Immunology and Cell Biology (1997) 75, 430-445

The role of CD45 and CD45-associated molecules in T cell activation

JOSEPH G ALTIN and ERICA K SLOAN
Division of Biochemistry and Molecular Biology, School of Life Sciences. Faculty of Science. The Australian National University, Canberra. Australian Capital Territoiy. Australia Summary CD45 (lymphocyte common antigen) is a receptor-linked protein tyrosine phosphatase that is expressed on all leucocytes, and which plays a crucial role in the function of these cells. On T cells the extracellular domain of CD45 is expressed in several different isoforms, and the particular isoform(s) expressed depends on the particular subpopulation of cell, their state of maturation, and whether or not they have previously been exposed to antigen. It has been established that the expression of CD45 is essential for the activation of T cells via the TCR, and that different CD45 isoforms display a different ability to support T cell activation. Although the tyrosine phosphatase activity of the intracellular region of CD45 has been shown to be crucial for supporting signal transduction from the TCR, the nature of the ligands for the different isoforms of CD45 have been elusive. Moreover, the precise mechanism by which potential ligands may regulate CD45 function is unclear. Interestingly, in T cells CD45 has been shown to associate with numerous molecules, both membrane associated and intracellular; these include components of the TCR-CD3 complex and CD4/CD8. In addition, CD45 is reported to associate with several intracellular protein tyrosine kinases including p56'^'^ and ^59^^'"' of the src family, and ZAP-70 of the Syk family, and with numerous proteins of 29-34 kDa. These CD45-associated molecules may play an important role in regulating CD45 tyrosine phosphatase activity and function. However, although the role of some of the CD45-associated molecules (e.g. CD45-AP and LPAP) has become better understood in recent years, the role of others still remains obscure. This review aims to summarize recent findings on the role of CD45 and CD45-associated molecules in T cell activation, and to highlight issues that seem relevant to ongoing research in this area. Key words: CD45, ligands, molecular associations, T cell activation, T cell receptor signalling.

Introduction
The interaction of a T cell with an APC such as a B cell, dendritic cell or a macrophage is crucial for eliciting an adaptive immune response. The response is initiated when a foreign antigen, presented as short peptides displayed by MHC class I or class II molecules on an APC, is specifically recognized and bound by the TCR. This event triggers a cascade of intracellular responses which results in the proliferation and differentiation of the T cell, a process known as T cell activation.'Apart from the TCR, a number of other T cell surface molecules are essential for efficient T cell activation. For example, evidence indicates that the CD4 and CD8 coreceptor molecules expressed on different T cell subsets (either CD4+ or CD8"^) may initiate or stabilize the T cell-APC interaction by binding to constant regions on the MHC class II and MHC class 1 molecules, respectively.-^-^ T cells also express various adhesion molecules including CD2 and LFA-1 (CD 1 la/CD 18) which bind to the cognate receptors CD58 (LFA-3), CD48 or possibly CD59, and ICAM-I, -2,-3. respectively, on APC.'^''' These receptors allow the T
Correspondence: Dr JG Altin, Division of Biochemistry and Molecular Biology, School of Life Sciences, Faculty of Science, The Australian National University, Canberra, ACT 2601, Australia. Email: <Joseph.Altin@anu.edu.au> Received 18 March 1997; accepted 2 June 1997.

cells to interact with APC encountered during circulation, and promote the appropriate interactions required for antigen recognition and T cell activation. Furthermore, the binding of the CD28 receptor on T cells to a molecule of the B7 family on APC, ensures that the TCR recognizes antigen in the correct cellular context by providing a costimulatory signal required for T cell activation.'-'' The primary function of these T cell surface molecules, therefore, appears to be that of supporting antigen-specific T cell activation, while at the same time preventing instigation of an immune response against a self-anligen expressed by a non-immune cell.'^'^ In addition to the molecules described above, considerable evidence indicates that the expression of molecules from the CD45 family'^"^*^ on T cells, is essential for efficient activation of these cells via the TCR and perhaps for the costimulating effects of other accessory molecules.^'"^^ Molecular interactions involving CD45, therefore, are most probably linked to the ability of the TCR and other molecules to transduce the signals required for induction of T cell activation.^^-^'' It has been established that the cytoplasmic region of CD45 possesses tyrosine phosphatase activity, and this activity is presumably crucial for CD45 function. The existence of isoforms of CD45 with different extracellular regions may reflect a potential for CD45 to interact with different ligands, and in recent years a number of intracellular and transmembrane molecules have been reported to associate with CD45. The aim of this review is primarily to sum-

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marize recent findings on CD45 and CD45-associated molecules in T cells, and to highlight issues that appear central to future research on the regulation of CD45 function, and the role of CD45 in T cell activation.

reports showing that CD45 isofomi switching can be re versed.

