Role of CD45 in Lymphocyte Activation

CD45 is an RPTP with a glycosylated extracellular domain, a single transmembrane segment, and two tandem PTP catalytic domains. The extracellular domain is thought to have a ligand binding function;

however, the ligand(s) for CD45 have not yet been identified. The expression of CD45 is limited to nucleated cells of hematopoietic origin, where it occupies about 10% of the cell surface. Relative to many other proteins with signaling functions CD45 is surprisingly abundant. The size of the extracellular domain varies in length from 400 to 550 residues because of alternative mRNA splicing. Splice variants differ at their N-termini and are expressed in a manner that depends on the cell type, developmental stage, and antigenic stimulation.

To carry out their immune function peripheral T and B lymphocytes must be activated to a fully functional state upon encountering antigen. Quiescent B lymphocytes that recognize antigen are activated to proliferate and differentiate into antibody secreting plasma cells or long-lived memory cells that persist after antigen is eliminated. Activation of B cells requires lymphokines produced by helper T cells. When T cells bind an antigen presented by the major histocompatibility complex (MHC) on a macrophage or target cell, they are also activated to secrete lym-phokines, proliferate, and differentiate into mature cells equipped to carry out their helper or cytolytic functions. Both T and B lymphocytes have multiprotein receptors on their surface that bind antigen and initiate a complex signaling cascade that culminates in their differentiation and proliferation. Antigen receptor signal transduction is also important for certain stages of lymphocyte development.

Genetic and biochemical analyses have demonstrated that CD45 is required for the development and antigen-dependent activation of T lymphocytes. This discussion focuses on the role of CD45 in T-cell activation. To describe how CD45 participates in this process, we will consider the basic structural organization of the T-cell antigen receptor (TCR) and the early signaling events that are triggered by antigen binding. The TCR is a large complex of several distinct integral membrane proteins that is not only responsible for antigen recognition but must also initiate signaling events leading to T-cell activation. Figure 9 shows the subunits and organization of the TCR. The disulfide-bonded aP heterodimers of the TCR are responsible for binding the MHC-antigen complex on an adjacent antigen-presenting cell. The subunits of the aP dimer are members of the immunoglobulin (IgG) superfamily and have large extracellular domains with very short cytosolic segments. The aP chains are tightly associated with a CD3 complex that is composed of y, 5, and e subunits, which have IgG-like extracellular domains and form noncovalently

Fig. 9. The role of CD45 and SHP-1 in T-cell activation. This schematic illustrates a model of the major signaling events that follow engagement of the TCR with antigen with an emphasis on highlighting the positive action of CD45 and the negative functions of SHP1. This is not a comprehensive depiction of TCR signaling and many components and steps have been omitted. (A) This diagram illustrates the components of the TCR and the associated Src family kinases. As the arrows show, CD45 targets a negative regulatory site in the C-termini of the Src family kinases, Lck and Fyn, and thereby activates them. Thus, CD45 opposes the action of the Csk kinase, which inhibits Src kinase activity by phosphorylating a C-terminal Tyr residue. Activated Lck and possibly Fyn then phosphorylate the ITAMs (open boxes) of the TCR subunits. (B) Results of ITAM phosphorylation on signal propagation as well as the action of SHP-1 in suppressing or negatively regulating this signaling pathway. Once phosphorylated, the ZAP-70 kinase is recruited to ITAMs of the TCR subunits as illustrated and activated via phosphorylation by the Src kinases. The CD4 coreceptor and the Src family kinases are not shown in (B) for clarity. As the large arrows indicate, activation of ZAP-70 leads to activation of multiple downstream pathways. There may be at least two mechanisms by which SHP1 down-regulates TCR signaling. SHP1 may be recruited directly to ZAP-70, as the arrows in the figure indicate, and directly dephosphorylate and inhibit its kinase activity. SHP1 may also be recruited to ITIM sequences in the cell surface receptor CD5. When recruited to CD5, ZAP-70 is one of the major targets for SHP1 as illustrated by the arrows. This scheme may not illustrate all the substrates or functions of CD45 and SHP-1 in T-cell activation.

