicating that
3
1-TM4SF complex is
associated with a PI 4-kinase with type II properties. This conclusion
was extended by Western blotting with an anti-PI 4-K polyclonal
antibody that detected a protein of 55 kDa, characteristic of PI 4-K
type II. Notably, the 55-kDa protein was present (Fig. 2C)
in anti-
3 (lane a) and anti-CD63 (lane
c) but not in anti-
5 or negative control
immunoprecipitates (lanes b and d). Compared with
the total lysate sample (Fig. 2C, lane e),
comparable levels of 55-kDa protein were detected in CD63 and
3 lanes that were derived from 20-fold more cell
equivalents. Thus, approximately 5% or more of the 55-kDa PI 4-K
protein may be present in a complex with
3 integrin
and/or CD63.
The amount and the activity of PI 4-K co-immunoprecipitated with
anti-CD63 mAbs was consistently greater than that detected with
anti-integrin or anti-CD81 mAbs (Fig. 2, A and
C), suggesting that the CD63 interaction with PI 4-K may not
require 3
1 integrin. Indeed, PI 4-K could
be co-purified with CD63 protein from K562 cells, which do not express
appreciable levels of
3
1 integrin (Fig.
2D, lane b). As expected for these cells,
1 integrins (predominantly
5
1) lacked associated phosphoinositide
kinase activity (Fig. 2D, lane c). However, when
the
3
1 heterodimer was expressed in K562
cells (after transfection of
3 subunit cDNA), PI 4-K was then co-immunoprecipitated with
1 integrins (Fig.
2D, lane g). Together these results suggest that
TM4SF proteins may link
3
1 integrin to PI
4-K.
Adhesion-dependent stimulation of phosphatidylinositol
4,5-bisphosphate production is an established biological phenomenon that may be controlled by members of the Rho family of small GTPases (35, 36). The present demonstration of physical association between
3
1 integrin and PI 4-K, an intracellular
enzyme that controls the first step in biosynthesis of
PIP2, suggests another link between integrin activation and
metabolism of phosphoinositides. The
3
1-CD63-CD81-PI 4-K-linked complex is
distinct from the conventional FAK-related pathway insofar as its
specificity for a particular
1 integrin (e.g.
3
1 but not for
5
1). Moreover, triggering of the
3
1-CD63-CD81-PI 4-K complex with anti-TM4 mAbs (to either CD63 or CD81) failed to induce tyrosine phosphorylation of 120-130-kDa cellular proteins (Fig. 3, lanes
e and f). In contrast, tyrosine phosphorylation of
120-130-kDa cellular proteins that probably correspond to FAK and Cas
(37-40) was induced by all three anti-integrin mAbs (Fig. 3,
lanes a, b, and d). Thus, we
hypothesize that the fraction of
3
1 in
3
1-CD63-CD81-PI 4-K complexes may be
distinct from that which signals through FAK or Cas.
What could be the function of the
3
1-CD63-CD81-PI 4-K complex in cells? The
formation of an integrin-TM4SF-PI 4-K complex is not
adhesion-dependent, because it is observed in K562 cells grown in suspension (e.g.. see Fig. 2D,
lane g). Rather, given its prominent clustering at the
periphery of spread cells, it is possible that an
3
1-CD63-CD81-PI 4-K adhesion complex may direct lamellipodial and filopodial protrusions during cell migration. Indeed, some properties of the complex may be well suited for this
purpose. First, the
3
1-CD63-CD81-PI 4-K
complex can be easily extracted from the cell membrane, thus suggesting
that its interaction with ECM substrate is not very strong (11). These
weak and transient interactions are particularly important at the
leading edge of lamellipodia because they allow a cell to sample the
substrate before deciding where to move. In this regard, the presence
in the complex of PI 4-K, an enzyme implicated in vesicular transport
(41, 42), and CD63, a protein that has a YXXM
internalization signal (43), could help to perpetuate the process of
sampling through the recycling of
3
1
within the leading edge. Second, the magnitude of the biochemical
signal (synthesis of phosphoinositides) produced by activated complex could be a decisive factor in determining the degree of actin polymerization at the leading edge and further guiding lamellipodial and filopodial protrusions. In this regard, TM4SF proteins that can
interact with one another (11, 44, 45) and with
3
1 integrin (11) may regulate lateral
clustering of the complex, thus affecting potency of the signal.
We thank Drs. O. Yoshie and D. G. Bole for generous gifts of antibodies and L. Cantley for helpful discussions.