Stimulation of CD45 induces responses CD45, its structure and expression

CD45, also known as leucocyte common antigen (LCA),'^ is a family of high molecular weight (170-220 kDa) transmembrane glycoproteins which are expressed in different isoforms on all nucleated cells of the haemopoietic lineage.'^^^-'^ On these cells CD45 is an extremely abundant protein, with more than lO'' molecules per cell occupying up to 10% of the cell surface.-"^-^ Potentially eight isoforms of CD45 are produced from a single gene by alternative splicing of the three adjacent exons which encode the external aminoterminal domain; so far seven of these isoforms have been identified on various cells. CD45 has been shown to possess an extracellular domain of 400-550 amino acids (depending on the isoform), a single transmembrane pass of 22 amino acids and a cytoplasmic domain of 705 amino acids. "*"-'^'The extracellular domain Is highly glycosylated and contains Ihree structural regions that are conserved among vertebrate species. These include a region containing potential 0-linked carbohydrate sites, a cysteine-rich region, and a region containing three fibronectin type III domains.-**"'" The different isoforms of CD45 are generated by alternative splicing of three exons that encode a portion of the region containing the potential 0-linked glycosylation sites.'^'''-" The extracellular domain of CD45 also contains several potential sites for A'-linked glycosylation""'- and sulfation.*' Glycosylation of CD45 appears to be required for its cell surface expression and stability,-^-^^ and the pattern of glycosylation seen on resting cells undergoes changes upon induction of cell proliferation.^'-*- The variability provided by the potential differential splicing, glycosylation and sulfation of the CD45 extracellular domain may allow for interactions with a variety of ligands expressed either on the same cell and/or on interacting cells.^"^-^^ The cytoplasmic domain of CD45 has been shown to have two catalytic regions, each with a sequence similar to the placental protein tyrosine phosphatase IB (PTP IB}.'**'''''*' Interestingly, both catalytic regions are required for PTP activity,-"-^^ and CD45 Is the major PTP of lymphocytes accounting for more than 90% of their total PTP activity.-^ On T cells the pattem of CD45 isoform expression varies with the subpopulation of cell,''^''^ its stage of development**'^"* and antigenic stimulation.'^'^"^'^ Evidence indicates that the expression of several CD45 isoforms is reduced on T cells from HIV-infected patients; and this may be relevant to the diminished potential for activation of these cells in HIV infection.'"*'^'' Interestingly, normal T cells expressing different isoforms of CD45 possess different activation requirements.^*'"^- Early studies have shown that naive T cells (either CD4+ or CD8'^) predominantly express the high molecular weight form of CD45 (CD45RA), whereas memory T cells express a higher level of the low molecuhir weight form (CD45RO). These observations have led to the hypothesis that CD45RA and CD45RO mark virgin and memory T cells, respectively, in both CD4* and CD8* T cell populations.^^ This hypothesis has since been challenged, however, by As reviewed previously,'^'^-''^ a number of studies indicate that the binding of mAb to CD45, which results in the crosslinking of CD45 molecules on the T cell surface, can modulate T cell responses. Reported effects of CD45 mAb crosslinking on T cells include the induction of proliferation of resting human peripheral blood T cells, and the modulation of proliferation and IL-2 secretion in human peripheral blood T cells stimulated via CD3 or CD2.-'*^ " Interestingly, the coligation of CD45 with either CD3 or CD2 was found to inhibit intracellular signalling as reflected by changes m protein tyrosine phosphorylation, activation of phosphoHpase Cyl, and mobilization of intracellular calcium; this was also accompanied by a decrease in T cell proliferation and lymphokine expression.*'*'"^' Other studies indicate that ligation of CD45 alone can induce apoptosis in thymocytes and mature T cells and that this is potentiated by cross-linking of CD3.'^-''' Studies using CD45-negative cell lines, in contrast, have shown that the expression of CD45 on T cells is essential for the signalling of T cell activation through the TCR-CD3 complex, CD4/CD8 and CD2.^^^ These findings are consistent with CD45 playing a central role in T cell activation. T^is aspect of CD45 function will be discussed in more detail in later sections of this review. In addition to the effects described above, evidence indicates that the stimulation of CD45 with specific mAb may alter T cell adhesiveness. Thus, stimulation of CD45 with CD45-specific mAb is reported to induce the homotypic adhesion of human thymocytes and various human T cell lines with an immature phenotype,''^ and the adhesion of peripheral blood T cells to monocytes.''^ In contrast, stimulation with CD4,'i mAb was also found to inhibit the adhesion and homotypic aggregration of resting human peripheral blood T cells,"*''* suggesting that the effect of CD45 on adherence may depend on the activation state of the cells. These adhesion events appear to be mediated largely through the ability of CD45 to regulate a tyrosine kinase-dependent adhesion pathway involving the engagement of LFA-I with its ligands ICAM-1,-2,-3.^-'*-^^'^'' Other studies report that cross-linking of CD45 with mAb can induce dramatic morphological changes in a variety of T cell lines including CD8^ T cell clones,^'^' and the down-regulation of the surface expression of L-selectin (CD62L), an adhesion molecule important in lymphocyte aggregation and extravasation.^' The ability of CD45 to regulate T cell adhesion and signal transduction through the TCR, therefore, underscores its potential role in T cell activation.

Ligands for CD45

Perhaps the first suggestion of a ligand for CD45 was provided by studies which showed that CD45 from lysates of murine thymocytes binds to heparin sulfate,'- thereby indicating that CD45 may potentially interact with glycosaniinoglycan-containing molecules on the surfaces of cells.^^-"^^ Subsequently, evidence was provided that CD45RO interacts

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with CD22p,'''^ an isoform of the B cell lineage-specific glycoprotein CD22 which promotes the binding of B cells to T cells.^-''"^^ This evidence initially was based on the finding that the binding of COS cells transfected with CD22p to the human T cell line Molt-4, and the binding of a soluble CD22p-immunoglobulin fusion protein (CD22.Rg) to T cells, can be blocked with CD45RO-specific mAb.^'' Additional studies showed that CD22 interacts with several isoforms of CD45, and that the binding of CD22.Rg to T cells inhibits TCR signalling.^''^'^'^ Immunochemical evidence for the CD45-CD22 interaction was also provided by the finding that CD22.Rg can immunoprecipitate CD45RO,'''''^^ and murine CD22 associates with CD45RA in digitonin lysates of cells.^'^ The finding ihat CD22 recognizes a number of A'linked oligosaccharides with a2,6-linked sialic acids,^^-^"^^however, has questioned the specificity of the CD22-CD45 interaction.^-^ Concerns were also raised by the finding that CD22.Rg immunoprecipitates a number of other proteins from cell lysates, and that CD22.Rg binds to only a small fraction of the total a2,6-linked sialic acids on cells. These observations raised the possibility that a2,6-linked sialic acids may be necessary, but not sufficient, for the binding of CD22 to CD45.^' and has led to the suggestion that the CD45-CD22 interaction may require components in addition to a2,6-linked sialic acids.^^-^"^ Interestingly, the search for CD45 ligands has recently produced an additional potential candidate. Thus, a uniquely glycosylated form of CD45 (a hybrid type high in mannose and containing A'-linked oligosaccharides) from immature murine thymocytes was found to carry binding sites for serum mannan-binding protein.^'' The physiological relevance of this is presently unknown, but the presence of binding sites on thymocytes, and not on mature T cells, suggests an involvement in thymocyte maturation. The observations that forms of CD45 may interact with CD22 and other a2,6linked sialic acids,^*^ with serum mannan-binding protein,^-^ and potentially with glycosaminoglycan-containing molecules on cells,^^ therefore, clearly are consistent with an ability of CD45 on T cells to be involved in receptor-ligand recognition and cell-cell interactions.

