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Immunity, Vol. 7, 315–324, September, 1997, Copyright 1997 by Cell Press The CD38 Differentiation Review Marker: New Insight into Its Ectoenzymatic Activity and Its Role as a Signal Transducer

George Shubinsky and Michael Schlesinger* expression of p190, a high-molecular-weight oligomeric The Paul Ehrlich Center form of CD38 (Umar et al., 1996). The short cytoplasmic for the Study of Normal and Leukemic WBC domain of CD38 contains no known motifs (Src homol- The Hubert H. Humphrey Center ogy domain 2 or 3 [SH2 or SH3], antigen receptor activa- for Experimental Medicine and Cancer Research tion [ARAM], or pleckstrin homology [PH]) that could The Hebrew University—Hadassah Medical School mediate interactions with other signaling and Jerusalem 91120 seems to have no enzymatic activity. The cytoplasmic Israel portion of both murine and rat CD38 contains one tyro- sine residue that is not conserved in human CD38. The intracellular part of CD38 contains two conserved serine During the past two decades significant progress was residues within consensus sites recognized by cyclic made in understanding the molecular mechanisms of guanosine monophosphate (cGMP)–dependent intracellular signaling that control the growth and devel- kinases (Figure 1). cGMP-dependent serine/threonine opment of . Numerous cell surface recep- kinases in sea urchin eggs modulate the activity of ADP- tors were shown to trigger the phosphorylation of intra- ribosyl cyclase, an that displays a functional cellular proteins either via their intrinsic cytoplasmic homology to CD38 protein (Lund et al., 1996). The cyto- kinase domain or via associated intracellular protein ty- plasmic tail of CD38 might therefore serve as a regula- rosine kinases (PTKs). Protein phosphorylation plays a tory subunit of the CD38 ectoenzyme rather than as a key role in the initiation and propagation of various sig- tool for transduction of signals into the cell interior. naling cascades (Chan et al., 1994; Weiss and Littman, A number of CD38-related proteins, such as RT6, 1994; Kim et al., 1993a). The transmembrane signaling BST-1, and Yac-1, are GPI-anchored (Itoh et al., 1994; by surface receptors with ectoenzymatic activity and the Okazaki and Moss, 1996). In contrast, CD38 is a trans- mechanisms of their signaling function remain, however, membrane protein, and no GPI-anchored CD38 mole- poorly understood. Regulatory signals may be gener- cules have been found on the cell surface. The trans- ated by membrane-associated peptidases CD10/NEP, membrane domain of human CD38 includes 18 residues CD13/APN, CD26/DPPIV, and yBP-1. Glycosylphospha- of hydrophobic amino acids that are located between tidylinositol (GPI)–anchored ecto-5Ј nucleotidase (CD73), two short spacers that contain hydrophylic amino acids. which mediates lymphocyte-vascular adhesiveness, may Each of these spacers includes a triad of small amino also participate in the transduction of signals into the acids. It is noteworthy that analogous amino acid se- cell interior (Shipp and Look, 1993). quences determine GPI-cleavage/anchor sites and are A novel family of multifunctional membrane ectoen- found in the C terminus of primary translation products zymes has recently attracted attention. These ectoen- of all known GPI-anchored proteins (Englund, 1993). It zymes convert nicotinamide adenine dinucleotide (NAD) remains to be seen, therefore, whether this site is impor- into cyclic adenosine diphosphate ribose (cADP ribose), tant for any functional property of CD38 protein. hydrolyze cADP ribose to ADP ribose, and in addition Analysis of the extracellular part of CD38 indicates display NAD:(amino acid) protein–ADP-ribosyl trans- that it may function in attachment to the extracellular ferase activity. The CD38 surface protein is a member matrix. Thus, human CD38 proteins contain three puta- of this family and seems to play an important but not tive hyaluronate-binding motifs (HA motifs). Two of these yet fully understood role in the regulation of lymphocyte HA motifs are localized in the extracellular domain of function. CD38 proteins were detected in humans, mice, CD38 (amino acid positions 121–129 and 268–276), and and rats (Reinherz et al., 1980; Howard et al., 1993; one in the cytoplasmic part of the molecule. Murine Koguma et al., 