The tyrosine kinase connection: proteins activate T

Deborah Brown

how GPI-anchored cells

State University of New York at Stony Brook, Stony Brook, USA

How can crosslinking of ceil-surface glycosylphosphatidylinositol-anchored proteins activate T cells when the proteins do not reach the cytosol? Recent

results show that glycosylphosphatidylinositol-anchored proteins associate with tyrosine kinases of the src family. Kinase activity is stimulated when

glycosylphosphatidylinositol-anchored proteins are crosslinked. How the

proteins are linked across the membrane, however, remains an intriguing

mystery.

Current Opinion in lmmunolgy 1993, 5:349-354

Introduction

Some cell-surface proteins are anchored in membranes by covalent attachment to glycosylphosphatidylinositol (GPI) rather than by conventional transmembrane spans. T cells express GPI-anchored proteins abundantly, and antibody-mediated clustering of these proteins activates the cells by a mechanism that requires the GPI anchor [VI. GPI-anchored proteins are restricted to the outer leailet of the bilayer, and are integrated into membranes only by phosphatidylinositol. The ability of these proteins to stimulate T cells is surprising and it is not known how the activation signal is transmitted across the membrane. This topic has been reviewed recently [3,4]; for general reviews of GPI-anchored proteins, see [ 5,6]. This review will focus on recent studies that suggest a possible mech- anism for this puzzling phenomenon.

Tyrosine phosphorylation

Phosphotylation of various cgllular proteins on tyro- sine residues plays a crucial early role in T-cell activa- tion [7-Io,II*-14.,15**,16.,17’], reviewed in [18*]. Two non-receptor protein tyrosine kinases of the src family, ~56’~~ and p59fy”, have been implicated in this process. p56lck binds to CD4 and CD8 [19,20], whereas p59fy” binds to the T-cell receptor (TCRxD3 complex (211. CD4 acts as a co-receptor with the TCR, and associates closely with it during activation (reviewed in [22]). AS- sociation of CD4 with the TCR depends on the binding of CD4 to p56lck, and is required for optimal stimula- tion [12*,23*]. Ligation of the TCR or of CD4 leads to rapid stimulation of the bound kinase [16.,24,25*]. Ty- rosine phosphorylation is required for activation [E&9].

The precise roles of the two kinases in mature T cells are not clear [13*,26.,27,28] (reviewed in [lS*]>. It has recently been shown that mature T cells (but not thy- mocytes) from mice lacking p59ti can be activated to some extent [ 14*,15**], whereas thymocyte development in mice lacking p56tck is severely perturbed [29-l.

GPI-anchored proteins and tyrosine kinases

Antibody-mediated crosslinking of GPI-anchored pro- teins also induces rapid phosphorylation of several sub- strates on tyrosine residues [ 71. This finding suggests that GPI-anchored proteins might interact in some way with src-like kinases such as p59fy” or p56ick.

Three groups have now demonstrated an interaction di rectly, by co-immunoprecipitating kinases with GPI-ar- chored proteins. Stefanova et al. [30] surveyed a van- ety of cell types, including human T cells and mono- cytes, human and murine lymphoid and myeloid cell lines, and a human colon carcinoma line. Antibodies di- rected against the GPI-anchored proteins CD59, CD55, CD48, CD24, CD14, Thy-l and LY-6 co-precipitated ki nases that were active in immune complex kinase as- says. Phosphorylation substrates among proteins in the complexes included proteins of 40, 60, and 80 kDa. The authors found p56ICk in the complexes immunoprecipi- tated from T cells and T-cell lines. The kinase responsible for in vitro phosphorylation in complexes isolated from the other cells was not identified.

Although p56lck could be recovered with either CD4 or CD59 in co-precipitates, there was no detectable asso- ciation between the two cell-surface proteins. This may indicate that separate p56tckc~4 and p56lckcD59 com- piexes are present in T-cell membranes. In vivo tyrosine phosphotylation of a number of proteins was stimulated

Abbreviations DAF4ecay accelerating factor; GPI-glycosylphosphatidylinositol; IL-interleukin; MDCK-Madin-Darby canine kidney;

TCR-T-cell receptor.

@ Current Biology Ltd ISSN 0952-7915 349

350 Lymphocyte activation and effector functions

by antibody-mediated crosslinking of CD55, CD59, CD48 or CD24 on cells expressing these proteins.

