[Frontiers in Bioscience 2, d207-221, May 1, 1997]

	[Frontiers in Bioscience 2, d207-221, May 1, 1997] Reprints PubMed CAVEAT LECTOR
	RECENT ADVANCES IN LYMPHOCYTE SIGNALING AND REGULATION Chun Kung and Matthew L. Thomas Department of Pathology, HHMI, Washington University, 660 S. Euclid Avenue, St. Louis, MO 63110 Received 4/4/97; Accepted 4/24/97; On-line 5/1/97 3. Protein tyrosine kinases The Src family of protein kinases contains nine family members: Src, Blk, Yes, Yrk, Fgr, Hck, Fyn, Lyn and Lck (1). The primary structure of the Src kinases can be subdivided into several interaction domains (Fig. 3). The amino-terminal region contains a Src homology 4 (SH4) domain for myristylation, palmitylation and interactions with acidic phospholipids. Adjoining the SH4 domain is a 50-80 amino acid stretch which acts as a putative cell surface protein binding region. This is followed in order by an SH3 domain and an SH2 domain, which bind to proline-rich sequences and tyrosine phosphorylated proteins, respectively. The kinase domain adjacent to the SH2 domain contains a tyrosine autophosphorylation site, that potentiates enzymatic activation. Another tyrosine phosphorylation site, located in the carboxyl-terminus, serves a negative regulatory function. Phosphorylation of these tyrosines regulates Src kinase activity. Figure 3. The domain structure of the Src protein tyrosine kinase family. The domain structure of the Src kinase family is shown, with the tyrosines in the autophosphorylation site and carboxyl-terminal domain indicated. The SH2, SH3, SH4 and kinase domains are shown. Nine members of the Src family are listed. T cells express three Src family members: Lck, Fyn and Yes. Lck interacts with the cytoplasmic tails of CD4 and CD8alpha, whereas Fyn associates with the cytoplasmic tail of CD3 chains and zeta chain (2,5,6). Upon TCR activation, Lck and Fyn initiate the phosphorylation of ITAMs on zeta chains, CD3eta, CD3gamma and CD3delta. In addition they are implicated in the phosphorylation and activation of ZAP-70 and/or Syk (6-8). B cells express Lyn, Lck, Fyn and Blk (1, 4). Similar to TCR activation, a cascade of phosphorylation events occurs upon BCR stimulation. The Src kinases are activated and presumably phosphorylate the ITAMs on Igalpha and Igbeta, which leads to the phosphorylation of Syk. Syk and ZAP-70 have similar primary structures that consist of two amino-terminal SH2 domains and a kinase domain linked by hinge regions (1,6,9). Antigen activation results in the recruitment of ZAP-70 and Syk to the TCR and BCR, respectively by interacting via their SH2 domains with the doubly phosphorylated ITAMs. Trans- and auto-phosphorylation by Src and/or ZAP-70/Syk increases kinase activity (6). 3.1 Signaling pathways involving protein tyrosine kinases Activation of the immune response in lymphocytes induces various cellular events such as cytoskeletal rearrangement, gene transcription and cell proliferation. The Ras/Rho family of GTPases are important in initiating these events. These GTPases convert extracellular stimuli into intracellular signals by regulating the activities of serine/threonine kinases, known as mitogen activated protein kinases (MAPK) (10). MAPK in turn controls gene expression important for many cellular functions, including cell growth and differentiation. The MAPK family can be subdivided into three subfamilies: extracellular signal regulated kinases (ERK), stress activated protein kinases (SAPK) or c-Jun N-terminal kinases (JNK) and p38 kinase (11). The Ras family controls ERK through the serine/threonine protein kinase, Raf. The Rho family of small GTPases is responsible for regulating stress activated protein kinases, SAPK or JNK. Along with the Ras and Rho pathways, mobilization of intracellular Ca²⁺ is also activated during the activation of the immune response. This is achieved through the activation of phospholipase C (PLC)-gamma1 and PLC-gamma2. IP₃ and diacylglycerol are formed from the hydrolysis of phosphoinositol bisphosphate (PIP₂) by PLCgamma. In due course, intracellular Ca²⁺ mobilization results from IP₃ stimulation. Concurrently, diacylglycerol activates various isoforms of protein kinase C, which gives rise to serine/threonine phosphorylation of selective substrates (3). To transduce the signal from the membrane receptors to any of the downstream pathways requires intermediary molecules known as adapter proteins. Many of these adapter proteins contain SH2 and/or SH3 domains making them fully capable of binding and recruiting numerous proteins (2). Upon stimulation of the immune response, adapter proteins are tyrosine phosphorylated conceivably by Src kinases or ZAP-70/Syk (1,2). In the Ras pathway, putative SH2 adapter proteins such as Vav and Shc are tyrosine phosphorylated. The 46-52kDa protein, Shc binds to the adapter protein Grb2, which is constitutively associated to Sos, the guanine nucleotide exchange factor for Ras. Shc can also interact directly with the phosphorylated zeta chains of the TCR, thereby coupling TCR stimulation to the Ras pathway (12). Vav, a 95kDa guanine nucleotide