1994). This family also includes the hu- CD38 contains two HA motifs (Nishina et al., 1994). In man, murine, and rat bone marrow stromal cell surface addition, four asparagine residues in the extracellular molecule BP3/BST-1 (Hirata et al., 1994; Itoh et al., 1994; part of CD38 serve as potential carbohydrate-binding Furuya et al., 1995); the rat differentiation marker sites. RT6 and its murine and human homologs; Yac-1 and The human CD38 molecule contains 12 conserved Yac-2 ADP-ribosyl transferases (Okazaki and Moss, cysteines, 11 of which are located in the extracellular 1996); ADP-ribosyl cyclase from sea molluscs (Tohgo domain. Purified human CD38 undergoes stable homo- et al., 1994); and other ADP-ribosyl transferases. oligomerization induced by thiol-reactive agents (Guida et al., 1995). It is tempting to speculate that thiol-depen- Functional Structure of CD38 dent interactions underlie the association of the extra- Human CD38 protein is a 45 kDa transmembrane glyco- cellular portion of CD38 with other receptors that may protein with a short N-terminal cytoplasmic part (21 be vital for the signaling function of CD38. Four cysteines amino acids) and a long extracellular domain (Jackson (Cys-119, Cys-160, Cys-173, and Cys-201) play an es- and Bell, 1990). The encoding human CD38 protein sential role in the cADP ribose synthetic and cADP ri- is located on 4p15 (Nakagawara et al., bose hydrolytic activity of CD38 (Tohgo et al., 1994). The 1995). Posttranslational modification may lead to the C-terminal part of CD38, including amino acid sequence 273–285 and particularly Cys-275, also contributes to * To whom correspondence should be addressed. the NAD glycohydrolytic activity of CD38 (Hoshino et Immunity 316

Figure 1. Functional Structure of the Human CD38 Molecule

al., 1997). Reducing agents such as dithiothreitol, 2-mer- on neurons of human brain (Mizuguchi et al., 1995), pan- captoethanol, or reduced glutathione inhibit the enzy- creatic cells (Kato et al., 1995), epithelial cells of the matic activity of CD38, suggesting that the disulfide prostate (Kramer et al., 1995), and other cells (cf. Mehta bonds are important for the catalytic activity of the CD38 et al., 1996). In the hematopoietic system, CD38 proteins proteins (Tohgo et al., 1994; Zocchi et al., 1995). Com- can be detected on , myeloid cells, T and B puted analysis of the structure of murine Rt6 and its lymphocytes, natural killer (NK) cells, follicular dendritic related proteins from rabbit, humans, and chickens cells, and even platelets (Alessio et al., 1990; Banche- showed that the amino acid sequence within the cata- reau and Rousset, 1992; Ramaschi et al., 1996). Dra- lytic domain and patterns of secondary structure motifs matic changes in the expression of CD38 correlate with predicted for different CD38-related NAD hydrolases the most critical events in the development of human were similar to those predicted for bacterial mono–ADP- hematopoietic cells. The early stage of maturation of ribosyl transferases (Koch-Nolte et al., 1996). In par- primitive CD34ϩ human hematopoietic progenitors into ticular, the conserved pair of amino acids Glu-146–Asp- committed bipotent erythroid/megakaryocytic (burst- 147 seems to endow ADP-ribosyl transferase activity to forming unit–erythroid/megakaryocytic), unipotent my- the CD38 protein (Grimaldi et al., 1995; Okazaki and eloid (colony-forming unit/granulocyte–), and Moss, 1996). lymphoid precursors is associated with the expression A number of leucines within the transmembrane and of CD38 (Terstappen et al., 1991; Debili et al., 1996; extracellular domains potentially form leucine zipper Oertel et al., 1996). Early T cell precursors that migrate motifs that can provide association of CD38 with other to the subcortical area of the thymus are CD38ϩ, proteins. Two dileucine (LL) motifs are located in the and likewise the majority of CD4ϩCD8ϩ double-positive middle of human CD38 proteins. One of these motifs cortical thymocytes express this antigen. Further matu- (Leu-149–Leu-150) is conserved for human, murine, and ration into either CD4ϩ or CD8ϩ single-positive medul- rat CD38. Intracellular targeting and internalization of lary thymocytes and peripheral T lymphocytes isaccom- different transmembrane proteins requires the LL motif panied by a significant decrease in CD38 