Thomas and Samelson [31**] used antibodies directed against Thy-l to recover kinase activity from mouse thy- mocytes, and from 2B4 and BW5147 T-cell lines. Proteins of 43,60,70-75, and 110 kDa from 2B4 cell lysates were phosphorylated when the complexes were subjected to an in vitro kinase assay. In vivo phosphorylation of the 43 kDa protein was reported to be stimulated following T-cell activation through the TCR. The 70 kDa substrate was determined to be the murine C-type virus envelope glycoprotein. In contrast to the results of Stefanova et al. [30], these authors found that the 60 kDa protein asso- ciated with the complexes isolated from each of these cell types was p59fy”. The association between Thy-l and p59fy” does not require the TCR, as the co-pre- cipitate can be isolated from BW5147 cells that do not express the antigen receptor.

Lublin and coworkers [32**] transfected the mouse T- cell line EL-4 with decay accelerating factor (DAF), a GPI-anchored protein, or with a transmembrane form of DAF. Cells expressing DAF, but not cells expressing the transmembrane form of DAF, produced interleukin (IL)-2 after antibody-mediated crosslinking. This treat- ment also stimulated in vivo tyrosine phosphorylation of a 40 kDa protein and of two 8595 kDa proteins in the DAF transfectants. Antibodies to DAF co-immunopre- cipitated kinase activity only from cells expressing DAF. Proteins of 40,56&O and 85 kDa were phosphorylated in immune complex kinase assays. The authors found both p561Ck and p59ti in these complexes.

Activation mechanisms

These lindings suggest that the mechanism of T-cell acti- vation through the TCR and through GPI-anchored pro- teins may be very similar. In both cases, binding of ligand (or antibody) to a cell-surface protein is followed rapidly by activation of intracellular kinases. Stimulation of tyro- sine kinase activity and cell activation through the TCR re- quire the leucocyte-specific protein tyrosine phosphatase CD45 [33,34], which activates p56Ick by removing an in- hibitory phosphate [35,36]. A close association between CD45 and the GPI-anchored protein Thy-l has been demonstrated by chemical crosslinking [37], and it seems likely that the phosphatase may activate kinases that are associated with GPI-anchored proteins. Downstream sig- naling events, including calcium flux and phospholipid hydrolysis, are similar in the two pathways. Activation of cells by GPI-anchored proteins requires the TCR, but the basis of this dependence is not known [ 38,391.

How do GPI-anchored proteins and kinases interact?

The binding of p561ck to CD4/CD8 has been very pre- cisely deiined [ 19,20,40], and studies characterizing the binding of p59h to the TCR are now emerging [18*]. In contrast, it is not clear how GPI-anchored proteins and src-like kinases associate. These proteins are re-

stricted to opposite leaflets of the bilayer and cannot bind each other directly. Presumably, a transmembrane ‘linker’ protein mediates the interaction between the two. As the GPI moiety is required for activation, this linker may bind directly to GPI (see below for an alternative). The sub- strates labeled in the immune complex kinase assays described above may serve as transmembrane ‘linkers’. Further characterization of these and other candidates for this role should proceed rapidly.

There may be a family of these linker proteins. Different members of the family may associate with the different src-like kinases in a cell type-specific manner. This might help explain the fact that in the three studies outlined above [30,31**,32**] either p56ICk, p59fy”, or both pro- teins were found to be associated with GPI-anchored proteins. Association of GPI-anchored proteins with the p62YeS kinase provides an additional example of this ap- parent cell-type specificity. Complexes from epithelial cells that are enriched in GPI-anchored proteins also contain p62YeS (D Brown, unpublished data), although the protein was absent from the similar complexes iso- lated by Thomas and Samelson [31**] from 2B4 cells.

Large co-immunoprecipitates contain lipid

Horejsi and colleagues [41**] have characterized their co-immunoprecipitated complexes in more detail, with surprising results. Using column chromatography, these authors found that all of the kinase-associated GPI-ar- chored proteins in T-cell lysates are present in structures of about 100 nM in diameter. Each of these contains sev- eral different GPI-anchored proteins, as well as p56Ick and epitopes commonly found on glycolipids. All of the CD59 that associates with kinase activity is present in the large complexes. The fraction that is not in these structures does not appear to associate with any other cell-surface proteins, as no labeled proteins are co-immunoprecipi- tated with CD59 from this pool after surface iodination.

