Signal transduction by T-cell receptors: mobilization of Ca and regulation of Ca-dependent effector molecules.

Signal transduction by T-cell receptors: mobilization of Ca and regulation of Ca-dependent effector mo lecules BRETT A. PREMACK AND PHYLLIS GARDNER Department of Medicine, Falk Cardiovascular Research Center, Stanford Stanford, California 94305-5246

University,

Premack, Brett A., and Phyllis Gardner. Signal transduction by T-cell receptors: mobilization of Ca and regulation of Ca-dependent effector molecules. Am. J. Physiol. 263 (Cell Physiol. 32): C1119-C1140, 1992.There have been major advances over the last several years in understanding the molecular basis of signaling by the T lymphocyte (T-cell) antigen receptor. In this article we discuss the early phases of T-cell activation with an emphasis on receptor-associated signaling molecules, mobilization of Ca, and on the possible roles of Ca in signal transduction. Ligation of the extracellular domains of the T-cell receptor activates receptor-associated tyrosine kinases that can phosphorylate the y-isoform of phospholipase C, increasing its catalytic activity. This leads to production of inositol 1,4,5trisphosphate, release of stored intracellular Ca, and activation of Ca-permeable plasma membrane channels. Many of the critical T-cell signal transducing enzymes such as phospholipase C and protein kinase C contain intrinsic Ca-binding domains, but for the most part the rise in cytoplasmic Ca is transduced by specialized Ca-binding proteins that lack catalytic domains. The Ca-binding proteins found in T-cells include members of both the EF-hand and annexin families, as well as other types of Ca-binding proteins. In T-cells, a number of important kinases, phosphatases, and cytoskeleton-modulating enzymes are functionally Ca dependent but have no Ca-binding domains and therefore must sense changes in the cytoplasmic Ca level through interactions with Ca-binding proteins. T lymphocyte; tyrosine kinase; fyn; lck; G protein; phospholipase; ion channel; calcium-binding proteins; calmodulin; calcineurin; calpain; annexin; calreticulin T LYMPHOCYTES ARE INDUCED to secrete cytokines, enter the cell cycle, and proliferate when an appropriate antigenic stimulus is presented to their surface membrane antigen receptors. Antigen receptor stimulation rapidly leads to activation of tyrosine kinases, hydrolysis of phosphoinositides, and a sustained elevation of intracellular free Ca. The Ca signal has two components, a transient release from storage organelles and a prolonged phase of extracellular Ca influx. After this rise in Ca, many critical Ca-dependent effector molecules either bind Ca directly or associate with specialized Cabinding proteins that act as Ca sensors. The regulation of these molecules by Ca is critical to the induction of early T-cell activation events such as serine-threonine phosphorylation cascades and cytoskeletal reorganization. Ca-dependent processes also regulate mitogenesis by controlling transcription of the gene encoding interleukin-2 (IL-2), an essential T-cell autocrine growth factor. In this review we summarize the early transmembrane signaling events that activate Ca-release and Cainflux channels and we examine the various signaltransducing and modulating roles of Ca-binding proteins in T lymphocytes. 0363-6143/92

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ANTIGEN RECEPTOR COMPLEX AND INITIAL TRANSMEMBRANE

SIGNALING

T-cells, unlike other cells of the immune system, respond mainly to protein antigens, and these must be processed and presented as peptide fragments on the surface of antigen-presenting cells (APCs). Under normal physiological conditions, T-cells are stimulated by molecular interactions between the T-cell receptor (TCR) and a peptide antigen bound to the class II major histocompatibility complex (MHC II) on APCs such as: B-cells, monocytes, or reticuloendothelial cells. Extracellular antigenic proteins are internalized by the APCs, degraded to 10-20 amino a.cids, processed intracellularly, and then expressed on the cell surface attached in the peptide binding site of the MHC II molecule. Only the MHC IIpeptide complex is recognized specifically by the TCR; interactions with the peptide or MHC II alone are not antigenic. Although the TCR has relatively low affinity for the MHC II-peptide complex (w 10 PM), this interaction is sufficient to induce a conformational change in the TCR and initiation of the intracellular signal transduction cascade (232). The following sections describe

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A Antigen .

B Presenting

Cell 1

. . .. . . . .. . . .. . . . .. . .. . . . . .. . .. . . . .. . . .. . . . .. . . . .. . . .. . ~..I..............

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T Cell Receptor Fig. 1. Diagram of T-cell receptor (TCR). A: extracellular, transmembrane, and intracellular domains of T-cell receptor subunits and accessory molecule CD4. Variable immunoglobulin domains (V) of a- and P-chains bind to antigenic peptide (Ag) that is presented by major histocompatibility complex (MHC) II of antigen-presenting cell. CD3 complex is shown as tetramer composed of 3 related immunoglobulin-like constant subunits (y, 6, c). Actual stoichiometry for CD3 is still uncertain; earlier data suggested a trimeric structure (see Ref. 5), but more recent experiments support a tetrameric grouping (see Ref. 116). Predominately intracellular, disulfide-linked t-chains complete TCR structure. T-cell CD4 molecule is thought to augment signaling through TCR by interacting directly with MHC II. B: possible arrangement for TCR subunits that would allow close association of charged hydrophobic transmembrane domains. Ti, T-idiotypic.

the structure of the TCR and the role of various early signal transduction molecules in T-cell activation. The T-Cell Receptor Complex Each human T-cell’1 expresses -lo4 cell surface TCRs. Each heterooligomeric TCR complex consists of at least seven, and probably eight, individual membranespanning protein chains (cu,/3, y, 6, t, 5; and 7) that interact to form the functional receptor (5, 53, 77, 116, 237). As shown diagrammatically in Fig. 1, the antigenic peptide interacts with a pocket formed by contributions from two predominantly extracellular, nonglycosylated, disulfide-linked chains termed a and p. The peptide binding site differs for each clone of T-cells and is composed of amino acids primarily from variable immunoglobulin-like domains. The structure of the peptide binding site defines the antigen specificity or idiotype. For this reason, (Yand fl are often referred to as the variable or T-idiotypic (Ti) chains. The CD3 complex, which is composed of constant immunoglobulin-like domains, is identical in nearly all T-cells. The CD3 complex is made up of three genetically related subunits, y and 6, which are glycosylated, and t, which is not. Recent experiments suggest that the stoi Due to the limited breadth of this review, we refer to CD4+CD8CD3+&+ helper T-cells as T-cells throughout this paper. We have not attempted to include other T-cell subtypes (6-r helper T-cells, cytotoxic T-cells, natural killer cells) in these disscussions. Many different T-cell preparations are used experimentally. We have tried to note the T-cell type used in each experiment where practical (i.e., human peripheral T-cells, murine thymic T-cells or cell lines).

ichiometry is probably one y-, one a-, and two t-chains per CD3 complex (116). Unlike the LY-and P-chains, which have very short cytoplasmic tails (5-12 amino acids), the y-, CL, and t-chains of CD3 have more substantial cytoplasmic tails (45-55 amino acids), which probably function in TCR transmembrane signaling. The final two protein chains of the TCR complex, { and the alternatively spliced form 77,are nonglycosylated and mostly cytoplasmic (113 of 142 amino acids). In most a&T-cells the TCR stoichiometry probably consists of one (Y-, /3-, y-, and a-, two t-chains, and two disulfide-linked {-chains, although in some cases a single {- pairs with one v-chain. The CD3 complex and the {s- or @chains are noncovalently linked to cy and p, possibly through charge interactions in the transmembrane domains. Oligomerization of the TCR subunits takes place in the endoplasmic reticulum (ER) before targeting of the complex to the plasma membrane, and the number of l-chains appears to be limiting (5). Models for overall TCR structure and subunit interactions are still evolving; a number of more detailed reviews discuss new data and some areas where there are still gaps in our understanding (5, 53, 62, 116, 237). Few biochemical studies have attempted to activate T-cells using physiological stimuli, because this requires clone-specific stimulation by MHC-restricted APCs bearing the appropriate peptide. Generally, the intercellular MHC II-peptide-TCR interaction is mimicked experimentally by polyclonal activators that can physically cross-link surface TCRs in the absence of APCs. In


