COMMUNICATION:
A Novel Link between Integrins, Transmembrane-4 Superfamily Proteins (CD63 and CD81), and Phosphatidylinositol 4-Kinase*

(Received for publication, September 17, 1996, and in revised form, November 13, 1996)

Fedor Berditchevski Dagger §, Kimberly F. Tolias par , Karen Wong **, Christopher L. Carpenter and Martin E. Hemler Dagger Dagger Dagger

From the Dagger  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

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

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 (alpha 3beta 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 alpha 3beta 1-CD63 complexes in alpha 3-transfected K562 cells, but also it could be co-purified from CD63 in untransfected K562 cells lacking alpha 3beta 1. Thus, TM4SF proteins may link PI 4-K activity to the alpha 3beta 1 integrin. The alpha 5beta 1 integrin, which does not associate with TM4SF proteins, was not associated with PI 4-K. Notably, alpha 3beta 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.


INTRODUCTION

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 beta 1 integrins, including alpha 3beta 1, alpha 6beta 1, and alpha 4beta 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 alpha 3beta 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.


MATERIALS AND METHODS

Antibodies

Anti-integrin mAbs used were: anti-alpha 3, A3-X8 (18), A3-IVA5 (18), and A3-IIF5 (18); anti-alpha 5, A5-PUJ2 (19); anti-alpha 6, A6-ELE (19); anti-beta 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).

Immunoprecipitation and Lipid Kinase Assays

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 [gamma -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.


RESULTS AND DISCUSSION

Immunofluorescent Staining of Integrins and TM4SF Proteins

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 alpha 6 (Fig. 1a) or anti-vinculin (Fig. 1b) mAbs. In addition, integrin alpha 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 alpha 6 integrin (10, 11) were detected in focal complexes but excluded from focal adhesions (Fig. 1, c and d). The alpha 3beta 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 alpha 3-transfected RD cells (18) plated on laminin-1, fibronectin, or a 40-kDa fragment of fibronectin, both alpha 3beta 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. alpha 6beta 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 alpha 3beta 1-CD63-CD81 complex because it is far more abundant in HT1080 cells than alpha 6beta 1-CD63-CD81.


Fig. 1. Distribution of integrin-TM4SF complexes. HT1080 cells in synthetic buffer (11) were allowed to spread on laminin for 90 min. Then cells were treated with a chemical cross-linker (250 µM dithiobis(succinimidyl propionate)) at 25 °C for 30 min. After three washes cells were fixed with 2% paraformaldehyde for 10 min and then permeabilized with 0.2% CHAPS for 2-3 min. Cells were then stained with mAbs against integrin alpha 6 (a), vinculin (b), CD63 (c), CD81 (d), integrin alpha 3 (e), or emmprin (f). Staining was visualized with rhodamine-conjugated goat anti-mouse Ig (b-f) or fluorescein isothiocyanate-conjugated goat anti-rat Ig (a). Focal adhesion-like structures are indicated by arrowheads (a and b).
[View Larger Version of this Image (116K GIF file)]


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 alpha 3beta 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 alpha 3, beta 1, CD63, and CD81 immunoprecipitates (Fig. 2A, lanes a and c-e) but not with alpha 5 or negative control P3 immunoprecipitates (Fig. 2A, lanes b and f). This result is consistent with previous results showing that even when alpha 5beta 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 alpha 3beta 1 integrin,3 did not exhibit associated phosphatidylinositol kinase activity (data not shown). This provides additional evidence for the specificity of interaction between alpha 3beta 1-CD63-CD81 and phosphatidylinositol kinase.


Fig. 2.

Specific association of PI 4-K with alpha 3beta 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-Kalpha , 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 alpha 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.


[View Larger Version of this Image (17K GIF file)]


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 alpha 3beta 1-TM4SF complex.

To determine the type of PI 4-K associated with the alpha 3beta 1-TM4 complexes, lipid kinase reactions on alpha 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 alpha 3beta 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-alpha 3 (lane a) and anti-CD63 (lane c) but not in anti-alpha 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 alpha 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 alpha 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 alpha 3beta 1 integrin. Indeed, PI 4-K could be co-purified with CD63 protein from K562 cells, which do not express appreciable levels of alpha 3beta 1 integrin (Fig. 2D, lane b). As expected for these cells, beta 1 integrins (predominantly alpha 5beta 1) lacked associated phosphoinositide kinase activity (Fig. 2D, lane c). However, when the alpha 3beta 1 heterodimer was expressed in K562 cells (after transfection of alpha 3 subunit cDNA), PI 4-K was then co-immunoprecipitated with beta 1 integrins (Fig. 2D, lane g). Together these results suggest that TM4SF proteins may link alpha 3beta 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 alpha 3beta 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 alpha 3beta 1-CD63-CD81-PI 4-K-linked complex is distinct from the conventional FAK-related pathway insofar as its specificity for a particular beta 1 integrin (e.g. alpha 3beta 1 but not for alpha 5beta 1). Moreover, triggering of the alpha 3beta 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 alpha 3beta 1 in alpha 3beta 1-CD63-CD81-PI 4-K complexes may be distinct from that which signals through FAK or Cas.


Fig. 3. Antibodies to TM4SF proteins fail to show conventional integrin-type signaling. Intact HT1080 cells were incubated with anti-integrin (lanes b-d), anti-TM4SF (lanes e and f), or with anti-CD109 control (lane g) mAbs and then with goat anti-mouse Ig polyclonal antibody. Clustering of the membrane proteins was induced at 37 °C for 15 min, and total cellular lysates were probed with anti-pTYR (A). As a control for loading, the filter was reprobed (B) with anti-BIP antibody (47). The arrow indicates the positions of the 120-125-kDa proteins (A).
[View Larger Version of this Image (37K GIF file)]


What could be the function of the alpha 3beta 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 alpha 3beta 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 alpha 3beta 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 alpha 3beta 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 alpha 3beta 1 integrin (11) may regulate lateral clustering of the complex, thus affecting potency of the signal.


FOOTNOTES

*   This work was supported by Grants GM38903 (to M. E. H.) and GM54387 (to C. L. C.) from the National Institutes of Health. 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.
§   Recipient of a Lady Tata Memorial Trust Fellowship. Present address: University of Birmingham, CRC Institute for Cancer Studies, Edgbaston, Birmingham B15 2TJ, UK.
par    Recipient of Ryan Fellowship
**   Supported by the Lucille P. Markey Charitable Trust.
Dagger Dagger    To whom correspondence should be addressed. Tel.: 617-632-3410; Fax: 617-632- E-mail: Martin_Hemler@DFCI.HARVARD.EDU.
1    The abbreviations used are: FAK, focal adhesion kinase; PI 4-K, phosphatidylinositol 4-kinase; PI 3-K, phosphatidylinositol 3-kinase; TM4SF, transmembrane-4 superfamily; mAb, monoclonal antibody; PtdIns, phosphatidylinositol; PIP, phosphatidylinositol phosphate; PIP2, phosphatidylinositol diphosphate; PIP3, phosphatidylinositol triphosphate; HPLC, high pressure liquid chromatography; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
2    F. Berditchevski, S. Chang, and M. E. Hemler, manuscript in preparation.
3    I. Tachibana, F. Berditchevski, J. Bodorova, and M. E. Hemler, manuscript in preparation.

Acknowledgments

We thank Drs. O. Yoshie and D. G. Bole for generous gifts of antibodies and L. Cantley for helpful discussions.


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