The complexes isolated by Lublin and coworkers (D Lublin; personal communication) have similar prop- erties. A fraction of detergent-solubilized DAF was recov- ered in the void volume from a Sepharose 4B column, indicating that it was associated with a large complex. The kinases only associated with the fraction of DAF that was in these structures.

Detergent-resistant membrane

Similar large particles have been isolated from cul- tured epithelial cells [42*]. These may provide a useful model for further study of the T-cell complexes. GPI- anchored proteins from lysates of Madin-Darby canine kidney (MDCK) cells were recovered in detergent-resis- tant membrane vesicles ranging in diameter from 100 nM to 1 PM. A lipid bilayer structure was visible in electron micrographs, and the complexes were found to contain a mixture of phospholipids, glycolipids and cholesterol in about equal proportions. The complexes are resis- tant to solubilization by a number of non-ionic deter- gents. Only octyl glucoside was found to solubilize the structures efficiently. The complexes are stable under

How GPI-anchored proteins activate T cells Brown 351

conditions that should disrupt protein-protein interac- tions, suggesting that the lipids might be important in maintaining their integrity. In agreement with this pre- diction, liposomes with a similar lipid composition are also resistant to detergent solubilization (R Schroeder, D Brown, unpublished data).

These results suggest that certain lipids spontaneously form membrane microdomains that are resistant to ex- traction by detergent, and are isolated from cells in the form of membrane vesicles, These membrane domains are rich in glycolipids, which can self-associate to form clusters in aritificial bilayers [43], This property sug- gests that they may also be important in forming the detergent-resistant microdomains in cell membranes. It has been proposed that some membrane proteins have an affinity for glycolipid clusters (441. GPI-anchored pro- teins are probably among these, and it appears that close association with detergent-resistant lipid clusters prevents their solubilization. The interaction is favored by the fact that both GPI-anchored proteins and glycolipids are re- stricted to the outer leaflet of the bilayer. (See [45] for a review of the interactions between GPI-anchored pro- teins and membrane lipids.)

There are’several indications that the complexes co-im- munoprecipitated with GPI-anchored proteins from T cells may be similar to the membrane complexes iso- lated from MDCK cells. HorejSi and colleagues [41*-l have found that their complexes contain lipid [46]. At least some of this is probably glycolipid, as antibod- ies directed against epitopes generally found on glyco- lipids recognize the structures. The complexes isolated by Thomas and Samelson [31**] are likely to contain lipid as well, as they move to a low density position after centrifugation of sucrose density gradients to equi- librium. Fully solubilized protein complexes would not be buoyant under the conditions used by these workers ( [42**] ; L Samelson, personal communication).

The MDCK-derived complexes are insoluble, and are eas- ily pelleted in a microfuge, whereas the complexes iso- lated from T cells remain in the supernatant. This differ- ence does not necessarily mean that the two complexes have very different structures. The large size of the com- plexes described by Cinek and HorejSi [41**] and by Lublin and colleagues (D Lublin, personal communica- tion) strongly suggests that they are not fully solubilized, and it seems likely that they are actually similar to the complexes derived from MDCK cells. Relatively minor cell type-specific differences between the two, such as the protein to lipid ratio, might alfect the ability to pellet in the centrifuge.

Role of lipids in linking proteins

GPI-anchored proteins from MDCK cells remain in detergent-resistant membranes even when harsh chao- tropes are included in the lysis buffer to disrupt protein-protein bonds, suggesting that GPI anchors may interact directly with membrane lipids [42*]. The T-cell complexes may be similar. T&o groups ( [ 31**] ; D Lublin, personal communication) found that octyl glucoside dis- rupted the interaction between GPI-anchored proteins and kinases. This detergent does not generally disrupt protein-protein interactions that are stable to Triton X- 100. However, the lipid clusters isolated from epithelial cells are dispersed by this detergent, leading to complete solubilization of both the lipids and the proteins. Disrup- tion of the T-cell complexes by octyl glucoside suggests that the lipids in these structures may play a crucial role in linking the proteins.