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situ,2 the most commonly used polyclonal activators are mitogenic lectins and anti-T& antibodies. Polymeric plant lectins such as concanavali n A or phytohemagglutinin bind carbohydrate residues present on the y- and b-chains of CD3 but may also bind nonspecifically to other glycosylated surface proteins. The exact mechanism by which lectins induce T-cell mitogenesis is not clear, but it is presumed to be primarily through activation of the TCR complex. More defined stimulation of the TCR can be obtained by using specific antibodies against constant regions on either CD3 (anti-CD3) or the CUPchains (anti-framework) or aga.inst clone-spec ific determinants present on the variable portions of the & chains (anti-idiotypic). TCR aggregation, or cross-linking, is critical for transmembrane signaling and subsequent Tcell activation. In most cases dimerization is not sufficient since soluble bivalent monoclonal antibodies fail to induce maximal signal transduction. Some monoclonal antibodies do form microaggregates in solution that can induce TCR signaling. Usually, however, bivalent antibodies immobilized on plastic or beads, polyvalent antibodies, or the addition of secondary cross-linking antibodies are needed to induce maximal TCR aggregation (127, 186). T-Cell Receptor Subunits and Transmembrane Signaling

The functional roles of each of the TCR subunits are still poorly understood. Based on the predicted membrane topology, it seems probable that the CY-and P-chains are mainly involved in extracellular binding of antigenic ligands, whereas the CD3y& complex and c-chains may be important for transmembrane signaling and interaction with downstream cytoplasmic components (reviewed in Refs. 5, 111, and 237). T-cell mutants lacking CD3y6~ fail to transduce normally (5). The TCR a-chain interacts with CD36 at a stretch of eight residues within the transmembrane domain near the COOH- terminus of the a-chain (140, 209). The antigenic signal is probably transmitted as a conformational change down the TCR @-chains to the CD3y6t complex. The most direct evidence supports the hypothesis that the r-chains actually couple the TCR signal to intracellular signaling pathways, but the CD3y& invariant chains may serve to mediate this process (62, 131, 151, 237). This has been demonstrated using the human leukemic T-cell line Jurkat, which expresses the TCR accessory molecule CD4 but lacks another accessory molecule termed CD8 In these cells, chimeric proteins that fuse the extracellular portion of the CD8 molecule to the cytoplasmic domain of the r-chain have been expressed successfully (102). When stimulated with anti-CD8 antibodies, the transfectants could transduce both early and late activation signals (see below) normally observed only with TCR stimulation. The construct apparently bypasses the need for Ti Cupchains and CD3y&-chains by directly coupling extracel2 Throughout this paper we use in vivo to designate experiments in which T-cells are stimulated in the animal by endogenous antigenpresenting cells. The term in situ refers to intact T-cells stimulated experimentally with antibodies or polyclonal activators. We use in vitro to describe biochemical experiments done with purified proteins, cell lysates, or membrane fractions.

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lular signals to the c-chain. Similar results have been reported for CD4l h. The Ca oscillations were dependent on external Ca influx, nels. but loading the cells with the Ca chelator BAPTA also Recent experiments using microfluorimetry techniques have showed that the Ca responses in single Jurkat T- depressed the oscillations, perhaps suggesting that release cells are remarkably heterogeneous (132,174). Many cells from stores initiates the oscillations. The Ca oscillations responded to lectin stimulation with a series of slow Ca in T-cells differ from those reported in other cells in that oscillations having a variable period of l-2 min. The they are very slow (period l-2 min), and the Ca waveform average Ca response calculated from a large number of is not highly stereotyped. The Ca changes in T-cells are more like noisy spikes of Ca overlaid on a plateau of single cells still showed an early peak and later a sustained plateau, much as was observed in the cell population stud- higher Ca, and it could simply be the mean Ca level that ies mentioned above. However, the peak resulted from the is sensed by intracellular Ca-binding proteins. It remains in phase first oscillation of many cells, followed by a Ca to be demonstrated whether the degree of TCR occuplateau that corresponded to out of phase oscillations in pancy is actually frequency coded as has been suggested the individual cells (132). Chelation of external Ca had no for some types of hormone receptors (220). In most other effect on the initial oscillation that was mostly release cell types, sustained Ca oscillations are observed for only from stores, but the later oscillations were blocked, sug- an optimal range of hormone concentrations; lower congesting that Ca influx drove these oscillations (132). centrations elicit a single peak and higher ones cause a

using laser confocal or other low-light video microscopy techniques. In the sections below we discuss data that have been obtained using each of these methods to study T-cell activation.


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sustained Ca rise (13,220). The biochemical mechanisms that lead to the production of repetitive Ca oscillations are not well understood, but possible models are discussed in Refs. 220 and 152. Microsomal

IP3 Receptor and Release of Stored Ca

The early release of Ca from internal stores that follows TCR stimulation results from a rise in IP3 (100,213) and can be elicited by application of exogenous IP3 in permeabilized T-cells (100). The Ca release transient has similar kinetics whether induced by TCR stimulation or by addition of excess IP3, suggesting that the duration of the transient is limited by either the release mechanism or the fullness of the stores but not by the kinetics of IP, production. This is supported directly by measurements of IP3 production, which show a transient peak, followed by a maintained lower level of IP3, lasting as long as TCR stimulation is present (discussed in Refs. 91, 101, and 236). Intracellular IP3 levels are very difficult to quantify because of uncertainties about the cytoplasmic volume and spatial distribution of IP3. In resting T-cells IP3 levels are probably -0.1 PM, rising to a transient peak of - 1-5 PM after TCR stimulation. This correlates fairly well with experiments using permeabilized T-cells, where the maximal Ca release from stores is seen at 1 FM IP3 (100). The Ca-releasing ability of other IPs has not been rigorously tested in lymphocytes, but it seems most likely that the stores release mechanism consists of a single protein that functions both as an IP3-specific receptor and a Ca-release channel, as has been described in other tissues (52). The IP3-binding protein from mammalian brain has been purified, cloned, and reconstituted into lipid bilayers (57, 208, and reviewed in Refs. 52 and 212). In planar lipid bilayers the purified protein (260 kDa) forms a Capermeable channel (26 pS) that is activated by micromolar concentrations of IP3 (138). The cloned cDNA codes for a single polypeptide (2,749 amino acids), which has been expressed in a fibroblast cell line and confers increased ability of IP3 to release Ca from stores (155). Hydropathy analysis suggests that each polypeptide has either four or eight transmembrane domains. Several of the transmembrane domains are negatively charged and have significant homology with the ryanodine receptor, suggesting that they may contribute to the Ca-permeable pore (52). Biochemical data for IP, binding, as well as structural information, suggest that the native receptor is a homotetramer with molecular mass of - 1,000 kDa (212). The IP3 binding site is in the NHz-terminus, and the putative Ca channel is at the extreme COOH-terminus; between is a span of ~1,400 amino acids that may function as a regulatory domain. There is a large number of putative regulatory sites, including binding sites for Ca, ATP, and calmodulin (CaM), and concensus sequences for phosphorylation by several protein kinases. PKC, adenosine 3’,5’-cyclic monophosphate (CAMP) kinase, and Ca/CaM-dependent protein kinase II can phosphorylate the purified protein reconstituted in lipid vesicles (51). Phosphorylation by CAMP kinase results in a diminished potency of IPs in releasing Ca (52, 207). Purified IP3 receptor-Ca channels reconstituted in lipid bilayers have a bell-shaped Ca dependence, with a max-