exchange factor, is implicated in Rho and Ras pathways. Moreover in the Ca²⁺ pathway, tyrosine phosphorylation of PLC-gamma1 and PLC-gamma2 leads to enhanced lipase activity, resulting in increased formation of IP₃ and diacylglycerol from PIP₂, and consequently elevated intracellular Ca²⁺ concentration. ZAP-70 and Syk are involved in phosphorylating components necessary in initiating the Ras signaling pathway (1,2). Vav interacts with and is phosphorylated by Syk and ZAP-70 in activated B and T cells, respectively (13). Syk interaction with Vav is dependent upon a catalytically active Syk, the SH2 domain of Vav and the phosphorylated tyrosine residues in the linker region of Syk. In addition, T cells transfected with Syk and Vav results in increased activation of the nuclear factor of activated T cells (NFAT). Hence Syk and ZAP-70 via Vav couple the antigen receptor to the Ras signaling pathway. Additional evidence associates Vav phosphorylation in COS-7 cells with the activation of JNK and the engagement of Rac-1 activity (14,15). Rac-1 is a member of the Rho family of GTPases, which are responsible for regulating JNK. Furthermore Vav signaling through JNK is down regulated in Rac-1 dominant negative mutants, supporting a relationship between Rac-1 and Vav (15). Other studies show Vav and Rac-1 involvement in mitogenesis, the Ras pathway and NFAT related T cell responses (16-19). Since Vav has SH2 and SH3 domains, it is possible that it can recruit proteins involved in both Ras and Rho pathways and couple them to antigen receptor-associated tyrosine kinases. Further clarification of the Vav signaling pathway is required. As noted above, Shc and Grb2 are linked in the Ras pathway. Upon BCR stimulation, Shc is tyrosine phosphorylated. B cells deficient in Lyn or Syk display a decrease in Shc phosphorylation and Grb2-Shc association is reduced (20). These findings suggest that Shc phosphorylation is dependent on Lyn and/or Syk. Immunoprecipitation studies of Shc in B cells and co-transfected COS-1 cells show that Syk associates with and phosphorylates Shc. Altogether these results point to an involvement for Syk and Lyn in coupling the antigen receptor to the Ras pathway via Shc and Grb2. SLP-76, a 76kDa SH2 adapter protein that is tyrosine phosphorylated during T cell activation, is preferentially phosphorylated by ZAP-70, and interacts with Grb2 and PLC-gamma1 (21). Over expression of SLP-76 in T cells results in a hyperactive receptor, whereas expression of a mutant SLP-76 that cannot be phosphorylated diminishes receptor function. Furthermore, decreased phosphorylation of SLP-76 is found in T cells expressing a catalytically inactive ZAP-70. These findings imply a role for SLP-76 in antigen receptor signaling which appears to require ZAP-70, and may involve the Ras and Ca²⁺ pathways. Fyn and Lyn phosphorylate c-Cbl, a 116 kDa product of a proto-oncogene, which binds to Grb2 and the p85 subunit of phosphatidylinositol 3'-kinase (PI 3-kinase) (22-25). Fyn, through its SH2 domain, associates with and tyrosine phosphorylates c-Cbl in activated T cells and IL-3 stimulated murine myeloid cells (23-25). Also in activated B cells, the phosphorylation of c-Cbl is dependent on interactions with Lyn but not Syk (25). However binding assays using GST-fusion proteins demonstrate that the SH2 domain of Fyn can bind to non-phosphorylated c-Cbl, suggesting that Fyn may also interact with c-Cbl in a phosphotyrosine-independent manner (24). These results indicate that Fyn probably binds to c-Cbl regardless of it phosphorylation state. Although the biological function of c-Cbl is not well defined, recent findings suggest that it may play a role in the Ras signaling pathway. Antigen stimulation causes Crk, a SH2/SH3 adapter protein, to bind to tyrosine phosphorylated c-Cbl, and results in the presence of c-Cbl-Crk complexes in the membrane particulate fraction (26). These Crk complexes are formed via interactions with SH2 domains of Crk. The SH3 domains of Crk preferentially bind C3G, a nucleotide exchange factor involved in the activation of Rap, the negative regulator of the Ras pathway. These results imply that Crk proteins may be responsible for co-localizing C3G to Rap at the membrane surface. As a result, Lyn or Fyn phosphorylates c-Cbl which in turn interacts with Crk, thereby engaging Rap mediated down regulation of Ras. Ca²⁺ mobilization requires PLC-gamma action on PIP₂ to form IP₃ However, studies on B cells deficient in tyrosine kinase show that there are possibly two pathways leading to Ca²⁺ mobilization (27). With activated B cells lacking Syk, PLC-gamma2 is not phosphorylated and, IP₃ production and Ca²⁺ mobilization are absent (27). Correspondingly, activated B cells that lack Bruton's tyrosine kinase (Btk) exhibit similar characteristics (28). In contrast, for BCR activation in B cells that lack Lyn, IP₃ generation remains unaffected and a slow Ca²⁺ mobilization occurs (27). Immuno-precipitation studies on activated B cells show that Syk is associated with PLC-gamma1 (29). Collectively, these findings point to tyrosine kinases directing two mechanisms for intracellular Ca²⁺ production. Syk regulates Ca²⁺ mobilization through PLC-gamma and IP₃ production, whereas Lyn affects Ca²⁺ mobilization through a different route. 