These results raise the unorthodox possibility that GPI- anchored proteins may not bind directly to transmem- brane linker proteins in order to associate with intra- cellular kinases. GPI-anchored proteins seem to have an affinity for clusters of associated lipids in the outer

/ GPI-anchored proteins

\

src-like. Putative transmembrane kinase kinase-binding protein

Fig. 1. Interaction of GPI-anchored pro- teins and src-like kinases. Antibodies directed against CPI-anchored proteins

can co-immunoprecipitate src-like ki- nases 130,31**,32**]. CPI-anchored pro-

teins are restricted to the outer leaflet of the bilayer, whereas the kinases are cy-

tosolic except for amino-teminal myris- tic acid. Thus, a transmembrane ‘linker’ protein is proposed to mediate the in-

teraction between the two. This pro- tein probably binds directly to myristy- lated src-like kinases. It may also bind

to CPI-anchored proteins, most likely to the CPI anchor. Alternatively, the trans- membrane proteins may associate with

patches of clustered lipids (shown with large, shaded head groups), as CPI-an- chored proteins appear to do. Asso-

ciation of both CPl-anchored proteins and putative ‘kinase receptors’ with the same lipid clusters could bring the CPI- anchored proteins and the kinases into

close association.

352 lymphocyte activation and effector functions

leaflet of the bilayer. Some tmnsmembrane proteins may also associate with these lipid domains. Binding of the ki- nases to the cytoplasmic domain of such proteins could lead to a close association with GPI-anchored proteins in the opposite leaflet of the bilayer. This model is shown in Fig. 1.

It is possible that kinases do not bind to transmembrane proteins, but associate directly with lipid microdomains. This seems unlikely, as pp60v-Src cannot associate with membranes except by binding to a saturable receptor [47]. Myristic acid is probably not hydrophobic enough to hold src-like kinases in the bilayer.

Internalization in caveolae?

Bamezai et al. [48] showed that immobilization of anti- bodies directed against GPI-anchored proteins (but not of TCR-specific antibodies) prevents activation, and that GPI-anchored proteins are internalized from the surface of T cells. These results were interpreted to suggest that the proteins must be internalized in order for activation to occur. More recently, these authors found that inter- nalization of LY-6~2 and Thy-l occur by a mechanism distinct from receptor-mediated endocytosis in clathrin- coated pits [49*].

GPI-anchored proteins can be transiently internalized via non-clathrin-coated membrane invaginations called cave- olae, in a process termed potocytosis by Anderson and colleagues [ 50.1. Using the uptake of folate after binding to the GPI-anchored folate receptor as a model system, these authors have proposed that potocytosis is a gen- eral mechanism for transport of small molecules into the cytoplasm. Thy-l and LY-6~2 may be internalized by this means. The fate of the proteins once in caveolae, and the role of internalization in signaling are still not clear. Perhaps GPI anchors are cleaved, leading to the genera- tion of signaling molecules that are transported into the cytosol. (See [3] for a full discussion of this model.) If so, tight regulation of cleavage and transport could explain why no cleaving activity of the GPI anchor has been detected in T cells, and why addition of exogenous anchor-cleaving phospholipase to T cells does not acti- vate them. Alternatively, internalization may be required in some undefined way in order for GPI-anchored pro- teins to activate tyrosine kinases.

Conclusion

Provocative new studies show that antibodies directed against GPI-anchored proteins can co-immunoprecipitate the src-like tyrosine kinases p561ck and/or p59fy” from T-cell lysates. The new results support previous data in strongly suggesting that tyrosine phosphorylation plays a key role in activation of T cells by GPI-anchored proteins. The finding that these proteins are physically associated is an important step toward determining a precise mech- anism, but also raises some new questions.

First, it is not clear how the GPI-anchored proteins and src-like kinases associate with each other. Transmem- brane proteins are almost certainly involved, and spe-

cialized lipid microdomains may also play a role. Thy-l artificially implanted in T-cell membranes cannot activate the cells [ 51.1; possibly signaling proteins must associate soon after synthesis. Secondly, the relative roles of p5WCk and p59fy” in activation are not clear. Recent studies that have started to clarify the functions of these kinases in activation through the TCR should provide models for future experiments with GPI-anchored proteins.