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imum open probability at 250 nM Ca in the presence of 2 PM IP, (17). This suggests that the cytoplasmic Ca level could provide either a positive or negative signal to IP3 receptors in the Ca storage organelle of intact cells. There has been considerable controversy over the identity and localization of the IP,-sensitive Ca-storage organelle in lymphocytes and other nonmuscle cells. The storage organelle should contain at least three characteristic proteins based on the known functions of the Ca store as follows: 1) the Ca-adenosinetriphosphatase (ATPase) needed to uptake cytoplasmic Ca 2) a highcapacity Ca-sequestering protein to keep luminal Ca from precipitating into crystals with metals or phosphate, and 3) the IP3 receptor-Ca channel that mediates release of Ca in the presence of cytoplasmic IPs. It has been generally believed that the lumen of the ER is the primary IP,-sensitive Ca store in nonmuscle cells, but direct demonstration of this has been lacking. This is particularly true in T lymphocytes where the ER itself is reduced to a thin band surrounding the predominant nucleus. Several recent studies on neutrophils, HL-60 cells, and other cell types have suggested that the IP,-sensitive Ca organelle may be distinct from the ER. In these cells, results obtained with fractionation studies and immunocytochemical analysis of ER marker proteins point to the existence of a separate non-ER Ca store termed “calciosomes” (227; and reviewed in Refs. 118 and 191). In electron micrographs the calciosomes appear as unique 50- to 250-nm microsomes, which immunoreact positively with antibodies against both the Ca-ATPase and calsequestrin-like Ca-binding proteins but are negative for traditional ER markers (227). So far, coassociation of the IP3 receptor has not been reported in calciosomes, but cell fractions containing calciosomes respond functionally to IP3 by releasing stored Ca. Calciosomes could represent a functionally discrete organelle that arises from a specialized ER subcompartment. Alternatively, they may represent normally contiguous regions of the ER that become separated artificially by fixation or fractionation techniques (153). These arguments are somewhat semantic, but it is important to try and localize the IP,-sensitive Ca stores because this may facilitate an understanding of how the stores interact with IP3-generating enzymes and Ca influx pathways. Ca Influx

Pathway

Mitogenic stimulation of T-cells, whether induced by APCs, lectins, or specific anti-TCR antibodies, leads to a sustained influx of extracellular Ca. This Ca influx is critical because treatments that reduce Ca influx also decrease IL-2R mRNA transcripts, IL-2R expression, and cell proliferation (reviewed in Refs. 59, 63, and 184). However, the Ca influx pathway differs from that present in neuronal and muscle cells, in that T-cells lack voltagedependent Ca channels. In T-cells, K-induced depolarization leads to reduced intracellular Ca levels, and reduced Ca influx on stimulation, apparently by reducing the electrochemical driving force for Ca (63,64). The absence of voltage-dependent Ca channels has been confirmed in numerous patch-clamp studies (reviewed in Refs. 58, 60, 133, and 180, but see Ref. 47). These results are also consistent with the observation that Ca influx in T-cells


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is not readily inhibited by known organic antagonists of voltage-dependent Ca channels such as verapamil, D600, nifedipine, nitrendipine, or diltiazem (Premack, unpublished observations). Extracellular application of inorganic Ca channel blockers such as Cd, Ni, and Co do inhibit Ca influx in T-cells, perhaps by physically occluding the pore of the channel. The evidence therefore points to involvement of one or more types of non-voltage-dependent, receptor-operated Ca channels in the TCRstimulated Ca influx. Receptor-operated Ca influx pathways have been characterized in T-cells using three variations of the patchclamp technique. In cell-attached patches on the surface of cloned human T-cells, mitogenic lectins and anti-CD3 or anti-CD2 antibodies activate a low-conductance (7pS), voltage-insensitive Ca- and Ba-permeable channel (60, 61, 121). A similar channel is activated by application of micromolar concentrations of IP3 to the cytoplasmic face of patches excised from the Jurkat cell line (120). In the maintained presence of IP3 and low-cytoplasmic Ca (-100 nM) the channels remain active, but raising the Ca to -1 PM inactivates the channels within a few seconds (61). Recently, a plasma membrane isoform of the IP3 receptor was localized to the plasma membrane of lymphocytes based on membrane fractionation studies (107). These results, and those discussed above, suggest that IP3 may have a dual role in T-cell signal transduction, possibly mediating both release of Ca from stores and activating Ca influx. The cytoplasmic Ca level negatively regulates both of these actions of IP, in a manner that would be expected to promote a return to Ca homeostasis. Another Ca-permeable mitogen-activated conductance has also been described in Jurkat cells using the whole cell and nystatin whole cell modes of patch clamp (132). This conductance may differ from the one described above, since the current develops with a distinct lack of noise or evidence of single-channel fluctuations, suggesting a very low, highly selective conductance to Ca. The current has several properties consistent with a role in T-cell activation, including activation by mitogenic lectin, lack of voltage dependence, and inhibition by Ni or Cd (132). The onset of the current precedes the rise in cytoplasmic Ca in the same cell, suggesting that the relatively small whole cell current (-6-12 PA) is sufficient to raise the cytoplasmic Ca level. A current with very similar properties is also activated in Jurkat cells by flash photolysis of intracellular caged IP3 (T. V. McDonald, B. A. Premack, and P. Gardner, unpublished observations) or by emptying Ca stores with microsomal Ca-ATPase inhibitors such as thapsigargin, 2,5-di-tert-butyl-hydroquinone, or cyclopiazonic acid (B. A. Premack, T. V. McDonald, and P. Gardner, unpublished observations). Taken together, these results suggest that the low-conductance Ca influx pathway may serve to replenish Ca stores that have been depleted by IP3 production during TCR stimulation. A Ca influx current with similar characteristics was recently shown to be activated in mast cells after release of stored Ca by IP3 or ionomycin (95). TCR stimulation, as well as application of microsomal Ca-ATPase inhibitors, induces Mn influx into lymphocytes, perhaps through a

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pathway similar to the one that normally carries the influx of Ca (75, 143, 196, 198). At present there is no clear molecular mechanism that would explain how refilling of the depleted IP,-sensitive Ca store by Ca influx might be regulated. Several hypotheses have been forwarded, including a “capacitive” model, which suggests that depletion of stores initiates an obligatory transmembrane Ca influx by an as yet unknown mechanism (182). EFFECTS MEMBRANE

OF

INCREASED VOLTAGE

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CYTOPLASMIC CA ION CHANNELS

ON

PLASMA

Stimulation of T-cells with lectin or anti-CD3 results in a rapid hyperpolarization from the resting membrane potential of -60 to -70 mV to about -80 mV, as measured by potential-sensitive oxonol dyes (221, 241). This finding is somewhat counterintuitive, since one might expect the influx of positively charged ions from the opening of receptor-activated Ca channels to depolarize a cell at rest. The expected depolarization appears to be overpowered by the nearly simultaneous opening of highaffinity Ca-activated K channels in the plasma membrane. This moves the membrane voltage toward the Nernstian equilibrium potential for K (about -80 mV). The rapid hyperpolarization is presumed to occur by opening of Ca-activated K channels, because it is dependent on both extracellular Ca and K concentrations, but is not affected by Na-K-ATPase inhibitors. Preloading cells with the Ca buffer BAPTA reduces the hyperpolarization, as does application of the K channel inhibitor charybdotoxin (139). Blocking the hyperpolarization unmasks the small depolarization induced by Ca entry (discussed in Ref. 63). The function of the Ca-induced hyperpolarization is not known. Earlier reports suggested that inhibition of K channels disrupts normal cell cycle progression, leading to inhibition of mitogenesis in lymphocytes (reviewed in Ref. 180). More recent studies with carybdotoxin, a relatively specific inhibitor of Ca-activated K channels, either show a small inhibition of mitogenesis (181) or no significant effects of the inhibitor at concentrations shown to completely block the antigeninduced hyperpolarization (65). It could be that the role of membrane hyperpolarization is to indirectly regulate Ca influx through Ca channels. In the absence of Caactivated K channels Ca influx would lead to a depolarization that in turn decreases the driving force for Ca into the cell. Ca-activated K channels may serve to counteract the depolarizing effect and thus help to maintain an increased Ca influx. More direct evidence for the presence of a Ca-activated K conductance was obtained recently, using the cell-attached patch-clamp technique to record from single Caactivated K channels in rat thymocytes activated by lectin (139; method discussed in Ref. 180). In these experiments, opening of the Ca-activated K channels was dependent on the external application of agents known to increase cytoplasmic Ca (lectin and ionophore). Two distinct small-conductance (6-7 and 17-18 pS) Ca-activated K channels were noted; both were only weakly voltage dependent, unlike the voltage-activated K channels previously reported in T-cells (39, 145; reviewed in Refs. 58, 60, and 133). The cellular Ca levels were not measured,