3.2 Regulation of protein tyrosine kinase activity The activity of Src kinases is regulated by phosphorylation and dephosphorylation of specific tyrosines (1). Src kinases contain an autophosphorylation site within the kinase domain that serves to potentiate kinase activation. The carboxyl-terminal negative regulatory tyrosine when phosphorylated, interacts intramolecularly with the SH2 domain thereby decreasing kinase activity (30-32). The crystal structures of c-Src and Hck indicate that Src kinase inactivation resulting from these intramolecular interactions arises from a conformational change in the molecule (31,32). The family of non-receptor protein tyrosine kinases, which consist of Csk and Ntk, have been shown to phosphorylate the negative regulatory domain tyrosine, thereby decreasing Src kinase activity (33,34). Studies using Csk/Ntk deficient cells suggest that Csk/Ntk are required for inactivating Src kinases. Fyn and Lyn in Csk-deficient mice are constitutively activated and exhibit increased phosphorylation, suggesting that Csk is required to repress tyrosine kinase activity (35,36). Csk cannot only phosphorylate Lck and Fyn but has also been shown to phosphorylate CD45, and thus increase phosphatase activity (37). Apart from the negative regulation by Csk/Ntk, Src kinases are positively regulated by the protein tyrosine phosphatase, CD45 which has been shown to dephosphorylate the negative regulatory domain, thus increasing the kinase activity necessary for TCR activation (1). However, CD45 may also negatively regulate Src kinases by dephosphorylating the autophosphorylation site on Src kinases (38). Yac-1 T cells deficient in CD45 exhibit Lck hyperphosphorylated at both the autophosphorylation site and negative regulatory domain, but to a higher degree at the latter. In addition, the phosphatase domain of CD45 can dephosphorylate the autophosphorylation site of active Lck in vitro. All in all, these results point to a role for CD45 in negatively and positively regulating Lck activity. It is also possible that Lck may be regulated by sequestration as an inactivated pool within a glycolipid enriched membrane domain (39). Lck within the glycolipid enriched membrane domain has its negative regulatory domain in a hyperphosphorylated state. It is postulated that this results from the absence of CD45 which is excluded by the glycolipid enriched membrane domain. Another protein tyrosine phosphatase that is important in the negative regulation of protein tyrosine kinase activity is SHP-1. In T cells, SHP-1 interacts through its SH2 domains with ZAP-70 and dephosphorylates ZAP-70 (40). Thymocytes from motheaten (me) mice, which have a deficiency in SHP-1, exhibit elevated tyrosine phosphorylation after TCR stimulation due to increased activation of Src kinases (41). This implies that SHP-1 may also dephosphorylate members of the Src kinase family. In B cells, SHP-1 binds to CD22 and FcgammaRIIB to negatively regulate BCR signaling (42,43). Altogether, SHP-1 is a vital negative regulator of antigen receptor mediated signaling in both B cells and T cells. 3.3 Lymphocyte development Thymocyte development occurs as a series of selection stages, where only those meeting a defined criteria pass into mature lymphocytes (44). Immature thymocytes begin as multipotent cells which do not express CD4 or CD8, and as such are designated double negative (DN) (CD4^- CD8^-). Upon expansion, alphabeta gene rearrangement and expression of the TCR, the thymocytes express TCR, CD4 and CD8, and enter the double positive (DP) stage. At this point they are designated CD4⁺ CD8⁺ TCR^low. The thymocytes then undergo selection for self recognition which is dependent on the avidity of the TCR for antigen presented by MHC molecules within the thymus. During the final development stage, DP thymocytes become single positive (SP) in either CD4 or CD8 with specificity for MHC class II or class I, respectively. Those that survive have undergone positive selection and are ready for maturation. In the bone marrow, the process of B cell development comprises several stages where the preB cells are selected to develop into long-lived mature B cells (45). At the same time the B cells are screened for tolerance against autoantigens. Initially, the pro-B/preB cells undergo rearrangement in their Heavy-chain gene loci. Those that contain the proper in frame gene rearrangement undergo Light-chain gene rearrangement to form immature B cells. After further secondary Light-chain gene rearrangement and selection against autoantigens, immature B cells exit the bone marrow to become mature B cells. To examine the biological relevance of the Src protein tyrosine kinases in lymphocyte signaling and maturation, mice expressing mutations in a Src kinase or a deficiency in Src kinases are analysed. In mice deficient in Lck, thymocytes are generally blocked at the DP stage, but a small number of single positives