Fig. 9. The role of CD45 and SHP-1 in T-cell activation. This schematic illustrates a model of the major signaling events that follow engagement of the TCR with antigen with an emphasis on highlighting the positive action of CD45 and the negative functions of SHP1. This is not a comprehensive depiction of TCR signaling and many components and steps have been omitted. (A) This diagram illustrates the components of the TCR and the associated Src family kinases. As the arrows show, CD45 targets a negative regulatory site in the C-termini of the Src family kinases, Lck and Fyn, and thereby activates them. Thus, CD45 opposes the action of the Csk kinase, which inhibits Src kinase activity by phosphorylating a C-terminal Tyr residue. Activated Lck and possibly Fyn then phosphorylate the ITAMs (open boxes) of the TCR subunits. (B) Results of ITAM phosphorylation on signal propagation as well as the action of SHP-1 in suppressing or negatively regulating this signaling pathway. Once phosphorylated, the ZAP-70 kinase is recruited to ITAMs of the TCR subunits as illustrated and activated via phosphorylation by the Src kinases. The CD4 coreceptor and the Src family kinases are not shown in (B) for clarity. As the large arrows indicate, activation of ZAP-70 leads to activation of multiple downstream pathways. There may be at least two mechanisms by which SHP1 down-regulates TCR signaling. SHP1 may be recruited directly to ZAP-70, as the arrows in the figure indicate, and directly dephosphorylate and inhibit its kinase activity. SHP1 may also be recruited to ITIM sequences in the cell surface receptor CD5. When recruited to CD5, ZAP-70 is one of the major targets for SHP1 as illustrated by the arrows. This scheme may not illustrate all the substrates or functions of CD45 and SHP-1 in T-cell activation.

linked e-y and e-รด heterodimers. The third component of the TCR is the disulfide-linked Z-Z homodi-mer. It is the CD3 complex and the Z-Z homodimer that are involved in transmitting a signal across the membrane upon antigen engagement despite the fact that their cytoplasmic segments all lack intrinsic enzymatic activity.

One of the earliest signaling events observed following antigen binding is the activation of PTKs and a concomitant increase in the tyrosine phosphorylation of multiple intracellular proteins. Subsequently, phospholipase Cy is activated, releasing inositol trisphosphate and diacylglycerol leading to elevated Ca2+ and activation of protein kinase C. Other distal signaling events include the activation of Ras and the

MAP kinase signaling cascade. Through mechanisms that are not yet completely understood these signaling events ultimately lead to T-cell proliferation and the changes in gene expression that result in T-cell differentiation.

The initial tyrosine phosphorylation that occurs upon antigen binding to the TCR results from the rapid activation of Lck and Fyn, two PTKs of the Src family (see Fig. 9). The Fyn kinase is thought to be associated with the e-chains of the CD3 complex, whereas Lck associates with either the CD4 or CD8 coreceptors. The primary targets of these PTKs are the immunoreceptor tyrosine-based activation motifs (ITAMs) located in the intracellular domains of the Y, 5, and e CD3 subunits and the Z-Z homodimer.

Tyrosine phosphorylation of ITAMs in the TCR results in the recruitment of ZAP-70, a protein kinase of the Syk PTK family. Once recruited to the TCR, ZAP-70 is phosphorylated and activated by Lck or Fyn. Activation of the Src family PTKs and ZAP-70 is essential for normal T-cell activation and development. The downstream targets of ZAP-70 include SLP-76 and other adaptor proteins. Phosphorylation by ZAP-70 leads to activation of phospholipase Cy and other distal signaling pathways that ultimately induce differentiation and proliferation. Effective TCR-mediated lymphocyte activation also requires signals initiated by the CD28 cell surface protein and may also involve signaling pathways activated by adhesion molecules that engage their ligands when T cells adhere to antigen presenting cells.

Numerous studies have shown that CD45 is required for TCR-mediated signal transduction. The strongest evidence comes from the analysis of CD45-T cells, which fail to hydrolyze phosphatidylinositol, elevate intracellular Ca2+, secrete cytokines, or proliferate in response to antigen ligation. More importantly, CD45- cells do not respond to antigen binding with the rapid increase in tyrosine phosphorylation observed in normal cells. Because tyrosine phosphorylation is one of the earliest signaling events following antigen stimulation, this finding suggested that CD45 was involved in steps that were immediately downstream of the TCR. Studies in CD45-deficient mice revealed that CD45 was required for normal T-cell development, a process requiring TCR signal trans-duction. Thus, the results from knockout mice also indicate that CD45 has a crucial role in TCR signaling.

The lack of tyrosine phosphorylation in CD45-cells is a paradoxical finding, as PTPs reverse the action of PTKs. However, the regulatory properties of the Src family kinases such as Lck can explain how dephosphorylation results in enhanced tyrosine phosphorylation. A PTK, known as Csk, inhibits Src family kinases by phosphorylating a C-terminal regulatory tyrosine (Tyr505 in Lck). Thus, CD45 is postulated to oppose the action of Csk and up-regulate Lck and Fyn activity during TCR signaling (see Fig. 9). This notion is supported by data showing that the regulatory site in Lck is hyperphosphorylated in CD45- cell lines and CD45 effectively dephosphory-lates this site in vitro. Moreover, Lck has been found to physically interact with CD45. This model predicts that Lck activity in CD45-deficient cells should be suppressed. In many studies, decreased Lck activity has been observed in CD45- cells. However, some researchers have found that Lck activity is actually increased in the CD45 mutant cells. CD45 is also capable of negatively regulating Src kinases by dephosphorylating a Tyr residue located in the activation loop of the catalytic domain.