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absence of Ca, whereas the other three isotypes have negligible activity under the same conditions (188, 193). Shifting the Ca concentration from 100 nM to 10 PM in vitro gives an increase in catalytic acti vity of approximately five - to sixfold for the ,01-, a-, and y-isofo rms with PIP:! as a substrate (see Ref. 193; calculated from their Fig. 6B). It should be noted that in vivo PLC activation will produce IP3 and increase cytosolic Ca, which will provide positive feedback to PLC, but the initial activation of PLC by receptor stimulation precedes the rise in Ca. The mechanism by which receptor stimulation leads to increased PLC activity at low Ca levels is not well understood. It has been widely suggested that G proteins couple receptor occupancy to phospholipase activity, but this has been shown directly only for activation of PLC-@l by the PTX-insensitive G, protein (202). The regulation of PLC-71 by tyrosine phosphorylation has been implicated in growth factor receptor signaling, as discussed above. The pathway that leads to activation of PLC cy- and &isotypes is not yet known. PLC CY-,@l-, yl-, and y2-isoforms are found in T lymphocytes, but the &isoforms do not appear to be present (69, 172). The relative amount of each isoform varies in different T-cell preparations. Pooling the data from sevTRANSDUCING THE RISE IN CYTOPLASMIC CA: eral studies on Jurkat T-cells indicates that, in this cell EFFECTS ON EARLY ENZYME CASCADES protein) me, 71 1s the most prevalent (~800 protein), In this section we examine the regulatory effects of the followed bY much lower levels of ,N (-35 rise in cytoplasmic Ca on early signal transduction mol- cy,and y2 (69, 172). ecules such as PLC, PKC, and other major T-cell CaThe TCR-stimulated activation of PLC-71 is regulated binding proteins. Although much of the current data are both directly and indirectly by the rise in cytosolic Ca. not conclusive, it is useful to present a synopsis of what is The early steps leading up to tyrosine phosphorylation do known about the role of Ca-binding proteins, because not appear to be Ca regulated because Ca ionophore alone these molecules are undoubtedly crucial to T-cell activadoes not increase phosphorylation on tyrosine residues tion. Throughout this section we have concentrated on (173, 238). Ca has a direct stimulatory effect on PLC in Ca-binding proteins whose activity is modulated by Ca situ; in Jurkat cells and some peripheral T-cells, treatchanges over the physiological Ca range, i.e., 100 nM to ment with Ca ionophore rapid .ly leads to inositol phos10 PM free Ca. We have omitted some proteins whose phate hydrolysis (30). This could result from a direct activity is dependent on Ca binding in the high-microCa-dependent activation of the predominant yl -isotype molar to millimolar range. For most of the proteins we of PLC, but other isotypes are also Ca sensitive. Because have tried to give an approximate affinity for Ca binding, PLC-71 is thought to be regulated by tyrosine phosphorbut in many cases this value is dependent on protein ylation, it seems possible that Ca-modulated tyrosine conformation, presence of substrate, ionic strength, phos- phosphatase activity might influence PLC-71 activity. pholipid environment, and other experimental factors. Evidence for this was obtained recently in a study of the These numbers should be used for comparison only; the lymphocyte-specific surface membrane tyrosine phosactual affinities in vivo may vary widely. phatase,- CD45 (171). Influx of Ca induced by the Ca ionophore ionomycin, caused decreased phosphatase acRegulatory Effects of Ca on PLC Activation tivity in CD45 immunoprecipitates from mouse thyFour major types (8 distinct isoforms) of inositol phos- mocyte membranes (171). CD45 does not appear to bind pholipid-specific PLC termed ~1,0, y, and 6 have been Ca directly; rather, the decreased phosphatase activity described in mammalian tissues. The different enzymes results from slower secondary reactions, leading to dephosphorylation of CD45 on serine residues. The enzyme all have significant homology in two catalytic domains responsible for dephosphorylation of CD45 is not known. (termed X and Y) but otherwise have very distinct structures and regulatory properties (41, 187, 188). All four These findings are very important, because previous studtypes of PLC have similar substrate preferences and will ies have shown that CD45 modulates TCR signal transhydrolyze three common membrane phospholipids, PI, duction, possibly by tyrosine dephosphorylation of p59fY”, p561ck, or PLC-yl (117, 128,129,142,163,170,177,219). inositol4-monophosphate, and inositol 1,4-bisphosphate (41, 188). The catalytic rates are Ca dependent for all of Taken together, these results suggest that a complex seand dephosphorylation the PLC isoforms. The molecular basis for Ca binding is ries of tyrosine phosphorylation events control PLC-71 activation. The regulatory roles of not known, and the PLC isoforms do not have significant Ca in these processes are not clearly established but are sequence homology to other Ca-binding proteins. PLC-a is reported to have -30% of maximal activity in the likely to be multiple and complex.

therefore the absolute Ca dependence of these Ca-activated K channels is not known. A similar small-conductance (4-7 pS) Ca-activated K channel has been described recently in Jurkat cells using the whole cell patch clamp (81). In this study the cells were perfused with solutions containing known free Ca concentrations from 100 nM to 1 PM. The Ca-activated K channels were steeply dependent on the internal Ca concentration over this range, with half-maximal activation at -500 nM cytoplasmic Ca. Plots of the Ca concentration vs. the Ca-activated K conductance were best fit with a Hill coefficient of 4-5, suggesting that several Ca ions must bind to the channel (or possibly an associated molecule) to open the pore (81). With the use of similar methods, the opening probability of Ca-activated K channels in other cells has also been shown to be highly dependent on Ca over the range of 100 nM to 1 PM (92, 126). The Hill coefficient for large conductance “maxi” Ca-activated K channels is between 2 and 4 when calculated from Ca concentration vs. probability of opening curves (126). The structural basis for Ca binding to small- and largeconductance Ca-activated K channels has not yet been elucidated.