expression within the C terminus of their cytoplasmic domains (Reinherz et al., 1980). Mature human T lymphocytes, (Aiken et al., 1994). The LL motif that is located within with low or undetectable levels of CD38, can be induced the extracellular part of 45 kDa monomeric CD38 is, of to express CD38 molecules by antigenic and mitogenic course, not accessible to intracellular targeting. How- stimuli (Hercend et al., 1981). Thus, the modulation of ever, high-molecular-weight oligomers of CD38 might CD38 on the T cell lineage strikingly coincides with the contain monomeric subunit(s) within the cell interior, ren- two most important events during T cell ontogenesis. dering the LL motif accessible for intracellular targeting. T cells lose their surface CD38 molecules during the acquisition of antigenic specificity and intrathymic negative selection (von Boehmer, 1992), while mature T Pattern of the Expression and Immunoregulatory cells gain expression of CD38 in response to various Activity of CD38 stimuli. The expression of CD38 declines in parallel with Although CD38 was initially discovered as a differentia- the differentiation of activated CD45RAϩ T cells into tion marker of T lymphocytes (T10), it is now clear that CD45ROϩ memory lymphocytes (Prince et al., 1992). various levels of CD38 can be discovered in a large The distribution of murine CD38 among T cell subsets number of cells and tissues. Thus, CD38 has been found differs from the distribution of its human homolog (Lund Review 317

et al., 1995b). In particular, only a minor subset of murine of CD38 antigen (Oliver et al., 1997), develop potent thymocytes displays CD38, and the expression of CD38 proliferative response to anti-CD38 antibodies and IL-4 seems to be the hallmark of the independent lineage of (Lund et al., 1995b). Murine spleen B lymphocytes T cell receptor (TCR) ␣␤ϩCD4ϪCD8Ϫ thymocytes, which undergo differentiation into IgM-producing cells upon possess unique regulatory functions (Bean et al., 1995). stimulation by anti-CD38 and IL-5 (Kikuchi et al., 1995). Similarly, modulation of CD38 in the human Ligation of CD38 on mature murine spleen B cells also lineage synchronizes with critical stages of B cell devel- prevents their apoptotic death elicited by either irradia- opment. CD38 is expressed on B cell progenitors (pro-B tion or dexamethasone (Yamashita et al., 1995). and pre-B cells) that display no surface immunoglobu- These and other studies indicate that CD38 is an im- lins (sIg) (Banchereau and Rousset, 1992). The ligation of portant regulatory molecule on human, murine, and rat CD38 on the surface of immature B cells by monoclonal lymphocytes. In addition to regulation of apoptotic antibodies (MAbs) inhibits their proliferation and elicits death in the B cell lineage, CD38 molecules transduce apoptosis (Kumagai et al., 1995; Kitanaka et al., 1996). mitogenic signals. Anti-CD38 MAbs elicit proliferation -induced down-modulation of CD38 on the of human thymocytes, mature T cells, and NK cells cells of an immature B cell lymphoma line was accompa- (Malavasi et al., 1994). In cultures of human peripheral nied by increased resistance to agents that elicited cell blood mononuclear cells, anti-CD38 MAbs induced the death (Shubinsky and Schlesinger, 1994). The matura- synthesis of mRNA of multiple (Ausiello et tion of B cell progenitors into sIg-positive antigen-reac- al., 1995). Exposure to anti-CD38 MAbs increased the tive B lymphocytes is accompanied by loss of CD38. expression of the ␣-chain of the IL-5 receptor in murine Mature sIgDϩMϩ naive B cells are characterized by a spleen cells (Kikuchi et al., 1995), while murine leukemic lack of or very low level of CD38. Such cells are predomi- B cells transfected with recombinant CD38 responded nant in the circulating B lymphocyte pool and in the to anti-CD38 MAbs by production of IL-2 (Lund et al., mantle zone of lymphoid follicles (Banchereau and 1996). Rousset, 1992; Lagresle et al., 1993; Pascual et al., Recently, a 120 kDa protein, Moon-1, was identified as a natural ligand for human CD38 (Deaglio et al., 1996). 