molecules of 29-34 kDa.iO5.io&-iio jj,e physiological significance of some of these associations has been questioned as their reproducibility may depend on the particular technique(s) used in the experiments.^^ Clearly, further studies are required to establish any potential physiological role for the observed molecular associations with CD45; such studies may await the development and use of techniques that are more suited to study low-affinity molecular interactions than those that have been used to date. It is interesting, nonetheless, that CD45 is also found to associate with the B ceil antigen receptor on B cells,'"""^ and with CD16 (the low-affinity receptor for IgG) on human natural killer cells,"''"^ suggesting that an association of CD45 with molecules important in immune function also occurs in cell types other than T cells. Although the precise role of each molecular association with CD45 remains to be established, it is clear that such associations may potentially regulate the ability of CD45 to interact with specific ligands on other cells. Alternatively, it is possible that at least some of the CD45-associated molecules may utilize the transmembrane signalling ability of CD45 to mediate their effects. This is exemplified by the association of CD45 with Thy-1, a glycosyl-phosphatidylinositol-anchored molecule that lacks a transmembrane region, but which is capable of transducing T cell activation signals.^^'^^ The association of Thy-1 with CD45 on murine T cells suggests that signalling through Thy-1 may involve the intracellular tyrosine phosphatase activity of associated CD45, as it is unclear otherwise how Thy-1 can transduce a transmembrane signal. This notion is supported by the observation that CD45-negative cells fail to respond to Thy-1 stimulation."^ In this regard, it is relevant also that molecular associations on the T cell surface may depend on the activation state of the cell and may correlate with immunological memory.^^"^ Therefore, through modulation of PTP activity and/or by altering the ability of either CD45 or any associated molecule to interact with substrates, molecular associations with CD45 may endow the associated molecule(s) with the capacity to transduce transmembrane signals or to modulate their signalling ability. "^-"^

CD45 associates with other molecules

Apart from interacting with potential ligands on different cells, considerable evidence indicates that on T cells CD45 can interact with other molecules located on the surface and/or intracellularly. Thus, the use of co-capping techniques and/or the immunoprecipitation (with CD45-specific mAb) of CD45 from lysates of T cells pretreated with chemical crosslinkers or from cell lysates made using mild detergents like digitonin, have found that CD45 associates with a number of other molecules on murine and human T cells. These include an association of CD45 with: Thy-l,^^-**^ CD2^o,9i (although an association of CD2 with LFA-1 has also been sugCD28,i LFA-1 (CD58),96-^^ and CDIOO.'"^' Moreover, CD45 is also reported to associate with the cytoskeletal proteins fodrin and spectrin,'"^"^-^ the intracellular tyrosine kinases p56''^^ and p59^"^''**-^ components of the Ras signalling pathway,'^^ a 116 kDa phosphoprotein,'*^'' and several

The role of CD45 in TCR signalling

The TCR expressed on the majority of T cells consists of an aP heterodimer, a non-covalently associated CD3 complex comprising the y, 5 and e chains, and a disulfide-linked ^-chain homodimer or a i^T) heterodimer (for reviews see -119-121^ xhe ap dimer determines the antigen-binding specificity of the receptor while the CD3 complex and ^ and ri chains are involved in the functional expression of the TCR and its coupling to intracellular signalling mechanisms. Stimulation of the TCR alters the phosphorylation state of a number of intracellular proteins involved in signal transduction. However, unlike many other receptors, the TCR-CD3 complex does not have intrinsic kinase activity."''"'--'' but instead mediates its effects by interacting with various intracellular tyrosine kinases (Fig. 1)."23-I26 g^ch interactions usually involve specific amino acid sequences such as the immunoreceptor tyrosine-based activation motifs (ITAM)'^^ which are contained in the cytoplasmic regions of components of the

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CD3 complex, and which upon their phosphorylation create temporary binding sites for Src-homology 2 (SH2) containing signalling tyrosine kinases and other molecules operating downstream of the TCR. Engagement of the TCR is known to induce a rapid and sequential activation of the tyrosine kinases p56'^'^ and p59'^^" of the src family,'^^'-^^ and Syk and ZAP-70 of the Syk famjly.131-133 ^i^g activity of these kinases is essential for normal T cell development and function. Evidence suggests that in resting T cells the TCR e-chain is associated with p59'>", and the TCR ^ chain is constitutively phosphorylated on tyrosines and is associated with ZAP-70.'-'-"'^-'' Upon engagement of the TCR, p56''^^ phosphorylates and activates ^-chain-associated ZAP-70,'^''"'^^ an event promoted by the recruitment of p56''^'' to the TCR complex as a consequence of the CD4 or CD8 receptor binding to the MHC (Fig. I).i3q-i4i Recent evidence suggests that activation of ZAP-70 and its physiological substrate SLP-76, a tyrosine-phosphorylated protein that interacts with the proteins Vav, Grb2 (a component of the Ras pathway) and phosphoiipase C7I,'''-'"'-^ is required

for the phosphorylation and activation of phosphoiipase Q^i 144-147 ^5 shown in Fig. 1, activation of phosphoiipase C7I is responsible for generating inositoi 1.4,5-tnphosphate (IPj) and diacylglycerol (DG), second messengers which increase cytoplasmic levels of calcium and activate protein kinase C, respectively.'"*' It appears that increases in intracellular calcium and activation of protein kinase C are both necessary and sufficient, for induction of T cell activation.^^''*' Thus, increases in calcium (through the activation of downstream effectors like calcineurin) and activation of protein kinase C can, either directly or indirectly, alter the phosphorylation state and activity of transcription (actors like Fos. Jun, NF-ATp and N F - K B , which in turn results in the induction of genes for lymphokine production and cell proliferation.-^'^^'^'' Effective T cell activation, however, also requires a costimulatory signal through CD28, as T cells stimulated through the TCR (but deprived of signals through CD28) can enter a state of anergy or undergo apoptosis. While engagement of the TCR predominantly initiates signalling via the calcium and protein kinase C pathways. CD28

CD28

CD4/CD8

T cell receptor

CD45

T cell membrajie

Cytoplasm
PKC, Ras, Rac, JNK, Protein tyrosine phosphorylation 7 Raf MAP kinases Changes in protein phosphorylaUon

Transcription factors
Fos, Jun, AP-1, NFKB,

Responses
Lymphokine genes (eg. IL-2) T cell proliferation

NFATp, NF-ATl

Figure I Signal transduction by the TCR-CD3 complex and the role of CD45. Engagement of the TCR by antigen leads lo the phosphorylation of the ^-chain and other components of the CD3 complex. Phosphorylation provides docking sites for proteins containing Src-homology units (SH2 domains) including ZAP-70. SLP-76, p59fy", and p56'*^'' (which also associates with CD4/CD8). It is thought that activated ZAP-70 and SLP76 couple directly to the activation of phosphoiipase C7I (PLC), which triggers the production o!" 1,2-diacylglycerol (DG) and inositoi phosphates (IP,. iPj). SLP-76 has also been implicated in the activation oi Ras. These events couple with signals from CD28 to activate downstream cytoplasmic and nucleic signalling events, including the activation of transcription factors, leading to the induction of genes for T cell proliferation and activation.