are found (46). However, those that do develop exhibit only partial signaling in response to TCR stimulation. In contrast, thymocytes from mice deficient in Fyn are able to mature but SP thymocytes are hyporesponsive to TCR ligation (47,48). Thus unlike Lck, Fyn contributes to TCR signaling but is not critical for thymopoiesis. For mice deficient in both Lck and Fyn, thymocyte development is blocked at the DN stage and no mature alphabeta T cells are observed in the peripheral lymphoid organs (49,50). However, there are normal numbers of natural killer cells which have normal cytolytic activity (50). In all, these results point to a possible redundancy effect of Fyn for Lck in T cell development. Expression of a gain of function Fyn (Y528F) transgene in lck^-/- mice restores DP thymocyte development and enhances the DP to SP transition of thymocytes, further supporting a redundancy between Lck and Fyn (49). However, the Fyn transgene only marginally affects RAG1^-/- mice whereas expression of a constitutively active Lck restores normal DP thymocyte development (49,51). In addition, a dominant negative Fyn does not affect T cell development while a dominant negative Lck abrogates DP thymocyte development (52-54). Taken together, these results show that Fyn and Lck do not have identical functions. Nonetheless, Fyn can transduce signals required for positive selection of DP thymocytes and can subserve Lck in some aspects of T cell development. It is apparent that Lck is required for positive selection of thymocytes. In support of this, a catalytically inactive Lck expressed in DP thymocytes unambiguously blocks positive selection (55). However, other defects present in DP thymocytes deficient in Lck can also influence positive selection. It is noted that thymocytes deficient in Lck are blocked at the DP stage and have decreased CD4 dependent signaling but relatively unaffected TCR signaling (41). In addition, these thymocytes display decreased CD5 expression and increased TCR expression. CD5, an accessory signaling molecule, is important since it negatively regulates TCR and BCR signaling, and is required for the positive selection of thymocytes (56,57). Similarly, CD4/TCR co-aggregation is essential for ZAP-70 activation in DP thymocytes (58). Hence, altered CD5 or TCR expression can affect the positive selection of DP thymocytes deficient in Lck. Nevertheless expression of the Fyn transgene (Y528F) normalizes CD5 and TCR expression (49). Lyn is critical for BCR signal transduction and this is supported by the findings from mice deficient in Lyn (59). These mice exhibit decreased numbers of B cells, which may result from a failure in B cell expansion. It is noteworthy that the lymph nodes of mice deficient in Lyn have deformed germinal centers. B cell function is also impaired as shown by their poor response to lipopolysaccharide stimulation. However, their response to CD40 stimulation is normal. In addition, these mice exhibit elevated levels of serum IgM due to increased numbers of plasma cells producing IgM, circulating autoreactive antibodies and symptoms characteristic of an autoimmune disease. Therefore, these results demonstrate that Lyn is vital for proficient B cell signaling and establish Lyn dependence in B cell selection. ZAP-70 and Syk are also important for lymphocyte development. A portion of humans or mice with severe combined immunodeficiency (SCID), display a defective ZAP-70 or a deficiency in ZAP-70 (60-62). The normal number of CD4⁺ cells are present but they are non-functional. No CD8⁺ thymocytes are present in the periphery. Mice deficient in ZAP-70 have a thymocyte block at CD4⁺ CD8⁺ TCR^low stage but can be rescued with human ZAP-70 (63). Deficient mice exhibit elevated numbers of normal DP thymocytes. Mice deficient in Syk exhibit normal thymopoiesis (64,65). Therefore ZAP-70 but not Syk is vital for thymocyte development. However, mice deficient in Syk have decreased numbers of mature B cells and signaling through the BCR is impaired (64-66). Furthermore, they display impaired gammadelta T cell development (67). ZAP-70 and Syk have similar structural and functional properties, which could imply a redundancy between these two kinases. However the different expression patterns of ZAP-70 and Syk may affect their roles (68). ZAP-70 is not expressed in peripheral B cells but is restricted to T cells, natural killer cells and thymocytes. However, Syk is expressed in thymocytes and predominantly in peripheral B cells but down regulated in peripheral T cells. Recent findings attempt to address the functional overlap between ZAP-70 and Syk. In B cells deficient in Syk, the BCR is non-functional. Nonetheless, expression of ZAP-70 in these cells reconstitutes BCR signaling (69). Functionally competent SH2 and catalytic domains of ZAP-70 are essential for full BCR activity. ZAP-70, like Syk, binds to the phosphorylated Igalpha and Igbeta subunits, with affinities similar to their interactions with the CD3eta subunit. Therefore under these conditions, ZAP-70 can substitute for Syk in its role in BCR signal transduction.