CD45-deficient mice have been employed to better understand the discrepancies in Src family kinase activity measured in CD45- cells. As described previously, T-cell development, which requires TCR-mediated signaling, is blocked in CD45 knockout mice. In recent studies, an Lck transgene in which Tyr505 was replaced by Phe was introduced into CD45-mice. T cells developed normally in these transgenic mice, indicating that the Lck Y505F mutant rescues the defect in TCR signal transduction. This study provides strong support for a model in which CD45 couples antigen stimulation to downstream signaling pathways by dephosphorylating the negative regulatory site of Lck and/or Fyn (see Fig. 9). Once activated, these Src kinases are able to phosphorylate ITAMs when antigen is bound, initiating the signaling pathways that lead to T-cell activation. How antigen stimulation influences CD45 activity, if at all, is not known. It has been suggested that by opposing the Csk kinase CD45 dephosphorylates the negative regulatory site in Src family kinases prior to antigen liga-tion. In this model, CD45 primes the Src kinases and sets the threshold for TCR signaling. The preactivated Src kinases are then recruited to the TCR when antigen is bound resulting in the rapid phosphorylation of ITAMs. An attractive feature of this model is that the Src kinases and their substrates are physically separated from CD45, which is not recruited to the TCR upon antigenic stimulation. This physical separation prevents the immediate dephosphorylation of ITAMs and other targets by CD45. Besides the Src family PTKs, little is known about other potential CD45 substrates. There is evidence that CD45 can dephosphorylate the Z-chains of the TCR. It is possible that CD45 targets the Z-subunits and other substrates as part of a negative feedback mechanism that terminates or limits TCR signaling.

The role of CD45 in the activation of B cells resembles its function in T-cell signaling. Antigen binding to the B-cell antigen receptor (BCR) also triggers an initial increase in PTK activity that ultimately leads to differentiation and proliferation. In the absence of CD45 expression, BCR-mediated signaling in response to antigen binding is much less efficient but not abrogated. In CD45- B cells, the antigen-induced activation of Lyn kinase and Ca2+ mobilization are significantly reduced, indicating that CD45 acts as positive regulator of B-cell activation just as it does in T cells. Little is known about the function of CD45 in other cells of hematopoietic origin such as mast cells or macrophages. Its presence in a variety of immune cells and its extraordinary abundance on the cell surface suggests that it has multiple functions.

The function of the extracellular domain of CD45 has not been defined. The external domain is believed to be the binding site for ligands, but thus far no physiologic ligands have been identified. Such ligands may be cell surface proteins residing on interacting cells or in the same membrane; however, a circulating macromolecule or low molecular weight compound cannot be excluded. Surprisingly, several studies have shown that the external segment of CD45 is not essential for lymphocyte activation and that it is only necessary for the internal catalytic segment to be located in proximity to the membrane. Although the external domain is not essential, there is evidence that the splice variants of CD45, which possess distinct N-termini and patterns of glycosylation, have differential effects on the intensity of TCR signaling. It is possible that the external domains of CD45 influence TCR-mediated signal transduction in ways that cannot be detected with the current techniques used to monitor lymphocyte activation. It is likely that ligand binding to the external domain will prove to be crucial for yet undiscovered functions.

Relatively little is known about how CD45 is regulated during lymphocyte activation or other immune cell functions. Phosphorylation appears to be an important mode of regulation, as CD45 is a phospho-protein in vivo and contains both phosphoserine and phosphotyrosine in its intracellular domains. Mito-genic stimulation results in phosphorylation of both Ser and Tyr residues, suggesting that phosphorylation is a potential mechanism for regulating CD45 during lymphocyte activation. Treatment of T cells with ionomycin or phorbol esters has been reported to elicit changes in Ser phosphorylation and the activity of CD45. Whether the modest alterations in CD45 activity are physiologically relevant and a direct result of changes in phosphorylation is not known. There are additional reports that phosphorylation of CD45 in vitro by both tyrosine and Ser/Thr kinases can enhance activity; again, it is not clear if these effects are of physiologic significance. An acidic insert in the distal PTP domain (D2) of CD45 is multiply phosphorylated in vivo by casein kinase 2, but the effects of this phosphorylation on CD45 function are not known. Much remains to be learned about how phosphorylation modulates CD45 function(s).

Protein-protein interactions also appear to be an important means for regulating CD45. A 30-kDa protein termed CD45-AP associates with CD45 from lymphocytes. CD45-AP is an integral membrane protein with a single transmembrane segment, a 145-residue cytosolic tail, and a very short external segment. CD45-AP and CD45 interact directly with one another through their transmembrane segments. T and B cells of mice lacking CD45-AP exhibited reduced proliferation on antigen stimulation, indicating that CD45-AP was not essential for antigen receptor signaling but was necessary for achieving full lymphocyte activation. Recent studies have suggested that CD45-AP might link Lck and either the CD8 or CD4 coreceptors together with CD45. Such interactions may facilitate the activation of Lck when antigen receptors are stimulated.

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