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activates signalling through the effector phosphoinositol-3 kinase which is thought to affect downstream signalling through Ras, Rac, protein kinase C, ^li)^^^ and JNK.'^"^ Both TCR and CD28 also appear to be linked to the activation of the shared effector Ras,^^"'^^ which activates the serine/threonine kinase Raf and gene expression via the MAP kinase pathway (Fig. 1). T cell activation is closely regulated by cross-talk between all these signalling pathways, and also by signals generated by the engagement of various adhesion and accessory molecules like CD2 and CD4/CD8 and LFA-1 during the interaction of the T cell with the APC. Some of these signals may feed into the TCR signalling pathway at an early stage, and the ultimate effect of TCR engagement is critically dependent on the integration of all these signalling events by the T cell.'" A number of recent studies have shown that CD45 is an essential component of the TCR signalling mechanism. Thus, evidence indicates that CD45-deficient T cells fail to induce the necessary signals, namely the increase in intracellular calcium and activation of protein kinase C, required for T eel! activation in response to TCR engagement.-''^'^"^'^^ In contrast, CD45* revertants regain their ability to respond to antigen stimulation.-^^'^'^^'-''^ Interestingly, the pathway of phosphoinositide hydrolysis from the human muscarinic type 1 receptor when expressed in CD45~ T cells is intact,'^^ suggesting that at least part of the function of CD45 is to link the TCR complex to the activation of phosphoiipase c.'^^"'^As indicated above, the activity of the kinases p56''^'' or p59^>''' is required for the tyrosine phosphorylation of a number of cellular proteins including phosphoiipase Cyl upon stimulation of the XCR.'--*'^'*"'^-'' These kinases possess a carboxy-terminal tyrosine which is highly conserved in the irefamily of proteins and has been implicated in the regulation of their kinase activity (for review see '-^}. Evidence shows that tyrosine phosphorylation of the carboxy-terminal tyrosine residues Y^^^^ and Y531, of p56''-''' and p59f5"', respectively, provides a negative regulatory mechanism which suppresses the kinase activity of these protein tyrosine kinases.'^^''"'^ This is thought to occur through the ability of phosphorylated carboxy-terminal tyrosine to bind to the SH2 domain of the respective enzyme, thereby keeping it in an inactive state.'-'' Accordingly, dephosphorylation of the negative regulatory tyrosine residue of these kinases leads to their activation: further stimulation of the kinase activity can occur by phosphorylation of their positive regulatory phosphorylation site.'-^ Considerable evidence indicates that CD45 regulates the activity of p56'^^^ and p59fy" (Fig. 2). Thus, in CD45-negative cells the phosphorylation of p56''=^ and p59'>'" on their conserved tyrosine residues was found to be increased eight-fold and two-fold, respectively,'^-^ and p56'''^ kinase activity was undetectable. Since the phosphorylation of these conserved sites correlates with an inhibition of their kinase activity, the findings suggest that CD45 is required for the optimal activity of p56'<''' and p59''>'".'^^-'" Consistent with these observations, co-precipitation studies have shown thai p56''^^ forms a molecular complex with CD45 and CD4/CD8,^5.96.i68,i69 and that p59*y" associates with the - and ^-chains of the TCR-CD3 complex.5^ In addition, recent studies suggest that CD45 co-distributes with ZAP-70 in intact cells, and that CD45 is required for the dephosphorylation and activation of

0,'^^-'''* and for the regulation of the phosphorylation state of components of the TCR-CD3 complex. As intimated above, evidence indicates that ZAP-70, and its substrate SLP-76, are both essential for the coupling of the TCR to activation of phosphoiipase Cyl and the Ras signalling pathway.''*^"''*^ Therefore, through its ability to regulate the phosphorylation states of components of the TCR-CD3 complex, and the activities of the protein kinases p56''^'', p 5 9 ^ , ZAP-70, SLP-76 and possibly other signalling molecules like Ras-GAP, the function of CD45 is crucial for the proper emanation of activation signals from the TCR and other receptors involved in T cell activation.''*'''''^-''^ Regulation of CD45 function Crucial to regulating the activity of kinases involved in signal transduction via the TCR is the question of how the PTP activity of CD45 itself is regulated, especially as the PTP activity of CD45 may be inactive in naive T cells.^^ In addition to likely changes in the levels of surface expressed CD45 that may accompany the activation of T cells.'^'''^^ several different mechanisms by which CD45 PTP activity may be regulated have been proposed (for review see ^^). These are also considered below in light of additional findings in this area.