1994). The activation of mature virgin B cells induces The strong expression of Moon-1 on vascular endothe- reexpression of CD38 (Stashenko et al., 1981; Galibert lial cells provides them with -type adhesiveness et al., 1996). In lymphoid follicles the expression of CD38 to CD38ϩ hematopoietic cells (Dianzani and Malavasi, distinguishes the subset of B lymphocytes that are local- 1995). Various levels of expression of Moon-1 were ized within germinal centers (GCs). CD38ϩ B cells in found on monocytes, platelets, NK cells, and even T GCs include a small number of progenitors of GCs and and B lymphocytes. Thus, CD38 may be involved in the a large number of activated cells. In the dark zone of homotypic and heterotypic adhesiveness of lympho- the GC, CD38ϩ centroblasts undergo intensive mitosis cytes and in the cooperative interactions between T and activation of the somatic mutation machinery. Divid- ϩ cells, B cells, and . Ligation of CD38 en- ing centroblasts give rise to CD38 centrocytes, which hances antigen-presenting function in monocytes (Lund accumulate within the zone of the GC. Isotype- et al., 1995b) possibly by increase of the expression switching recombination and substitution of the synthe- of major histocompatibility complex class II molecules sis of IgM by IgG, IgA or IgE seems to take place in (Santos-Argumedo et al., 1993). ϩ CD38 centrocytes (MacLennan, 1994; Pascual et al., A number of other CD38-related proteins are found 1994; Liu et al., 1996). Selection of B cells in GCs is on the surface of hematopoietic cells. RT6.1 and RT6.2 accompanied by apoptotic death of the majority of are two allelic proteins expressed on mature rat T lym- ϩ CD38 B lymphocytes. Study of the microenvironmental phocytes and on intraepithelial lymphocytes. RT6ϩ T ϩ factors that regulate the selection of CD38 B cells cells prevent the development of autoimmune diabetes showed that ligation of B cell receptors (BCRs) and in the BB rat (Waite et al., 1996). Rt6, a murine homolog CD40 proteins prevents apoptosis of CD38ϩ B cells and of rat RT6, is also a T cell–restricted membrane protein. enhances their proliferation (MacLennan, 1994; Galibert Defective expression of Rt6 seems to constitute part et al., 1996). Various combinations of soluble CD23, in- of the genetic disposition of the NZW mouse to the terleukin 1␣ (IL-1␣), IL-2, IL-4, IL-5, and IL-10 caused autoimmune lupus-like disease in (NZB ϫ NZW)F1 ani- terminal differentiation of activated B cells into plasma mals (Koch-Nolte et al., 1995). Human BST-1 and murine blasts (Banchereau and Rousset, 1992). This process is BP3/BST-1 proteins are widely expressed in different apparently accompanied by the continuous expression tissues and, particularly on bone marrow stromal cells, of CD38 (MacLennan, 1994). In contrast, the develop- reticular cells of lymphoid organs and endothelial cells ment of activated B lymphocytes into memory cells is (Itoh et al., 1994). In murine T-lineage cells, BST-1 serves characterized by loss of CD38 (Lagresle et al., 1993; as an early marker of immature TCR␣␤ϩ T cells, and Pascual et al., 1994). Direct interactions of CD38ϩ B MAbs against BST-1 enhanced the proliferative response cells with activated T lymphocytes seems to provide the of these cells by CD3 MAbs (Vicari et al., 1996). Two signals required to generate memory B cells (Lagresle additional NAD-dependent ADP-ribosyl transferases, et al., 1995). The exposure of mature human B cells Yac-1 and Yac-2, were recently cloned in murine lymphoid isolated from GCs to anti-CD38 MAbs also prevented cells (Okazaki and Moss, 1996). their death, although it had no effect on division of the cells (Zupo et al., 1994b). This finding indicates that Intracellular Signaling Triggered via CD38 CD38 proteins may be involved in the regulation of B Little is known about the biochemical reactions that cell differentiation within GC. This is also true for murine mediate CD38-triggered signaling. Ligation of CD38 in- GC B cells, which, while expressing only a low level creases the concentration of intracellular Ca2ϩ in murine Immunity 318