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this case more p translocates than CY(124). This suggests that lectin-induced Ca influx may regulate translocation The importance of PKC as a mediator of TCR signalof the cu-isoform, although this was not tested directly. ing has been recognized for many years, since it was first These studies also do not address the question of whether shown that the combination of phorbol ester plus Ca translocation is synonymous with increased catalytic acionophore could mimic several of the early events in T- tivity. A recent study in permeabilized Jurkat cells shows cell activation, including transcription of the fos, jun, and that PKC phosphorylation of the CD3y-chain can be IL-2 genes. The critical targets of PKC have not been induced by using ionophore to raise the Ca concentration defined, but a large number of important signaling mol- to 500 nM in the absence of added phorbol ester (135). ecules are phosphorylated on serine or threonine residues This argues that a Ca rise alone is sufficient to activate after activation of PKC. These include the y-subunit of PKC but does not address the possibility that endogenous the CD3 complex, cytoplasmic domains of the CD4 and DAG is produced secondarily, perhaps by Ca-dependent CD45 molecules, and the tyrosine kinase ~56~“~. These activation of PI-PLC. Because the PKC cu-isoform is prefindings have been reviewed in numerous articles (X,50, dominant in these cells, it seems most likely to be respon183,236) and will not be discussed in detail here. We will sible for the Ca-dependent phosphorylation of the CD3rsummarize some of the recent data, with emphasis on chain. This is supported indirectly by studies of the those studies that have characterized the Ca requirement catalytic activity of brain PKC isoforms in vitro, which for activity of the various PKC isoforms in T-cells. have demonstrated that the cu-isoform is more Ca sensiSeven distinct PKC isoforms have been identified by tive than the ,&isoform (167). molecular cloning, and these fall into two distinct groups. Besides the stimulatory effects of PKC on T-cell actiThe first group includes the most common isoforms (cy, vation already mentioned, PKC activity also inhibits the ,01, p2, and y); these are structurally related but show rise in cytoplasmic Ca through several negative-feedback differences in regulation by Ca and DAG. All four of these loops. Phosphorylation by PKC seems to control the isoforms have a conserved Ca-binding domain and can be cellular Ca response at two levels. First, Ca release from substantially activated in vitro by high concentrations of stores is diminished, and second, the Ca level during the Ca (10 PM) even in the absence of DAG when Hl histone later-sustained phase is reduced. In T-cells short (2-min) is the phosphate acceptor (167). However, under these applications of phorbol esters or membrane-permeable conditions, the kinase activity is maximal at supraphysDAG analogues inhibit anti-CD3-induced release of Ca iological Ca concentrations (-50 PM). With DAG from stores and the later Ca influx (196,216). This is not present, the Ca dependence shifts to lower Ca concentracaused by PKC-induced downregulation of CD3, as is the tions, so that maximal activity is observed at - 10 PM Ca. case with long applications of phorbol esters (26), but is This effect of DAG binding on the Ca sensitivity of the more likely to be secondary to rapid inhibition of PLC kinase differs for the PKC isoforms, with a and y show- activity and decreased IP3 production (87). The decrease ing the greatest degree of Ca dependence and p the least. in IP3 is probably a result of PKC phosphorylation of one In the presence of DAG, but absence of Ca, the P-isoforms of the early signal-transducing molecules upstream of have 60% maximal activity, whereas CYand y have only PLC activation. Possible candidates are either the CD3y15-25% activity (see Ref. 167, their Fig. 4). The second chain or ~56 lck, both of which are targets of PKC (173). group of PKC isoforms (6, 6, {) lacks the Ca-binding re- Application of phorbol esters after TCR stimulation, gion, differs in substrate preference, and may therefore when the release of Ca from stores is complete, results in have very different biological properties (84,96,167,168). a rapid decrease in the Ca level during the later sustained Because many cell types do express more than one iso- phase (196, 216). The reduction in the Ca level could form of PKC, it seems likely that each isoform has a result either from a decrease in Ca influx through surface channels or from an increase in Ca efflux by stimulation special functional role in terms of activation by physiological stimuli and interaction with target proteins. of the plasma membrane Ca-ATPase, which is a major effect of PKC in other cell types (27, 82, 199, 201). NeuMurine thymocytes express mRNA for PKC isoforms CY,,& E, and c (156), but fl and c appear to be the most ronal and cardiac L-type Ca channels are substrates for highly expressed proteins based on immunoblotting with PKC, and phosphorylation has been reported to either isoform-specific antisera (205). Using biochemical frac- enhance or inhibit Ca channel activity (43,96, 168, 217). sites per channel, tionation and specific antisera, human T-cells were There are multiple phosphorylation shown to express mostly the CY-and @2-isoforms, with low and it has been suggested that up- or downregulation of L levels of fll and y absent (14,15,135). However, it should channel activity may be dependent on the PKC isoform be noted that the relative abundance of each PKC iso- that is stimulated (137). The possibility that phosphoryform can differ, depending on the T-cell system used. In lation by PKC directly effects Ca influx channels in Thuman T lymphoblasts @l is most highly expressed, cells has not been investigated, but such a Ca-dependent feedback mechanism could be important for maintenance whereas the Jurkat T-cell line expresses predominantly the a-isoform, possibly relating to the transformed state of Ca homeostasis. of the latter cells (135). THE RISE IN CYTOPLASMIC CA: In Jurkat cells stimulated with mitogenic lectin, the TRANSDUCING ACTIVATION OF CA-BINDING PROTEINS PKC cu-isoform is translocated to a greater extent and There are four major classes of intracellular Ca-binding remains membrane bound for much longer than the @isoform. In the same cells stimulated with phorbol ester proteins distinct from PLC and PKC. These are 1) the there is increased translocation of both isoforms, but in Ca-modulated signaling proteins of the EF-hand family,

Activation

of PKC by Ca and DAG


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2) the Ca- and phospholipid-binding proteins of the annexin (also called calpactin-lipocortin) family, 3) a diverse group of cytoskeletal Ca-binding and actin-modulating proteins, and 4) the high-capacity Ca storage proteins found in the microsomal compartment of the ER. Representatives of each of these classes of Ca-binding proteins are present in lymphocytes. Although few specific functions have been assigned to the lymphocyte isoforms, data from other cell types provide indirect evidence of a role for members of each group in signal transduction.

strictly dependent on CaM even for basal activity and those that are modulated by CaM to increase or decrease catalytic activity. The CaM-dependent enzymes include myosin light-chain kinase, caldesmon, CaM kinases, and a CaM phosphatase (calcineurin). The CaM-modulated enzymes include Ca-ATPases (discussed in CalCaM Modulation of Plasma Membrane Ca-ATPase), and IP3 kinase, cyclases, and phosphodiesterases, which are beyond the scope of this review.

EF-Hand

The organization of the lymphocyte submembrane cytoskeleton is altered rapidly on ligation of the TCR by appropriate mitogenic stimuli. Many cytoskeletal components are reorganized into a new dense structure that forms specifically under the area of focal contact with the APC (122). Surface membrane receptors, including the TCR, and subsurface microsomal vesicles are also actively concentrated into a “cap” at the site of cell-cell contact (16,21). The cytoskeletal reorganization is driven by actin-myosin interactions, with structural changes translated from actin to the membrane through fodrin or other spectrin-like linker molecules (12, 23, 130, 223). Fodrin (nonerythrocyte spectrin) may provide either a direct connection to integral membrane proteins (23) or interact with specialized proteins like ankyrin or band 4.1, which complex with the cytoplasmic domain of some membrane proteins (20, 22). In lymphocytes, receptor capping can be partly induced with Ca ionophores (19) and is regulated by several Ca/CaM-dependent processes (106, 210). CaM binding directly controls activity of the cytoskeletal myosin light-chain kinase, which in turn regulates actinomyosin interactions leading to surface receptor capping. Indeed, myosin light chains are phosphorylated during lymphocyte activation, and direct phosphorylation with a Ca-independent form of myosin lightchain kinase leads to receptor capping without need for an increase in Ca (see Ref. 228 and references therein). A high-molecular-weight form of the Ca/CaM-dependent actin binding protein, caldesmon, is present in T lymphocytes, and cells depleted of this protein are defective in their ability to collect surface receptors during capping (228). In nonlymphoid cells, CaM can modulate the activity of a number of cytoskeletal components including spectrin, fodrin, and tubulin (147, 210). In mouse Cl27 cells, overexpression of CaM leads to changes in cytoskeletal organization and cell morphology, as well as alterations in cell cycle regulation (185). The exact regulatory role of CaM in most of these interactions is not known, but it seems likely that many of the Cainduced cellular conformation changes in T-cells are coupled to cytoplasmic Ca levels through CaM-dependent mechanisms.