Regulation by ligands
Evidence indicates that the extracellular regions of CD45 contain a cysteine-rich motif which is conserved in a number of ligand-binding receptors capable of transducing intracellular signals,'^*" raising the possibility that CD45 function may also be regulated by ligands. Consistent with this notion is the fact that antibodies directed against extracellular regions of CD45 can modulate the function of murine and human T cells, although it appears that chimeric proteins containing the cytoplasmic region of CD45 alone can rescue signalling via the TCR.'"-'^^ Regulation of CD45 function by ligands may occur through a direct effect of ligand binding on PTP activity (perhaps involving a conformational change in the CD45 molecule), or by ligand-induced changes in the accessibility of substrates to the CD45 PTP. Interestingly, different CD45 mAb display different abilities to activate CD45 PTP activity, and this effect appears not to depend on CD45 crosslinking.'^^"^"^ As pointed out by Trowbridge.-^^ a concern when using mAb to stimulate CD45 function is that it is unclear whether the action of a particular mAb mimics or suppresses the action of the physiological ligand(s). Any interpretation is also based on the assumption that a particular mAb binds to the same epitope, and/or induces the same effect as the binding of a natural ligand. Perhaps the most persuasive evidence supporting the regulation of CD45 function by ligands, however, is that CD22, a candidate ligand for CD45 modulates tyrosine phosphorylation of phosphoiipase Cyl and calcium flux changes that are induced by TCR/CD3 stimulation.^^

Regulation by expression of different isoforms

The assay of PTP activity in CD45 immunoprecipitates in vitro has indicated that all CD45 isoforms have a similar

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basal activity, however, specific isofonns of CD45 have been found to differ in their ability to modulate TCR signalling.-"^^ For example, low molecular weight isoforms of CD45 are purported to be more likely to associate with CD4,*'^'^' and to be more efficient in promoting T cell activation upon TCR stimulation, than high molecular weight isofonns.'^^~"^** Also of possible relevance to the notion that different CD45 isofonns may differentially affect TCR signalling are studies which indicate that increases in cytosolic calcium levels and protein kinase C activity are higher in CD45RO* than in CD45RA^ CD3-stimulated T cells,'^-^ and that CD45RO-^ and CD45RA"^ cells have a different ability to proliferate and to secrete IL-4 and IFN-y.'"''"*^ CD3-stimulated human CD45RA^ and CD45RO* T cells also display a different capacity to activate Ras.'**^ and to induce the tyrosine phosphorylation of the cellular proteins Vav and SLP-76.'**'^-'^' As discussed earlier, Ras is involved in signalling via the Raf pathway, and Vav interacts with other signalling molecules,''*^-'-'''*-'52 Clearly, while these latter findings may reflect differences in the intracellular signalling mechanism(s) that might exist in naive versus memory T cells, the observations nonetheless, are consistent with different CD45 isoforms being able to support different TCR downstream signalling events.^^"*''*'- The fact that expression of alternatively spliced forms of CD45 is under the influence of negative regulatory trans-acting splicing factors whose levels are dependent on the particular state of development or maturation of the cell,'^- therefore, also provides a mechanism by which CD45 PTP activity can be regulated.

cells treated with either phytohaemagglutinin or mAb to CD3, and that CD45 PTP activity is inhibited in T cells treated with ionomycin.'"^ Although CD45 PTP activity has been correlated with a decrease in the phosphorylation of CD45 on serine residues,'^^ it is unclear from these studies whether any observed change in PTP activity is the direct result of the phosphorylation of CD45 itself, or whether it is due to other regulatory mechanisms.^^ In vitro studies suggest that CD45 may be phosphorylated on specific tyrosines by the protein tyrosine kinase p50*^*^. which is thought to activate the tyrosine phosphatase activity of CD45 and provide a binding site for proteins like p56''^'" which contain specific binding sequences like the SH2 domain.'"^^ Interestingly, p50'-'''' may also phosphorylate the negative carboxy-terminal tyrosine in p56'''^, and hence oppose CD45 in the regulation of p56''^'^ (Fig. 2).'^-'^^ The phosphorylation of CD45, therefore, may play a role in regulating CD45 activity by providing docking sites for other regulatory molecules.'^** Clearly, as CD45 has a number of potential serine and tyrosine phosphorylation sites, the particular phosphorylation site(s) involved in regulating PTP activity, as well as the nature of the enzymes involved in their phosphorylation, will need to

CD45 CD4 CD4

Membrane CD45 dimerization

Evidence that dimerization of the epidermal growth factor receptor (FGFR) enhances its enzymatic activity, and the observation that the topographical arrangement of EGFR is similar to that of CD45, led to the suggestion that the PTP activity of CD45 may also be regulated through dimerization.''^^ Experiments in which T cell lysates were incubated with chemical cross-linkers to covalently cross-link associating molecules, followed by immunoprecipitation with CD45specific antibodies and analysis of the immunoprecipitates by SDS-PAGE indicated that dimers of CD45 can form on the cell surface.'**-^ However, while an in vitro PTP assay showed differences in PTP activity in different fractions of the immunoprecipitates separated by sucrose gradient ultracentrifugation, these differences could not be explained by CD45 dimerization.'"^-^ Other studies, nonetheless, suggest a possible role for CD45 dimerization in the regulation of CD45 tyrosine phosphatase activity.'''* Furthermore, the fact that the cross-linking of bivalent CD45 with mAb can affect the responses induced,'^'' is also consisent with a potential role for oligomerization in regulating CD45 function.

Cytoplasm

Inactive p56

lck

Active p56
Figure 2 A current model for the regulation of the src family of kinases by CD45. The diagram shows a current model of how the CD4.5 tyrosine phosphatase may regulate the activity of the tyrosine kinase p56'''''. Phosphorylation of p56''-'^ at position 505 (which is a negative regulatory tyrosine phosphorylation site) is thought to allow the carboxy terminus of the protein to fold around and bind to its own SH2 domain, thereby disabling the kinase. (Note thai other domains of p56''^^ including SH3 are not shown). Dephosphorylation of the carboxy terminal tyrosine by CD45 removes tlie restraint, leading to activation of the kinase. Further activation may occur upon phosphorylation of the positive regulatory phosphorylation site (Tyr 394, not shown). Conversely, inactivation of the kinase can occur upon phosphorylation of the carboxy terminus (Tyr 505) by the cytoplasmic tyrosine kinase p50"''. An analogous mechanism is thought to be involved in the CD45 regulation of the kinase activity of the related tyrosine kinase p59f''", which associates with the TCR-CD3 complex.