[Frontiers in Bioscience 2, d207-221, May 1, 1997]
Reprints
PubMed
CAVEAT LECTOR

RECENT ADVANCES IN LYMPHOCYTE SIGNALING AND REGULATION

Chun Kung and Matthew L. Thomas

Department of Pathology, HHMI, Washington University, 660 S. Euclid Avenue, St. Louis, MO 63110

Received 4/4/97; Accepted 4/24/97; On-line 5/1/97

3. Protein tyrosine kinases

The Src family of protein kinases contains nine family members: Src, Blk, Yes, Yrk, Fgr, Hck, Fyn, Lyn and Lck (1). The primary structure of the Src kinases can be subdivided into several interaction domains (Fig. 3). The amino-terminal region contains a Src homology 4 (SH4) domain for myristylation, palmitylation and interactions with acidic phospholipids. Adjoining the SH4 domain is a 50-80 amino acid stretch which acts as a putative cell surface protein binding region. This is followed in order by an SH3 domain and an SH2 domain, which bind to proline-rich sequences and tyrosine phosphorylated proteins, respectively. The kinase domain adjacent to the SH2 domain contains a tyrosine autophosphorylation site, that potentiates enzymatic activation. Another tyrosine phosphorylation site, located in the carboxyl-terminus, serves a negative regulatory function. Phosphorylation of these tyrosines regulates Src kinase activity.

Figure 3. The domain structure of the Src protein tyrosine kinase family.

The domain structure of the Src kinase family is shown, with the tyrosines in the autophosphorylation site and carboxyl-terminal domain indicated. The SH2, SH3, SH4 and kinase domains are shown. Nine members of the Src family are listed.