Ca-Binding

Proteins

The largest and most well characterized family of Cabinding proteins is the EF-hand (or helix-loop-helix) proteins containing a characteristic motif that binds Ca selectively and with high affinity. All EF-hand proteins have two to eight paired Ca-binding repeats, each consisting of a loop of 12 amino acids flanked by two a-helices. Oxygen atoms from the side chains of the loop amino acids interact specifically to bind and coordinate a single Ca ion (90, 175). Included in this group are a number of well-known proteins with affinities for Ca in the nanomolar to micromolar range, such as troponin C, parvalbumin, and the SlOO proteins, none of which have been described in lymphocytes. Several of the EF-hand proteins including calmodulin (CaM), myosin light-chain kinase, calcineurin, and calpain are expressed in T-cells and are important for transduction of Ca changes through Ca-dependent interactions with a number of important target proteins. CaM

CaM is the major intracellular Ca receptor and is expressed in all eukaryotic cells as a 17-kDa acidic nonmyristylated cytoplasmic protein. CaM binds to and directly regulates the activity of a large number of enzymes, cytoskeletal proteins, ion channels, and other proteins. In some cases, CaM is also present as an integral subunit of enzymes such as phosphorylase kinase. Each CaM molecule is elongate and bilobed, consisting of a pair of Cabinding EF-hand repeats in each lobe, connected by an a-helix. The Ca ions bind cooperatively, and, in total, four Ca ions are bound with an average dissociation constant (&) of - l-4 PM (85). The Ca binding and dissociation kinetics are complex, consisting of rapid (650 s-l) and slow (9 s-l) phases. This has led to the prediction that all four Ca binding sites are not equivalent, with the COOH-terminal sites having an -lo-fold greater affinity. Nuclear magnetic resonance studies have confirmed this and further support the hypothesis that the first two Ca bind near the COOH-terminus, followed by binding to sites in the NH,-terminal one-half of the molecule (see Ref. 85). Ca binding leads to conformational changes that expose hydrophobic clefts within the molecule, and the exposed residues may interact with CaM-binding sites on target proteins (175). The activated Ca/CaM complex binds to and modulates the activity of a large number of enzymes that are important targets of biological signaling by Ca. These enzymes fall roughly into two groups, those that are

CalCaM-Dependent Lymphocyte Cytoskeletal Organization

CalCaM-Dependent

Protein

Kinases

Although PKC isoforms do have some catalytic activity in the presence of micromolar levels of Ca, it is the Ca/CaM-dependent protein kinases I through IV that are responsible for most of the purely Ca-dependent phosphorylation of cellular substrates. However, some of the


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CaM kinases have a very limited number of substrates and a fairly narrow tissue distribution. The expression of CaM kinases I, III, and IV has not been demonstrated in lymphocytes, and possible protein targets have not been defined. On the other hand, CaM kinase II, also termed multifunctional CaM kinase, has many potential substrates and a widespread tissue distribution (33,86). The proteins of the CaM kinase II family have very complex biochemical and structural characteristics. Each functional kinase consists of 6- 12 noncovalently associated subunits with a total molecular mass of 300-700 kDa. Four distinct subunit isoforms (termed CY,& y, and 6) with molecular masses of 54-60 kDa have been identified from rat brain by cDNA cloning. Both the total number of subunits and the subunit composition of the holoenzyme vary between tissues and across species. CaM kinase II is essentially inactive (~1% of maximal activity) in the absence of Ca/CaM and is half-maximally activated at 0.5-1.0 PM free Ca when CaM is saturating (86). The Ca/CaM binds to a hydrophobic regulatory domain with a stoichiometry of one CaM per kinase subunit (33). CaM binding rapidly induces autophosphorylation at up to 40 sites per holoenzyme, and this appears to precede measurable phosphorylation of exogenous substrates. Autophosphorylation also has the effect of converting the enzyme to a form that is active in the absence of Ca and CaM. The Ca/CaM-independent kinase activity persists until the enzyme is dephosphorylated by protein phosphatases, probably type 1 or 2A, both of which can dephosphorylate CaM kinase II in vitro. The multifunctional CaM kinase II is known to phosphorylate a large number of proteins containing the ArgXxn:- Yyy-Ser/Thr consensus sequence. This includes enzymes involved in cellular functions as diverse as lipid biosynthesis, carbohydrate metabolism, protein synthesis, and cytoskeletal organization. Most of the known substrates have only been identified in vitro, but several of these are likely to be important targets of the T-cell CaM kinase II after TCR stimulation in vivo. We have recently identified and cloned a lymphocyte-specific isoform of CaM kinase II in Jurkat T-cells, where it phosphorylates a wide variety of proteins in response to stimulation with lectins or ionomycin (P. Nghiem, H. Schulman, and P. Gardner, unpublished observations). TCR stimulation induces CaM kinase II to undergo autophosphorylation and subsequent long-term Ca-independent kinase activity in situ, suggesting that it may have a special role in transducing the mitogen-activated Ca rise. The cDNA sequence for the lymphocyte CaM kinase II isoform is unique but has 95% sequence identity with the rat brain y-isoform. The holoenzyme in T-cells is composed of six to eight of these y-like subunits. We recently identified one of the cellular substrates for the lymphocyte CaM kinase. Using patch-clamp experiments we demonstrated that plasma membrane Cl channels are a target for CaM kinase II in Jurkat T-cells (166). Ca ionophores increase a whole cell Cl current when applied extracellularly, but this activation is not direct, because the current can be inhibited by a specific peptide inhibitor of CaM kinase II. Furthermore, single 40-pS Cl channels can be activated in excised patches by direct

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application of purified CaM kinase II to the cytoplasmic face of the patch. These results provide the first evidence that phosphorylation by CaM kinase II can upregulate Cl channel activity and imply that such regulation would occur during T-cell stimulation by mitogens. Calcineurin, a CalCaM-Dependent Protein Phosphatase

Calcineurin is a Ca-dependent protein phosphatase (type 2B) that acts mainly on phosphoserine and phosphothreonine residues. The active enzyme is composed of two subunits; the larger A subunit (59-61 kDa) has CaMbinding and catalytic domains, and the smaller (17-18 kDa) myristylated B subunit has four functional EF hands (108, 114, 175). However, Ca bound to the B subunit in the absence of CaM has little effect on catalytic activity; therefore the enzyme can be considered to be functionally Ca/CaM dependent (discussed in Refs. 3 and 114). The Ca/CaM complex binds to the A subunit with 1:l stoichiometry and an affinity of 200 PM) and does not bind Ca in the physiological signaling range and so will not be discussed here. The catalytic properties of calpain I are quite interesting. It is believed that the inactive protease is anchored to the membrane by a hydrophobic region in the NH,-terminal of the regulatory subunit. This glycine-rich domain is cleaved by autoproteolysis in the presence of Ca, releasing the cytosolic form of the active protease (150). Many CaM-binding proteins, such as myosin light-chain kinase, CaM kinase, and calcineurin are substrates for calpain (229). Although >30 protein substrates for calpain I have been described in vitro, only a few of these are likely to be bona fide targets in vivo (35). Besides the CaM-binding proteins, the evidence discussed below suggests that in lymphocytes PKC and cytoskeletal proteins are also likely to be physiological targets of calpain I. In a number of cell types including lymphocytes and neutrophils, surface receptor stimulation causes a rise in intracellular Ca, activation of calpain I, and partial proteolysis of PKC to an active cytosolic kinase termed protein kinase M (PKM) (15, 35, 178, 179). PKM is a fragment of PKC, which retains a high level of kinase activity and no longer requires Ca or DAG for activation. Under these conditions PKM is capable of phosphorylating a variety of cytosolic substrates with much greater efficiency than membrane-bound PKC (discussed in Ref. 211). This change in substrate preference may be important for signal transduction. No direct evidence for this is available in T-cells, but in murine splenic B cells PKM may convey a mitogenic signal to the nucleus after crosslinking of surface immunoglobulin receptors. In these cells, PKC is cleaved to PKM by calpain I, and upregulation of expression of the immediate/early gene c-myc is blocked specifically by both PKC inhibitors (which also inhibit PKM) and calpain inhibitors (178). Calpain I is known to cleave a variety of cytoskeletal proteins in vitro; a partial list includes myosin light chains, tubulin, filamin, fodrin, spectrin, talin, and ankyrin (35). The susceptibility of so many of these proteins to Ca-dependent proteolysis has raised the suggestion that cleavage by calpain I may represent a general degradative pathway for cytoskeletal proteins (35). It seems probable that some of the cytoskeletal proteins will turn out to be important substrates of calpain I in lymphocytes where the cellular activation process is tightly coupled to changes in the cytoskeleton. Summary:

Calpain: Ca-Activated

Neutral

Protease

Calpain (also called Ca-activated neutral protease) is a widely distributed cytosolic cysteine protease found in all

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EF-Hand

Ca-Binding

Proteins

Clearly, high-affinity Ca-binding proteins of the EFhand family comprise the majority of physiologically important sites of cytoplasmic “Ca sensing” in lymphocytes.