Pho.sphorylation ofCD45
As reviewed previously.^*" post-translational modifications such as the phosphorylation of the intracellular domain of CD45 on serines''^-'' and tyrosines''"' arc also potential mechanisms for regulating CD45 function. Evidence suggests that CD45 is transiently phosphorylated on tyrosines in Jurkat

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be elucidated for a better understanding of this potential regulatory mechanism. CD45-associated molecules Another potential mechanism for regulating CD45 activity is through an association with other molecules either at the cell surface, or inside the cell. In mouse lymphoma cells CD45 associates strongly with the cytoskeletal proteins fodrin and spectrin,'"-'^'^^ and the binding of these proteins to isolated CD45 is reported to stimulate its PTP activity in vitro.^^^ It would seem that an association of CD45 with cytoskeletal proteins could occur in normal T cells, suggesting a mechanism by which CD45 function may be regulated in vivo. Also, it is conceivable that any CD45-associated cell surface receptor(s) like CD2, TCR/CD3, or CD4/CD8, as described earlier can regulate CD45 function by affecting the accessibility of specific substrates to the CD45 PTP.^6-2^2O2 j^js could occur through steric constraints imposed by the presence of the cytoplasmic regions of these receptors and any associated molecule(s), and/or through any effector function of these molecules. Of potential relevance to the regulation of CD45 function is the finding that CD45 associates with several phosphorylated molecules of 29-34 kDa.^'^-'os.ios-iio These molecules have been found only on CD45-expressing cells and appear to possess properties characteristic of molecules involved in protein-protein interactions: these features make them potential candidate molecules for the regulation of CD45 function. CD45-associated molecules CD45-AP and LPAP Several studies using CD45-specific mAb to immunoprecipitate CD45 from digitonin lysates of T cells have reported the co-precipitation of molecules of 29-34 kDa; the co precipitation was specific for CD45. as mAb to other lymphocyte antigens failed to co-precipitate the 29-32 kDa molecules.'"*^ "" The cloning and amino acid sequencing of a 30 kDa CD45associated molecule from the murine T cell line YAC-1 revealed a transmembrane protein of 186 amino acids, and was designated CD45-associated protein (CD45-AP)."" In addition, the cloning and sequencing of a 32 kDa CD45-associated protein from human Jurkat T cells revealed a protein of 207 amino acids, and was designated lymphocyte phosphatase-associated phosphoprotein (LPAP).'** CD45-AP and LPAP were shown to have 64% amino acid similarity, but screening of several computer databases showed no homolo-

gy to other proteins.'^'^"0 CD45-AP and LPAP, therefore, appear to represent novel proteins that specifically associate with CD45, and that may regulate its function. Both LPAP and CD45-AP were found to consist of a short (7-10 amino acid) extracellular domain, a single transmembrane pass of 21-23 amino acids and a larger (158-174 amino acid) cytoplasmic domain."'^'"^ The amino acid sequences of LPAP and CD45-AP predict proteins of a molecular weight of 19 kDa, which is 11-13 kDa lower than their corresponding molecular weights as indicated by SDS-PAGE. However, in vitro translation of each protein from the cloned genes produced proteins which migrated at 30-32 kDa indicating that the cloned sequence was complete. The 13 kDa discrepancy has been attributed to post-translational changes including glycosylation."^^-'"^ Interestingly, initial studies using naive T cells indicated that the LFAP protein band (32 kDa) could be further separated into 29 and 32 kDa forms after analysis by SDS-PAGE using a high percentage (18%) polyacrylamide gel.'^''^'-'' Moreover, activation of the T cells resulted in the association of CD45 with molecules of 30 and 31 kDa, instead of 29 and 32 kDa. The incubation of these molecules with alkaline phosphatase followed by SDS-PAGE analysis resulted in all four molecules migrating as a single 32 kDa species, indicating that the separate bands represent hyperphosphorylated forms of the same protein. That a functionally intact protein tyrosine kinase pathway was required for the phosphorylation of LPAP (and hence conversion of the 29 and 32 kDa molecules to the 30 and 31 kDa form) was suggested by the fact that no change in the migration pattern occurred when the cells were activated in the presence of tyrosine kinase inhibitors. "^-"^^ Differences between LPAP and CD45-AP
The characterization of the CD45-AP and LPAP molecules has also highlighted differences in phosphorylation states and consensus sites between the mouse and human proteins; these differences are summarized in Table 1. Like CD45, both proteins are heavily phosphorylated on serine residues in vivo, indicating an ability to interact with one or more serine kinases.'<'^-"o However, LPAP and CD45-AP vary in their consensus sites for serine kinase recognition, suggesting that their phosphorylation on serines may be mediated through different kinase enzymes. Thus, CD45-AP contains a consensus phosphorylation site for glycogen synthase kinase-3," whereas LPAP contains consensus recognition sites for protein kinase C. cAMP-dependent serine kinase and casein

Table 1 A comparison of the phosphorylation states and consensus sites of the murine CD45-AP and the human LPAP CD45-associated proteins Protein Origin of cells Phosphorylation (in vivo) Phosphorylation [in vitro) Serine consensus recognition sites GTP consensus sequences Molecular weight CD45-AP Murine thymocytes, murine T cell lines, YAC-1 Serine Serine Glycogen synthase kinase-3 Absent 32-33 kDa LPAP Human T cell line, Jurkat Serine and tyrosine Tyrosine PKC. cAMP-dep. serine kinase, casein kinase-2 Absent
29-32 kDa

LPAP, lymphocyte phosphatase-associated pho.sphoprotein; PKC. protein kinase C; GTP. guanosine 5'-triphosphate.

The role of CD45 and CD45-associated molecules

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kinase-2.'*' Also, despite early suggestions that the CD45associated molecules are G-proteins,'**-"'^-^''^ consensus sequences characteristic of GTP-binding proteins are not present in either CD45-AP"^ or LPAP.'"** Interestingly, LPAP, but not CD45-AP, was found to be phosphorylated on tyrosine residues, both after in vivo labelling of cells with [-^P]-Ojand after in vitro kinase assay of CD45 immunoprecipitates."^^ Consistent with this, the consensus tyrosine phosphorylation site present in LPAP is absent in CD45-AP."'^-"" In vitro. LPAP has been shown to be tyrosine phosphorylated by p56''^'', and rapidly dephosphorylated by CD45. TTie phosphorylation of LPAP on tyrosine residues in vivo increases rapidly following treatment of the T cells with pervanadate which inhibits the PTP activity of CD45.""* These findings suggest that LPAP may be a physiological substrate of CD45 in vivo.