lymphocytes (Santos-Argumedo et al., 1993; Kirkham et and with Fc␥RIII (CD16) on NK cells (Funaro et al., 1993). al., 1994). Conflicting results were obtained in studies The signaling pattern induced in human B cells via CD38 of the effect of ligation of CD38 on tyrosine phosphoryla- is similar to signaling reactions in various lymphocyte tion of proteins in murine B cells. Following the ligation populations activated via the BCR complex, the CD19/ of CD38, no detectable tyrosine phosphorylation of pro- CD21 complex, Fc␥receptors, or the TCR/CD3 complex. teins was observed in cells of a mature B cell lymphoma One common denominator in the activation of T and B line that was transfected with the CD38 expression vec- cells is the role of p72syk PTK. In activated cells, phos- tor (Lund et al., 1996). In contrast, the ligation of CD38 phorylated p72syk is associated with PI3K and PLC␥ and on splenic murine Bcells significantly increased tyrosine seems to enhance their catalytic activity (Weiss and phosphorylation of proteins with molecular weights of Littman, 1994; Satterthwaite and Witte, 1996). c-Cbl pro- 170, 130, 90, and 80 kDa (Kirkham et al., 1994). One of tooncogene and other phosphoproteinswere also found these phosphoproteins was identified as p77 Bruton’s in complexes with p72syk (Panchamoorthy et al., 1996). tyrosine kinase (Btk) (Kikuchi et al., 1995). Exciting re- In contrast to CD38-mediated signaling, biochemical sults were obtained in CD45-null transgenic mice. The cascades induced via other membrane receptors in- knockout of CD45 protein tyrosine phosphatase (CD45- volve phosphorylation and activation of PTKs from the PTP) significantly reduced but did not abrogate the pro- Src family, which seem to act upstream to p72syk (Weiss liferation of B cells following CD38 cross-linking (Byth and Littman, 1994; Satterthwaite and Witte, 1996). et al., 1996). These data indicate that CD38-triggered Activation of murine splenocytes via CD38 results in signaling can involve CD45-PTP and/or target PTKs reg- the phosphorylation of p77btk, which is a characteristic ulated by CD45-PTP (Chan et al., 1994). trait of the signaling cascade triggered via BCRs. Mature Pathways involved in CD38 signaling were studied in B lymphocytes from X-linked immunodeficient (xid) mice different human cell lines. Ligation of CD38 on Jurkat are unresponsive to CD38 stimulation. Since the xid and MOLT-4 human T leukemic cell lines failed to elicit gene encodes Btk, this finding supports the conclusion tyrosine phosphorylation of intracellular proteins (Kon- that p77btk is involved in CD38-mediated signal transduc- tani et al., 1996a). In contrast, in cells of human immature tion (Santos-Argumedo et al., 1995; Yamashita et al, B cell lines anti-CD38 induced rapid and transient ty- 1995). Using B cell lines transfected with CD38, co- rosine phosphorylation of numerous proteins. These in- cross-linking of CD38 and the BCR lowered the thresh- clude the tyrosine kinase p72syk, the p120c-cbl protoonco- old for signaling through the BCR. The coexpression of gene, the p85 subunit of phosphatidyl inositol 3-kinase the BCR, and particularly of the cytoplasmic tails of (PI3K), phospholipase C␥ (PLC␥), and nonidentified pro- Ig␣ and Ig␤ domains, was required for CD38-mediated teins with molecular masses of 145 and 65 kDa. CD38- . In these cells the anti-CD38–induced mediated phosphorylation of p72syk was accompanied response was characterized by influx of extracellular by activation of this enzyme. Ligation of CD38 on imma- calcium but was not accomplished by tyrosine phos- ture human B cells did not elicit phosphorylation of p59fyn phorylation of cellular proteins. Thus, while the BCR and p53/p56lyn PTKs, Vav, and p25rasGAP, and in contrast complex must be present in B cells to render them re- to results in mice, had no effect on the phosphorylation sponsive to anti-CD38 induced signaling, the expression of p77btk (Silvennoinen et al., 1996). A similar pattern of of the BCR and CD38 may not suffice for CD38-induced phosphorylated proteins was found in cells of the HL- tyrosine phosphorylation, seen in murine splenic B cells 60 myeloid and THP-1 monocytic cell lines after expo- (Lund et al.,1995a, 1996). CD38 may use the cytoplasmic sure to anti-CD38 (Kontani et al., 1996b). The possibility tail of the BCR for intracellular signaling, but additional that p72syk PTK plays a role in CD38-mediated activation receptors and pathways, not related to the BCR, seem is supported by the finding that during lymphocyte de- to be required for the full signaling activity of CD38. In velopment, p72syk is expressed preferentially in cells that human B cells, CD38-induced phosphorylation of pro- presumably display high levels of CD38 (Chan et al., teins did not require at all the expression of either the 1994; Satterthwaite and Witte, 