Finally and most importantly, the physiological role of ac- tivation by GPI-anchored proteins is still a complete mys- tety, as is the relationship between signaling through lipid-linked proteins and through the TCR. It appears that signal transduction complexes containing GPI-an- chored proteins, transmembrane linkers, intracellular src- like kinases and possibly CD45 are present in the T-cell membrane. p56 Ick, p59fy” and CD45 are also associated directly or indirectly with the TCR during activation. De termining the relationship between these two signaling complexes remains a major challenge for the future

Acknowledgements

I thank Douglas Lublin for communicating results prior to publica- tion. Work in the author’s lab is supported by grant GM47897-01 from the NIH.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as . . . 1.

2.

3.

4.

5.

6.

7.

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9.

10

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An excellent recent review of the role of tyroslne kinases, and of tyrosine-based activation motifs found in the cytoplasmic domains of the CD3 subunits, in T-cell activation

Crossllnking of CD3 and CD4 together gives enhanced signaling com- pared with crosslinking either alone. Using a mutant cell line defective in surface expression of CD3-( the authors test the role of the CD3 c-chain in the enhancement of early signaling events by CD4 Signaling after crosslinking CD3 and CD4 is defective in this line, showing the importance of CD3-6.

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Cl% associates with the TCR-CD3 complex upon cell stimulation. This requires the cytoplasmic domain of CD4 Using point mutants and de- tecting molecular association by fluorescence resonance energy trans- fer, the authors show that CD4 must bind p561ck before it can associate with TCR-CD3 and to engage in proper signaling.

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During stimulation of T cells, two src-like tyrosine kinases associate with TCR-CD3: p59Iv” by direct binding, and p5Wck bound to CD4 that is recruited to the complex. Although stimulation of tyrosine kinase activ- ity is known to be crucial to signaling, it is not yet clear what kinase is responsible. This report of defective signaling in a mutant line of Jur- kat cells that lacks p56lck, and correction of the defect by exogenous expression of the kinase, establishes a clear role for lck in activation through the TCR.

Janeway and colleagues have proposed that CD4 acts as a co-recep- tor with the TCR and plays an obligatory role in signaling. A panel of TCR-specific antibodies is used to show first that interaction be tween CD4 and the TCR must occur with a specific orientation, and, more importantly, that CD4 must associate with the TCR in order for the TCR <-chain to be phosphotylated on tyrosine. Strongly supports the co-receptor hypothesis.

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354 Lymphocvte activation and effector functions

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Complexes that are imrnunoprecipitated with antibodies against GPI- anchored proteins also contain p56u [30]. Each complex is very large (100 nM), and contains several different GPI-anchored proteins and gly- coUpids. These may be similar in structure to complexes described in [42*] and may arise from specialized membrane microdomains.

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port to the Apical Cell Surface. Cell 1992, 68: 533544. GPI-anchored proteins from epitbelial cells are recovered from lysates in detergent-resistant membrane vesicles that are rich in glycolipids. These complexes may provide a model for studying the interactions between the lipids and proteins in the complexes isolated by other groups [31**,32**,41**].

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As antibodies against GPI-anchored proteins cannot activate T cells when immobilized, the lipid-linked proteins may require internalization to stimulate the cells. Here, GPI-anchored proteins are shown to be in- ternalized by a mechanism distinct from receptor-mediated endocytosis. Internalization may occur by potocytosis, or transient sequestration of GPI-anchored proteins into structures specialized for transport of small molecules into the cytosol [50*].

50. ANDERSON RGW, KAMEN BA, ROTHBERG KG, LACE?’ SW: Po- . tocytosis: Sequestration and Transport of SmaU Molecules

by Caveolae. Science 1992, 255: 410-411. GPI-anchored proteins in many cells are transiently internalized into caveolae, which can be specialized for the transport of small molecules into the cell. It has been reported that GPI-anchored proteins must be internalized to activate T cells, and the internalization pathway [48] is similar to that, used in potocytosis. Signaling may require exposure of GPI-anchored proteins to the interior of caveolae following binding of antibody.

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linked Protein Thy-l Into Cell Membranes. Proc Nat1 Acud Sci USA 1992, 89: 5231-5235.

Fluorescence recovery after photobleaching (FRAP) was used to show that the diffusion rate and immobile fraction of Thy-l artificially im- planted in ceU membranes was normal. However, crosslinking of the protein did not activate the cells. Trivial explanations were not ex- cluded, but the results could imply that Thy-l must interact with other proteins before reaching the cell surface to be competent in signaling.

D Brown, Department of Biochemistry and Cell Biology, Stony Brook, State University of New York at Stony Brook, New York 11794-5215, C’SA.