T cells express three Src family members: Lck, Fyn and Yes. Lck interacts with the cytoplasmic tails of CD4 and CD8alpha, whereas Fyn associates with the cytoplasmic tail of CD3 chains and zeta chain (2,5,6). Upon TCR activation, Lck and Fyn initiate the phosphorylation of ITAMs on zeta chains, CD3eta, CD3gamma and CD3delta. In addition they are implicated in the phosphorylation and activation of ZAP-70 and/or Syk (6-8).

B cells express Lyn, Lck, Fyn and Blk (1, 4). Similar to TCR activation, a cascade of phosphorylation events occurs upon BCR stimulation. The Src kinases are activated and presumably phosphorylate the ITAMs on Igalpha and Igbeta, which leads to the phosphorylation of Syk.

Syk and ZAP-70 have similar primary structures that consist of two amino-terminal SH2 domains and a kinase domain linked by hinge regions (1,6,9). Antigen activation results in the recruitment of ZAP-70 and Syk to the TCR and BCR, respectively by interacting via their SH2 domains with the doubly phosphorylated ITAMs. Trans- and auto-phosphorylation by Src and/or ZAP-70/Syk increases kinase activity (6).

3.1 Signaling pathways involving protein tyrosine kinases

Activation of the immune response in lymphocytes induces various cellular events such as cytoskeletal rearrangement, gene transcription and cell proliferation. The Ras/Rho family of GTPases are important in initiating these events. These GTPases convert extracellular stimuli into intracellular signals by regulating the activities of serine/threonine kinases, known as mitogen activated protein kinases (MAPK) (10). MAPK in turn controls gene expression important for many cellular functions, including cell growth and differentiation. The MAPK family can be subdivided into three subfamilies: extracellular signal regulated kinases (ERK), stress activated protein kinases (SAPK) or c-Jun N-terminal kinases (JNK) and p38 kinase (11). The Ras family controls ERK through the serine/threonine protein kinase, Raf. The Rho family of small GTPases is responsible for regulating stress activated protein kinases, SAPK or JNK. Along with the Ras and Rho pathways, mobilization of intracellular Ca²⁺ is also activated during the activation of the immune response. This is achieved through the activation of phospholipase C (PLC)-gamma1 and PLC-gamma2. IP₃ and diacylglycerol are formed from the hydrolysis of phosphoinositol bisphosphate (PIP₂) by PLCgamma. In due course, intracellular Ca²⁺ mobilization results from IP₃ stimulation. Concurrently, diacylglycerol activates various isoforms of protein kinase C, which gives rise to serine/threonine phosphorylation of selective substrates (3).

To transduce the signal from the membrane receptors to any of the downstream pathways requires intermediary molecules known as adapter proteins. Many of these adapter proteins contain SH2 and/or SH3 domains making them fully capable of binding and recruiting numerous proteins (2). Upon stimulation of the immune response, adapter proteins are tyrosine phosphorylated conceivably by Src kinases or ZAP-70/Syk (1,2). In the Ras pathway, putative SH2 adapter proteins such as Vav and Shc are tyrosine phosphorylated. The 46-52kDa protein, Shc binds to the adapter protein Grb2, which is constitutively associated to Sos, the guanine nucleotide exchange factor for Ras. Shc can also interact directly with the phosphorylated zeta chains of the TCR, thereby coupling TCR stimulation to the Ras pathway (12). Vav, a 95kDa guanine nucleotide exchange factor, is implicated in Rho and Ras pathways. Moreover in the Ca²⁺ pathway, tyrosine phosphorylation of PLC-gamma1 and PLC-gamma2 leads to enhanced lipase activity, resulting in increased formation of IP₃ and diacylglycerol from PIP₂, and consequently elevated intracellular Ca²⁺ concentration.

ZAP-70 and Syk are involved in phosphorylating components necessary in initiating the Ras signaling pathway (1,2). Vav interacts with and is phosphorylated by Syk and ZAP-70 in activated B and T cells, respectively (13). Syk interaction with Vav is dependent upon a catalytically active Syk, the SH2 domain of Vav and the phosphorylated tyrosine residues in the linker region of Syk. In addition, T cells transfected with Syk and Vav results in increased activation of the nuclear factor of activated T cells (NFAT). Hence Syk and ZAP-70 via Vav couple the antigen receptor to the Ras signaling pathway.