Interaction of CD45 with CD45-AP and LPAP

The stretches of amino acids required for the interaction of CD45 with LPAP and CD45-AP were assessed using CD45/MHC chimeras and truncated forms of the CD45-associated proteins.^^'^"^'^^ These studies showed that the transmembrane regions of both CD45. and of LPAP and CD45AP proteins, are essential to facilitate interaction of these proteins. Interestingly, the cytoplasmic regions of LPAP and CD45-AP, each contain the highly conserved tryptophan residues which are characteristic of a WW domain.-'^ Although not required for the interaction of LPAP or CD45AP with CD45, this domain has previously been shown to mediate protein-protein interactions in a variety of proteins involved in cell signalling functions,-*^''^*^^ suggesting that the associated proteins may mediate the interaction of CD45 with other proteins. Interestingly, studies employing northern blot analysis have shown that LPAP mRNA is expressed at normal levels in CD45-deficient cells, but that the presence of LPAP protein is undetectable in lysates of these cells by immunoprecipitation with LPAP-specific antibodies.""* The ability to detect the 32 kDa protein and its association with CD45 was restored, however, by transfection of these cells with fulllength CD45 cDNA."^'* Pulse chase experiments also showed that in CD45-deficient cells LPAP is synthesized at a rate similar to that in wild-type cells, but that it is degraded ten times more rapidly,""* These findings indicate that while LPAP can be produced in the absence of CD45. its stability in cells is dependent on an association with CD45.

initially reported that the four phosphorylated forms of the 32 kDa protein that co-precipitated with CD45 reacts with G-protein antiserum. subsequent studies and cloning of the proteins indicated that these proteins did not bind or hydrolyse GTP, nor did they contain G-protein-specific sequences. These findings, therefore, support a case for the 32 kDa GTF-binding associated with CD4/CD8 and p56'^'' being distinct from LPAP. Studies employing cell surface biotinylation followed by the immunoprecipitation of CD45 with specific mAb have also shown that CD45 associates with 32 and 33 kDa molecules on murine thymocytes and the T cell clone DIO."^ Interestingly, while the 32 kDa CD45-associated molecule was detected in digitonin lysates of surface biotinylated cells. experiments carried out using digitonin lysates prepared from cells that were biotinylated after permeabilization with lysolecithin indicated that CD45 also associates with a protein of 33 kDa.'"'' These findings provide evidence for the existence of a pool of 33 kDa protein which is not accessible to cell surface labelling, and which is presumably located intracellularly. The intracellular location of a 30 kDa molecule associated with CD45 was first suggested by studies employing surface radio-iodination of the murine T cell line YAC-l.'"^'The detection of the 32 kDa protein in experiments employing cell surface biotinylation.'"'* is consistent with the transmembrane structure predicted from the amino acid sequence of CD45-AP.'"' It is unclear, however, whether the 32 kDa molecules detected by cell surface biotinylation, and the 33 kDa molecule of potentially intracellular localization, represent different forms of the same protein, and whether either of the two molecular species is identical to CD45-AP. A very recent report showing that CD45-AP can exist in an isoform containing an additional 12 amino acids that may constitute part of an N-temiinal signal peptide and potentially affect the cellular distribution of CD45-AP,-" nonetheless, has provided a likely candidate for the 33 kDa molecule detected only when the cells were biotinylated after permeabilization.'*'^ Phosphoamino acid analysis of CD45 immunoprecipitates from lysates of murine thymocytes and DIO ceils after in vitro kinase assay also indicated that both the 32 and the 33 kDa proteins are phosphorylated exclusively on serine residues; and no change in the rate of migration of these molecules was detected when the proteins were isolated from activated, rather than resting, DIO cells."'** Moreover, neither of the two proteins bound GTP or reacted with G-protcinspecific antibodies, suggesting that they are unlikely to be G-proteins. Interestingly, however, in vitro kinase assay indicated that phosphorylation of botli proteins is significantly inhibited by preincubation of the immunoprecipitates with GTP-y S, a non-hydrolysable form of GTP.'''*^ This suggests that phosphorylation of the proteins nuiy be regulated by a G-protein-dependent mechanism(s), and raises the possibility that CD45 immunoprecipitates also contain a GTPbinding protein. This GTP-binding protein may be a homologuc of the protein reported to associate with CD4/CD8 and p56''^'^ in human Jurkat cells.-'^ Alternatively, as the TCR I, chain migrates at 32 kDa under certain conditions.^''''^ and associates with CD45 and binds GTP, the results tnay also refiect an involvement of the ^ chain in regulating the phosphorylation of CD45-AP. Clearly, a definitive resolution of

Other 32-34 kDa CD45-associated molecules?

The association of CD45 or other surface receptors with molecules of molecular weight similar to that of LPAP and CD45-AP have been reported in other studies. Thus, a 32 kDa molecule was found to exist in complex with CD4/CD8 and p56''^'' in iysates of the human T cell line Jurkat; this molecule was immunoprecipitated with G-protein antiserum. and bound and hydrolysed GTP.-''^ Although LPAP associates with p56''^'* in the presence of CD45. the pool of p56''-'^ which associates with CD45 has been shown to be distinct from that which associates with CD4 and 168.210 Moreover, while Schraven and c

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these issues awaits further characterization of the CD4/CD8associated 32 kOa molecule, and perhaps other CD45-associated molecules of similar size.

Are CD45-AP and LPAP adaptor molecules?

The ability of CD45-AP and LPAP to associate with CD45 through their respective transmembrane regions, while leaving their WW domains free to interact with other molecules, suggests that they may be involved in bringing other molecules into contact with CD45. Recently, it was proposed that LPAP and CD45-AP could function as adaptor molecules which promote the interaction between CD45 and other molecules involved in signal transduction.-"^ According to this model. CD45 interacts with tyrosine kinases such as p56''^'' and pSg"^" through the associated LPAP or CD45-AP molecule which serves as an adaptor protein.'^'^-'-'"-'-'' As adaptors, CD45-AP and LPAP could also regulate the tyrosine phosphatase activity of CD45 by promoting the interaction of CD45 with specific substrates (Fig. 3). This model is supported by the fact that complexes consisting of CD45, p56''-"'' and LPAP are found in CD45 immunoprecipitates from lysates of resting human T cells,'**^''^^'^** and that the direct association of CD45 with pSG'*^^ is weak.-"^-'-' Studies using chimeric molecules have shown that the CD45 transmembrane region is essential for maintaining the levels of LPAP in T cells,-'^^ and transfection of CD45-negative T cells with chimeric molecules consisting of the extracellular and transmembrane sequences of an MHC molecule and the cytopiasmic sequence of CD45 restores TCR signal transduction, but with reduced efficiency.'^' Therefore, while the cytoplasmic portion of CD45 is sufficient to couple the TCR to the activation of phospholipase Qy\, the transmembrane and/or the extracellular region(s) are required to optimize the TCR signalling, potentially by allowing the actions of LPAP to enhance the interaction of CD45 with one or more protein tyrosine kinase. This conclusion is consistent with the observation that a similar MHC/CD45 chimera which is not able to associate with LPAP, also does not associate with

A role for CD45-AP and LPAP in T cell activation?