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Fig. 3. TCR signaling pathways. After production of IPs and release of stored Ca is a prolonged phase of Ca influx through PM Ca channels. Increased cytosolic Ca, in the presence of DAG, activates PKC, which provides negativefeedback signal onto CD3 chains, leading to decrease in DAG and IPs production. Increased Ca also binds to calmodulin (CaM), which can activate a number of target enzymes including myosin light-chain kinase (MLCK), Ca/CaMdependent protein kinase II (CaMKII), and phosphatase calcineurin (CaN). Solid arrows, intracellular messenger pathways. Dotted arrows, intramolecular phosphorylation pathways. Large boxes give a few representative substrates for major kinases and phosphatase.

granule release from adrenal chromaffin cells (46, 194). A number of studies suggest that annexins could regulate cytoskeletal interactions or phospholipase activity by controlling phospholipid availability in a Ca-dependent manner (36, 112). The nomenclature for the annexin Ca-binding protein family is rather daunting. Up to 40 different names have been used previously to describe members of the family. These include calpactins, lipocortins, chromobindins, calcimedins, endonexins, calelectrins, synexins, and the lymphocyte membrane-binding proteins. However, it is now recognized that a number of individual members of each subfamily were actually identical proteins, originally described by differing isolation-purification procedures Annexin Ca- and Phospholipid-Binding Proteins using different tissues in a number of laboratories (see Currently, 22 distinct proteins The annexin (also called calpactin-lipocortin) family of Ref. 37 for terminology). non-EF-hand proteins were initially described as a di- have been identified as belonging to the annexin supergene family (10). These have been divided, based on verse group whose binding to membrane phospholipid amino acid homology, into 10 classes that fall into 2 was Ca dependent (reviewed in Refs. 36 and 112). A number of members of this group also bind actin in the pres- major groups. The group of smaller proteins have molecular masses ranging from 28 to 36 kDa and include anence of Ca and phospholipid, hence the name calpactins. nexins I through V, VII, and VIII. The second group is The annexins are small, globular, amphipathic proteins approximately double the molecular mass (67-73 kDa) whose hydrophobicity is increased by Ca binding. This and includes the lymphocyte Ca-binding protein p68 (ancharacteristic gives all members of the family the ability nexin VI) and high-molecular-weight lipocortins. to translocate between cytosolic and membrane compartComplete or partial sequencing of all members of the ments within the cell. None of the annexins are myristylated; therefore it appears that membrane association is family shows a domain structure consisting of repeated caused by a Ca-induced conformational change in the sequences 70-80 amino acids long. The low-molecularprotein, which leads to an interaction between the pro- weight annexins have four of these repeats, whereas the forms have eight. In both cases tein and negatively charged phospholipid head groups. high-molecular-weight the repeats are joined by short linker regions of 5-10 Most of the annexins can facilitate the aggregation and fusion of phospholipid vesicles in vitro, leading to the amino acids. Within each repeat is a sequence of -17 amino acids that is highly conserved for all members of suggestion that they regulate Ca-dependent protein-mediated exocytosis in vivo (148). There is substantial evi- the group and may be important for Ca binding (10). The dence that several members of the family are involved in crystal structure for annexin V with three Ca bound has

This is reflected in the wide range of processes that are regulated. However, several EF-hand proteins that are abundant in brain and other tissues have not been reported in lymphocytes, including parvalbumin, calbindin, calcyclin, and calretinin. Also of interest, the endogenous CaM-inhibitor protein neuromodulin (or GAP43), which is highly expressed in neural cells (49), has not been reported in lymphocytes. However, neither are there studies that specifically show that these proteins are not expressed in T-cells. Therefore it remains to be determined whether these differences represent real tissue-specific distribution of the proteins or merely point to a lack of investigation in lymphoid cells.


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been resolved to 2.0 A and shows that the Ca-binding sites are distinct from the EF-hand motif, although the bound Ca are coordinated in a similar way by oxygen atoms from side-chain carboxylates and main-chain carbony1 groups (97). There is a high degree of sequence homology throughout the COOH-terminus and repeat regions of all the known annexins, whereas the NH2-terminus of each member of the family has the greatest variation in amino acid sequence and in length (10). The NH2-terminus therefore seems most likely to confer specialization of function to the individual Ca-binding proteins. In lymphocytes, three annexin family proteins have been described, p32 (annexin IV), p36 (annexin II), and p68 (annexin VI) (31, 162). Functional studies have not been done in lymphocytes, but in other cell types p36 homologues are substrates for both serine-threonine and tyrosine kinases. Phosphorylation regulates both Ca binding and membrane association, which could be of functional importance. In transformed 3T3 cells, ~60”‘” phosphorylates the NHz-terminus of ~36, causing a decreased affinity for Ca that results in decreased membrane association. The related molecule ~35 (annexin I) is one of the major substrates for the EGF receptor tyrosine kinase in A431 cells, but in this case phosphorylation increases the affinity for Ca fivefold (112). In neutrophils and chromaffin cells, degranulation is preceded by a rise in Ca that promotes association of annexin II with secretory granules (148). This association seems to mediate fusion of the vesicles with the plasma membrane at lower Ca levels than would be needed in the absence of annexin II. The T-cell p36 (annexin II) may have an analogous role, perhaps facilitating Ca-dependent vesicle fusion and

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regulating secretion of interleukins during T-cell activation. The lymphocyte-specific annexin p68 was recently sequenced and also has consensus sites for both tyrosine and serine phosphorylation (162). The possible functions of lymphocyte p68 (annexin VI) have not yet been examined, but a homologous annexin purified from bovine liver cells is localized on stress fibers at points of focal contact with the substrate, suggesting a role in membrane-cytoskeletal interactions (94). It seems possible that lymphocytes ~35, ~36, and p68 are phosphorylated in vivo by TCR-associated src-like kinases and that membrane association could be regulated by antigen-induced rises in cytoplasmic Ca, but this remains to be demonstrated. At this time, the functional significance of the annexin family of Ca-binding proteins in T-cell membranes is still open to speculation. Ca-Binding

Actin-Modulating

Cytoskeletal Proteins

In addition to the CaM-dependent interactions with cytoskeletal proteins discussed above, there are several actin-modulating proteins whose activity is controlled directly by high-affinity Ca binding. The best characterized are gelsolin and the smaller gelsolin-related actin-fragmenting proteins such as fragmin and severin (223, 233). These proteins regulate the state of the actin cytoskeleton by facilitating the Ca-dependent conversion of actin filaments into monomers, causing a gel-to-sol transition in the actin network, which may be critical for cytokinesis. Gelsolin also has other complex effects on actin capping and assembly that are important in pseudopod outgrowth (reviewed in Ref. 243). Gelsolin is a single 80- to 90-kDa polypeptide that contains six repeated actin- binding domains and two putative Ca-binding sites with -1 PM