Additional evidence associates Vav phosphorylation in COS-7 cells with the activation of JNK and the engagement of Rac-1 activity (14,15). Rac-1 is a member of the Rho family of GTPases, which are responsible for regulating JNK. Furthermore Vav signaling through JNK is down regulated in Rac-1 dominant negative mutants, supporting a relationship between Rac-1 and Vav (15). Other studies show Vav and Rac-1 involvement in mitogenesis, the Ras pathway and NFAT related T cell responses (16-19). Since Vav has SH2 and SH3 domains, it is possible that it can recruit proteins involved in both Ras and Rho pathways and couple them to antigen receptor-associated tyrosine kinases. Further clarification of the Vav signaling pathway is required.

As noted above, Shc and Grb2 are linked in the Ras pathway. Upon BCR stimulation, Shc is tyrosine phosphorylated. B cells deficient in Lyn or Syk display a decrease in Shc phosphorylation and Grb2-Shc association is reduced (20). These findings suggest that Shc phosphorylation is dependent on Lyn and/or Syk. Immunoprecipitation studies of Shc in B cells and co-transfected COS-1 cells show that Syk associates with and phosphorylates Shc. Altogether these results point to an involvement for Syk and Lyn in coupling the antigen receptor to the Ras pathway via Shc and Grb2.

SLP-76, a 76kDa SH2 adapter protein that is tyrosine phosphorylated during T cell activation, is preferentially phosphorylated by ZAP-70, and interacts with Grb2 and PLC-gamma1 (21). Over expression of SLP-76 in T cells results in a hyperactive receptor, whereas expression of a mutant SLP-76 that cannot be phosphorylated diminishes receptor function. Furthermore, decreased phosphorylation of SLP-76 is found in T cells expressing a catalytically inactive ZAP-70. These findings imply a role for SLP-76 in antigen receptor signaling which appears to require ZAP-70, and may involve the Ras and Ca²⁺ pathways.

Fyn and Lyn phosphorylate c-Cbl, a 116 kDa product of a proto-oncogene, which binds to Grb2 and the p85 subunit of phosphatidylinositol 3'-kinase (PI 3-kinase) (22-25). Fyn, through its SH2 domain, associates with and tyrosine phosphorylates c-Cbl in activated T cells and IL-3 stimulated murine myeloid cells (23-25). Also in activated B cells, the phosphorylation of c-Cbl is dependent on interactions with Lyn but not Syk (25). However binding assays using GST-fusion proteins demonstrate that the SH2 domain of Fyn can bind to non-phosphorylated c-Cbl, suggesting that Fyn may also interact with c-Cbl in a phosphotyrosine-independent manner (24). These results indicate that Fyn probably binds to c-Cbl regardless of it phosphorylation state.

Although the biological function of c-Cbl is not well defined, recent findings suggest that it may play a role in the Ras signaling pathway. Antigen stimulation causes Crk, a SH2/SH3 adapter protein, to bind to tyrosine phosphorylated c-Cbl, and results in the presence of c-Cbl-Crk complexes in the membrane particulate fraction (26). These Crk complexes are formed via interactions with SH2 domains of Crk. The SH3 domains of Crk preferentially bind C3G, a nucleotide exchange factor involved in the activation of Rap, the negative regulator of the Ras pathway. These results imply that Crk proteins may be responsible for co-localizing C3G to Rap at the membrane surface. As a result, Lyn or Fyn phosphorylates c-Cbl which in turn interacts with Crk, thereby engaging Rap mediated down regulation of Ras.

Ca²⁺ mobilization requires PLC-gamma action on PIP₂ to form IP₃ However, studies on B cells deficient in tyrosine kinase show that there are possibly two pathways leading to Ca²⁺ mobilization (27). With activated B cells lacking Syk, PLC-gamma2 is not phosphorylated and, IP₃ production and Ca²⁺ mobilization are absent (27). Correspondingly, activated B cells that lack Bruton's tyrosine kinase (Btk) exhibit similar characteristics (28). In contrast, for BCR activation in B cells that lack Lyn, IP₃ generation remains unaffected and a slow Ca²⁺ mobilization occurs (27). Immuno-precipitation studies on activated B cells show that Syk is associated with PLC-gamma1 (29). Collectively, these findings point to tyrosine kinases directing two mechanisms for intracellular Ca²⁺ production. Syk regulates Ca²⁺ mobilization through PLC-gamma and IP₃ production, whereas Lyn affects Ca²⁺ mobilization through a different route.