As discussed earlier, CD45-AP and LPAP are membraneanchored proteins which associate with all isofonns of CD45 and are found in association with the majority of CD45 molecules. ' '"-'^-'^ The fact that expression of CD45 Is essential for T cell activation, suggests that CD45-associated molecules like CD45-AP and LPAP also play an important role in this process, presumably by regulating CD45 function. This notion is supported by the finding that the CD45-associated molecules are phosphorylated, that their stability in the cell depends on the expression of CD4f>, and that (at least for LPAP) the state of phosphorylation changes with T cell activation.

CD45

Cell surface receptor

Membrane

Cytoplastn

Regulation of substrate accessibility?

Effectors ?
Figure 3 A current model of how CD45-associa[ed protein, lymphocyle phosphalase-associated phosphoprotein (LPAP). may rcguiaie CD45 function in human T cells. The cytoplasmic region of the LPAP has been shown to contain a WW domain, which is a region that has been implicated in prolein-protein interactions. Evidence Indicates that CD45 interacts with the associated protein LPAP via the transmembrane regions of botli CD45 and LPAP. This may leave the WW domain free to interact wiih specific effector molecules (denoted x? and y?) including membrane receptors and so bring them under the influence of the CD4,S [yrosine phosphatase. The association of CD45 with LPAP, therefore, may provide a mechanism for regulating CD45 function. A similar mechanism may operate in murine T cells with the murine CD45-associated protein, CD45-AP. The model as outlined in the diagram is aii extended form of that originally proposed by Kitamura el al.^^ The recent finding that CD45-AP exists with and without a 12 amino acid signal sequence, which may be involved in directing protein trafficking, has also raised the possibility that different isofomis of the CD4.'i-associated proteins may regulate the accessibility of substrates to the CD4.'> tyrosine phosphatase.

Evidence indicates that in resting T cells otily approximately 70% of CD45 is located at the plasma membrane, while the remaining 30% is retained in the Golgi. The fact that activation of T cells is accompanied by an exit of CD45 from the Golgi and a redistribution to other unidentified subcellular organelles, has led to the suggestion that the residence time of CD45 within the Golgi is regulated by signals from the TCR and that this, in tum, regulates access ofthe CD45 PTP to its substrates."'' The observation that tnurine T cells contain a pool of CD45-associated protein of slightly higher molecular weight than that which is asscssible by cell surface labelling,'"'* and the very recent report that CD45-AP can exist in an isoform that contains an additional 12 amino acids in the amino terminus, has also raised the possibility that CD45-AP is involved in regulating substrate accessibility.-" Confirmation of this involvement, however, awaits further study.

Conclusions and future directions

Considerable evidence indicates that expression of CD45 is essential for the activation of T cells, which highlights the

The role ofCD45 and CD45-associated molecules

439

importance of CD45 in regulating immune function. Despite this, a number of issues concerning the role of CD45 in T cell activation remain to be resolved. Among the most perplexing of these include: the nature of the ligand(s) for the different isoforms of CD45 expressed on T cells, and the role of the numerous CD45-associated molecules. Evidence indicates that CD45 may interact with glycosaminoglycans on cell surfaces and that the a2.6-linked sugar structure of CD45 is recognized by the B cell surface antigen CD22. However, the physiological relevance of these potential ligands remains to be established. Furthermore, a better understanding of the role of CD45 in immune function is also critically dependent on establi.shing the identity of the ligands for the different isoforms of CD45 expressed on the different T cell populations. The fact that the cytoplasmic region of CD45 possesses tyrosine phosphatase activity, points to the importance of determining whether any potential CD45 ligand(s) can directly alter CD45 tyrosine phosphatase activity, as this activity appears to be crucial for CD45 function. At present, it is unclear how the FTP activity of CD45 IS regulated, but potential mechanisms include: the direct effects of an interaction of CD45 with ligands, the expression of CD45 isoforms that differ in their ability to support TCR-induced responses, the ability of CD45 to associate with other molecules both membrane-associated and cytosolic, and the ability of CD45 to be phosphorylated and to undergo dimerization. A model of T cell activation in which CD45 activates the protein tyrosine kinases responsible for phosphorylating phospholipase Cyl, thereby leading to the transduction of signals through the hydrolysis of membrane phospholipids. is well supported by experimental evidence. It is conceivable that the LPAP and CD45-AP proteins may act as adaptors for linking CD45 to other proteins involved in signal transduction. It remains to be determined, however, whether the CD45-associated molecules regulate CD45 tyrosine phosphatase activity, or perhaps whether they are themselves crucial physiological substrates for CD45. Presently, evidence favours a model in which the 29-34 kDa CD45-associated molecules (LPAP and CD45-AP) function to couple CD45 to protein tyrosine kinases like p56'^^. p59f>^" and ZAP-70. but this needs to be confirmed. The relationship of LPAP and CD45-AP to the 32 kDa GTP-binding protein purported to associate with CD4/CD8 and p56''^'' is presently unclear. Future studies will also need to establish the role(s) of the different isoforms of CD45-AP and LPAP that exist,-"'^'^ and the significance of the possible differentiation-specific expression of these molecules in cells.-'^ Moreover, the significance of the phosphorylation of LPAP (on tyrosines and serines) and CD45-AP (exclusively on serines) lo T cell activation and the identity of the CD45-associated serine kinase(s) involved in the phosphorylation remain to be determined. Clearly, studies employing site-directed mutagenesis to determine the importance of the various phosphorylation sites, on the CD45-associated moleculc(s) and on CD45 itself, and the generation of T cells which are deficient in the production of CD45-associated proteins or that express mutated forms of the proteins, may in future provide important information concerning the role of LPAP and CD45-AP in regulating CD45 function.

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