Table 1. Major T-cell Ca-modulated proteins Ca-Modulated

Protein

Family

Protein

or Isoform

Apparent

Ca affinity,

PM

References

Ion channels Ion pumps

I K(Ca) 0.5 81, 139 PM Ca-ATPase* m 20 (No calmodulin) 27,28 ER Ca-ATPase* cl 199 Phospholipase C l-3 188, 193 a, PL YL 72 Protein kinase C 3-5 (DAG present) 135, 167 a, Y, PI, PII EF-hand Ca-binding proteins Calmodulin 1-4 85, 113, 175 Calcineurin B subunit 1004,000 109, 114, 175 Calpain l-20 35, 150 Fodrin (spectrin) ND 12, 23, 130 Calmodulin-binding proteins Calmodulin kinase II 33, 86 Calcineurin A subunit 109, 114 PM Ca-ATPase* 27,28 Myosin light-chain kinase 19, 228 Caldesmon 19, 228 Fodrin (spectrin) 12, 23, 130 Annexin Ca-binding proteins Annexin II (~36) 2-10 10, 36, 112 Annexin IV (~32) ND 10,36, 112 Annexin VI (~68) -1 31, 112, 162 Actin-modulating proteins Gelsolin* 223, 233, 243 Fragmin* 223 -1 Severin” 223 Calreticulin* Ca storage proteins - 1,000 (Low affinity sites) 8, 119 Apparent Ca affinity, as used here, is defined as concentration of Ca that produces half-maximal activation of protein. Affinity values were measured in vitro under differing experimental conditions and should not be taken as definitive. Actual dissociation constants (Kd)s) for Ca binding were not available for most proteins. Calmodulin-binding proteins listed are modulated by Ca through binding of Ca-calmodulin complex and therefore are likely to have apparent Ca affinities similar to that listed for calmodulin. However, in some cases affinity of calmodulin for Ca is known to be markedly increased when complexed with calmodulin-binding protein. * Specific T-cell isoforms of these proteins have not been characterized, but they are included because other leukocytes are known to express these proteins, and cellular distribution is considered to be widespread. ND, values not determined in cited references. PM, plasma membrane; ER, endoplasmic reticulum; DAG, diacylglycerol.


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affinity (fragmin and severin have only three actin-binding domains). The actin-binding and severing domain is in the NH2-terminus of the molecule and seems to be blocked in the absence of Ca. Ca binding in the COOHterminus is thought to induce a conformational change that exposes an active site in the middle of the molecule. It has been postulated that the COOH-terminus covers up the actin-severing sites and that Ca binding results in unblocking of the catalytic area (243). In intact macrophages and platelets, receptor-stimulated Ca mobilization regulates the translocation of gelsolin from the cytoplasm to plasma membrane, where it may interact with actin and phopholipids (88). In T lymphocytes gelsolin is likely to be a component of the complex pathway leading to actin rearrangement during receptor capping. Microsomal

Ca-Sequestering

Proteins

The function of Ca-sequestering proteins is to allow for the storage of high (approximately millimolar) concentrations of Ca in specific storage organelles in a stable but rapidly releasable form. Without stable buffering, the Ca would be likely to precipitate as biologically inert Ca phosphate crystals. The main Ca-sequestering protein of the Ca storage organelle in nonmuscle cells is calreticulin. Calreticulin has been described in leukocyte cell lines such as HL-60 cells where it copurifies with the IP3sensitive Ca storage organelle on Percoll density gradients (119). Histological studies suggest that in these cell types calreticulin is distributed in the lumen of a microsoma1 vesicular compartment that is either in part of the smooth ER (153) or in distinct Ca storage organelles termed calciosomes (118, 191). Calreticulin has also been called calregulin and is immunologically related to calsequestrin, a more highly studied Ca-sequestering protein of skeletal muscle sarcoplasmic reticulum. The primary sequence of calreticulin predicts a molecular mass of 47 kDa, but the protein migrates anomalously at ~60 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8). Calreticulin has -20 low-affinity and one high-affinity Ca-binding site when expressed in Escherichia cob (8). The high-capacity, low-affinity (l-2 mM) sites are clustered in a very acidic region near the COOHterminus, suggesting that the acidic residues may be involved in low-affinity Ca binding. The single high-affinity (6- 11 PM) site was found in the centrally located “P domain” of the molecule. The P domain has no sequence homology to the EF-hand or annexin families of Ca-binding proteins, although there is a region with a rigid turn structure repeated three times, which might be involved in high-affinity Ca binding. Calreticulin has not been specifically described in T-cells, but its expression is widespread in nonmuscle tissue, including leukocytes (119). SUMMARY

In this review we have tried to outline the signal transduction pathways leading from TCR antigen binding to the prolonged increase in cytoplasmic Ca that accompanies T-cell activation. Rapid progress has been made in defining the molecules that are physically associated with the TCR and that are likely to mediate transmembrane

REVIEW

signaling. These studies suggest a direct role of receptorassociated tyrosine kinases in the activation of PLC and the subsequent mobilization of Ca (Fig. 2). The release of stored Ca, in conjunction with influx of extracellular Ca, directly regulates several important enzymes as well as a large number of specialized Ca-binding proteins (Fig. 3). We have summarized most of the major Ca-modulated proteins discussed in Table 1. To our knowledge, this review is the first attempt at a thorough listing of the Ca-binding proteins in T-cells. However, it is clear that the list is far from complete because many of the Cabinding proteins that are present in T-cells have not yet been adequately characterized. This is especially true in terms of functional data, which are still lacking for many of the Ca-binding proteins that have already been identified in T-cells. For example, very few substrates are known for the major Ca-dependent kinase, CaM kinase II, and the same is true for the major Ca-dependent phosphatase, calcineurin. Similarly, although there is clear evidence that T-cell activation is accompanied by Cadependent cytoskeletal rearrangements, the complicated interactions between the proteins involved are not well understood. The evidence presented suggests that a sustained rise in Ca is necessary for T-cell activation, for transcription of the IL-2 gene, and, ultimately, for T-cell mitogenesis. We have briefly discussed Ca-dependent nuclear factors but await future experiments that should give a more detailed picture of the relationships between nuclear factors and cytoplasmic Ca-binding proteins. We thank John Imboden, John Ransom, and Mollie Meffert for sharing unpublished work and for valuable comments on earlier versions of the manuscript. The work in the authors’ laboratory was supported by American Cancer Society Grant CO313. P. Gardner is a recipient of the Faculty Scholar Award of the Burroughs Wellcome Foundation. Address for reprint requests: B. A. Premack, Falk Cardiovascular Research Building, Stanford University Medical Center, Stanford, CA 943053246.

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Signal transduction by P2-purinergic receptors for extracellular ATP.

Receptor-effector coupling by G proteins: implications for normal and abnormal signal transduction.

Suppression by staurosporine of Ca(2+)-mobilization triggered by ligation of antigen-specific receptors on t and B lymphocytes. An essential role of protein tyrosine kinase in the signal transduction.

Signal transduction mechanism of biased ligands at histamine H2 receptors.

Gastric chief cells: receptors and signal-transduction mechanisms.

Signal transduction for chemotaxis and haptotaxis by matrix molecules in tumor cells.

Signal transduction mechanisms of Ia induction in B cells by interleukin 4 and immunoglobulin receptors.

Signal transduction by guanylyl cyclases.

Exercise-induced signal transduction and gene regulation in skeletal muscle.

PRDM Proteins: Molecular Mechanisms in Signal Transduction and Transcriptional Regulation.

Regulation of autophagy by amino acids and MTOR-dependent signal transduction.

Signal transduction by T- and B-cell antigen receptors: converging structures and concepts.

Nerve growth factor mediates signal transduction through trk homodimer receptors.

Signal transduction and protein phosphorylation in the regulation of cellular metabolism by insulin.

Signal transduction by steroid hormones: nuclear localization is differentially regulated in estrogen and glucocorticoid receptors.

Regulation of Early Steps of GPVI Signal Transduction by Phosphatases: A Systems Biology Approach.

Signal transduction.