1996). CD38-mediated BCR or any of its components (Silvennoinen et al., 1996). phosphorylation of c-Cbl was accompanied by its asso- Moreover, coexpression of CD38 and the TCR/CD3 ciation with p85 PI3K and by a significant increase of complex by human T cell lines was not sufficient to the catalytic activity of PI3K. The activation of PI3K, in enable signal transduction via the CD38 molecule (Kon- turn, seems to play a critical role in the inhibition of cell tani et al., 1996a). These findings indicate that CD38- growth by ligation of CD38, since specific inhibitors of triggered signaling cannot be mediated solely via anti- PI3K effectively rescued normal and leukemic immature gen receptors that transmit signals through associated B cells from CD38-induced suppression (Kitanaka et Src kinases or other signaling molecules. It can therefore al., 1996). be suggested that CD38-mediated signals are propa- These findings indicate that the CD38-triggered sig- gated in different cells, either via distinct pathways or naling in human B cells involves activation of cellular via pathways shared with other receptors. PTK. A critical question that remains unresolved, how- ever, is how the CD38 molecule accomplishes this task even though it has no intrinsic capacity to generate Enzymatic Activity of CD38 and Related Proteins phosphorylation events. It can be envisaged that addi- CD38 is one of the first cell surface signaling molecules tional membrane molecules are involved in the transduc- shown to possess ectoenzymatic activity. It catalyzes tion of CD38-mediated signals. Indeed, surface CD38 the hydrolysis of NAD to ADP ribose and also generates comodulates with the TCR complex on the surface of cADP ribose (Howard et al., 1993; Takasawa et al., 1993). T cells, with the CD19/CD21 complex and sIg on B cells, One intracellular event elicited by the ligation of CD38 Review 319

can be satisfactorily explained by its ectoenzymatic ac- 40 kDa proteins were not biochemically characterized tivity. In murine B cells, anti-CD38 MAb induced a rela- in this study, it is tempting to speculate that the enzyme tively weak but prolonged influx of extracellular Ca2ϩ, is similar either to GPI-anchored RT6 or to BST-1 pro- which preceded the onset of cell division (Kirkham et teins, while the target 40 kDa protein is either identical al., 1994). cADP ribose generated by the enzymatic or similar to the ␣-subunit of GTP-binding (G) proteins. activity of CD38 is a strong inositol 3-phosphate–inde- pendent Ca2ϩ mobilizing agent, suggesting that modula- tion of the enzymatic activity of CD38 by its ligation Regulation of CD38 increases the influx of Ca2ϩ (Aarhus et al., 1995). Overex- The enzymatic activity of CD38 is affected by various pression of human CD38 seems to cause the accumula- chemical agents. Zinc ions stimulated the ADP-ribosyl tion of intracellular cADP ribose (Kato et al., 1995; Taka- cyclase activity of recombinant CD38, while dramatically hashi et al., 1995). The accumulation of intracellular inhibiting the NAD glycohydrolase activity of CD38 cADP ribose may in turn lead to the activation of ryano- (Kukimoto et al., 1996). Gangliosides inhibited glycohy- dine receptors and subsequently to the mobilization of drolase and ADP-ribosyl cyclase activities of CD38 Ca2ϩ from the intracellular store (Lee et al., 1994). CD38- (Yokoyama et al., 1996). Extensive dimerization of hu- mediated Ca2ϩ release from intracellular cADP ribose– man CD38 by reactive thiols diminished both the cyclase sensitive stores seems to play an important role in the and cADPR hydrolase activity of CD38 (Zocchi et al., activation of lymphocytes (Santos-Argumedo et al., 1995). The prolonged exposure of Farage, a human B 1993, 1995) and in the regulation of insulin secretion by cell lymphoma line, to IL-4 reduced the amount of CD38 glucose in CD38ϩ pancreatic ␤ cells (Kato et al., 1995). antigen expressed on the surface of cells and in cell The mechanisms underlying the accumulation of intra- lysates. No evidence was obtained for accelerated cellular cADP ribose remain unclear. Cytoplasmic ex- breakdown or shedding of CD38 molecules following pression or internalization of membrane CD38 might IL-4 treatment, nor for the accumulation of CD38 mole- increase the synthesis of cADP ribose in the cell interior. cules in the cell interior. Using various inhibitors and It is possible that extracellular cADP ribose may be activators of protein kinases, the mediatory role of ser- transported into the cytoplasm via the cell membrane ine/threonine kinases in IL-4–induced down-regulation and that CD38 may play a role in the cellular uptake of of CD38 was demonstrated in these experiments (Shu- cADP ribose, a role similar to that of ecto-NAD glycohy- binsky and Schlesinger, 1997). The exposure of CD38Ϫ drolase in the uptake of extracellular noncyclic ADP human tonsillar B lymphocytes to phorbol ester, which ribose (Kim et al., 1993b). may affect serine/threonine kinases, resulted in the ac- In addition to generating ADP ribose and cADP ribose, celerated expression of CD38 (Zupo et al., 1994a). CD38 may function as a mono–ADP-ribosyl transferase. A number of other observations indicate that serine/ Mono–ADP-ribosyl transferases catalyze posttransla- threonine protein kinases are recruited in IL-4–triggered tional modification of proteins in which the ADP ribose signaling. Cytokine binding to IL-4 receptors moiety of NAD is transferred to cysteine, arginine, or induced significant generation of diacylglycerol, an acti- histidine residues of target proteins. CD38 and CD38- vator of protein kinase C (PKC), and was associated related proteins share within their catalytic sites amino with translocation of PKC␣ to a nuclear fraction (Ho et acid sequences similar to those found in eukariotic al., 1994). IL-4 increased the activity of PI3K in a human mono–ADP-ribosyl transferases and in bacterial toxin cell line and elicited the translocation transferases (Okazaki and Moss, 1996). Recombinant of PKC␨ from the to the membrane fraction (Iki- murine CD38 was shown to elicit autoribosylation and to zawa et al., 1996). Serine/threonine kinase Raf-1 was ribosylate a number of different proteins at their cysteine required for IL-4–induced proliferation of myeloid and T residues (Grimaldi et al., 1995). ADP-arginine ribosyl cell lines (Muszynski et al., 1995). The IL-4 signaling transferase activity was detected in the rat T cell differ- cascade involves activation, by tyrosine phosphoryla- entiation antigen RT6 and in its murine homolog Rt6. tion, of janus family kinases, and of two additional sub- Ligation of RT6 by specific antibody induced its autori- strates, a signal transducing factor (STF-IL-4) and 4PS/ bosylation. Murine Rt6 protein can catalyze ADP ribo- IRS2. Jak kinases can phosphorylate 4PS/IRS2, sug- sylation of the light chains of immunoglobulins (Koch- gesting a pathway by which cytokine receptors induce Nolte et al., 1996). A number of observations indicate the phosphorylation of 4PS (Johnston et al., 1995; Pernis that ADP ribosylation may affect signal transduction. et al., 1995). The mechanisms linking these events with ADP ribosylation of low-molecular-weight Rho p21 mes- activation of serine/threoninekinases followed by dimin- senger protein decreased the tyrosine phosphorylation ished expression of CD38 has yet to be defined. of p43 mitogen-activated protein kinase, of p125 focal Transretinoic acid (TRA) increases the expression of adhesion kinase, and of other proteins, and inhibited CD38 in hematopoietic cells (Drach et al., 1994). TRA the phosphorylation and catalytic activity of PI3K (Ku- also stimulates the synthesis of cADP ribose in renal magai et al., 1993). In cytotoxic T lymphocytes, a GPI- epithelial cells (Beers et al., 1995), suggesting the possi- anchored membrane ectoenzyme ribosylated 40 kDa bility that retinoids may induce the expression of CD38 proteins associated with p56lck tyrosine kinase. ADP ri- or related proteins in different cells. The augmentary bosylation of p40 adapter proteins inhibited the kinase effect of TRA on the expression of CD38 is mediated activity of p56lck and, concomitantly, diminished prolifer- by nuclear retinoic acid receptor ␣ (RAR␣), which acts ation and cytotoxic activity of T cells activated by cross- as a ligand-inducible transcription factor (Drach et al., linking of either TCR or CD8 receptors (Wang et al., 1994). Activation of PKC by phorbol ester inhibited the 1996). Although the ADP-ribosyl transferase and target expresson of RAR␣ (Kumar et al., 1994). Moreover, Immunity 320

Figure 2. Hypothetical Model for the Signal- ing Activity of CD38

PKC␣ may reduce the DNA-binding capacity of RAR␣ various stages of cell development. For instance, coor- by the phosphorylation of RAR␣ molecules and diminu- dinated expression of CD38 and PC-1, a nucleotide tion of their ability to form homodimeric and heterodi- phosphodiesterase, might allow activated lymphocytes meric complexes (Tahayato et al., 1993). These data to reutilize NAD and nucleotides from dead cells (Deterre indicate that down-modulation of CD38 by activated et al., 1996). The expression of CD38 can also provide serine/threonine kinases may result from reduced pro- activated cells with ADP ribose, which is utilized by duction and impaired binding of RAR transcription factor poly–ADP-ribosyl transferases to repair extensive DNA to DNA. damage induced by apoptogenic agents. Recently, it was shown that cytoplasmic transgluta- The signaling function of CD38 may result from en- minase may convert low-molecular-weight CD38 into a hanced Ca2ϩ influx in activated cells, which in turn is p190 high-molecular-weight form (Umar et al., 1996). elicited by cADP ribose. An intriguing hypothesis is that The expression of transglutaminase was augmented by the immunoregulatory properties of CD38 molecules are TRA (Zhang et al., 1995), while the activation of serine/ based on their ability to ribosylate other signaling pro- threonine kinase, and particularly of PKC, seemed to teins (Figure 2). Various intracellular may serve down-regulate the transcription of the transglutaminase as targets for the regulatory modification by ADP-ribosyl gene (Liew and Yamanishi, 1992). The involvement of transferases (Okazaki and Moss, 1996). Hematopoietic activated serine/threonine kinases in the down-regula- and lymphoid cells may accordingly employ mecha- tion of both CD38 and transglutaminase suggests that nisms whereby membrane ADP-ribosyl transferases, the IL-4–induced modulation of CD38 may result in part and particularly CD38, are internalized and modify intra- from lower level of the expression of transglutaminase. cellular substrates. No evidence, however, was obtained so far for the internalization of CD38. Alternatively, CD38 and related proteins may exert their effect on other How CD38 Can Contribute to the Regulation membrane proteins. Recent experiments with TRA- of Lymphocyte Development treated HL-60 cells give rise to the hypothesis that G CD38 is a lymphocyte differentiation marker the expres- proteins from the Gs family may be involved in CD38 sion of which coincides with critical stages in lympho- signaling. Cholera toxin is known to ADP ribosylate the cyte development and with activation of T and B cells. ␣-subunit of Gs proteins. The exposure of HL-60 human The expression of CD38 is regulated by cytokines and myeloid cells to TRA decreased the ADP ribosylation of by other regulatory molecules. The immunoregulatory Gs proteins catalyzed by cholera toxin (de Cremoux et activity of CD38 seems to result from its signaling func- al., 1991). Treatment of HL-60 cells with TRA dramati- tion. Various surface proteins were shown to employ for cally increased the expression of CD38 protein (Drach their signaling function two interlinked properties. They et al., 1994). The decreased availability of ␣-subunits recognize and bind specific ligands and mediate the to ADP ribosylation by bacterial toxins might therefore phosphorylation of intracellular proteins either by intrin- result from TRA-induced expression of CD38, which in sic catalytic domains or by associated intracellular en- itself exerted ribosylation of proteins, preventing them zymes. The CD38 molecules and CD38-related proteins from further ribosylation by bacterial toxin. ADP ribo- are unique since they possess not only receptor and sylation of Gs proteins in turn can cause intracellular signaling functions but also are endowed with ectoenzy- signaling via the activation of adenylate cyclase (Kahn matic activity. The relation between these three proper- and Gilman, 1984). ties of CD38 remains to be clarified. One possibility is Recent findings indicate that G proteins are associ- that the enzymatic activity of CD38 is not a prerequisite ated with various PTKs via PH domains and glycolipid- for its signaling function and contributes differently in enriched membrane domains. Particularly, p72syk PTK is Review 321

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