3.2 Regulation of protein tyrosine kinase activity

The activity of Src kinases is regulated by phosphorylation and dephosphorylation of specific tyrosines (1). Src kinases contain an autophosphorylation site within the kinase domain that serves to potentiate kinase activation. The carboxyl-terminal negative regulatory tyrosine when phosphorylated, interacts intramolecularly with the SH2 domain thereby decreasing kinase activity (30-32). The crystal structures of c-Src and Hck indicate that Src kinase inactivation resulting from these intramolecular interactions arises from a conformational change in the molecule (31,32). The family of non-receptor protein tyrosine kinases, which consist of Csk and Ntk, have been shown to phosphorylate the negative regulatory domain tyrosine, thereby decreasing Src kinase activity (33,34). Studies using Csk/Ntk deficient cells suggest that Csk/Ntk are required for inactivating Src kinases. Fyn and Lyn in Csk-deficient mice are constitutively activated and exhibit increased phosphorylation, suggesting that Csk is required to repress tyrosine kinase activity (35,36). Csk cannot only phosphorylate Lck and Fyn but has also been shown to phosphorylate CD45, and thus increase phosphatase activity (37).

Apart from the negative regulation by Csk/Ntk, Src kinases are positively regulated by the protein tyrosine phosphatase, CD45 which has been shown to dephosphorylate the negative regulatory domain, thus increasing the kinase activity necessary for TCR activation (1). However, CD45 may also negatively regulate Src kinases by dephosphorylating the autophosphorylation site on Src kinases (38). Yac-1 T cells deficient in CD45 exhibit Lck hyperphosphorylated at both the autophosphorylation site and negative regulatory domain, but to a higher degree at the latter. In addition, the phosphatase domain of CD45 can dephosphorylate the autophosphorylation site of active Lck in vitro. All in all, these results point to a role for CD45 in negatively and positively regulating Lck activity.

It is also possible that Lck may be regulated by sequestration as an inactivated pool within a glycolipid enriched membrane domain (39). Lck within the glycolipid enriched membrane domain has its negative regulatory domain in a hyperphosphorylated state. It is postulated that this results from the absence of CD45 which is excluded by the glycolipid enriched membrane domain.

Another protein tyrosine phosphatase that is important in the negative regulation of protein tyrosine kinase activity is SHP-1. In T cells, SHP-1 interacts through its SH2 domains with ZAP-70 and dephosphorylates ZAP-70 (40). Thymocytes from motheaten (me) mice, which have a deficiency in SHP-1, exhibit elevated tyrosine phosphorylation after TCR stimulation due to increased activation of Src kinases (41). This implies that SHP-1 may also dephosphorylate members of the Src kinase family. In B cells, SHP-1 binds to CD22 and FcgammaRIIB to negatively regulate BCR signaling (42,43). Altogether, SHP-1 is a vital negative regulator of antigen receptor mediated signaling in both B cells and T cells.

3.3 Lymphocyte development

Thymocyte development occurs as a series of selection stages, where only those meeting a defined criteria pass into mature lymphocytes (44). Immature thymocytes begin as multipotent cells which do not express CD4 or CD8, and as such are designated double negative (DN) (CD4^- CD8^-). Upon expansion, alphabeta gene rearrangement and expression of the TCR, the thymocytes express TCR, CD4 and CD8, and enter the double positive (DP) stage. At this point they are designated CD4⁺ CD8⁺ TCR^low. The thymocytes then undergo selection for self recognition which is dependent on the avidity of the TCR for antigen presented by MHC molecules within the thymus. During the final development stage, DP thymocytes become single positive (SP) in either CD4 or CD8 with specificity for MHC class II or class I, respectively. Those that survive have undergone positive selection and are ready for maturation.

In the bone marrow, the process of B cell development comprises several stages where the preB cells are selected to develop into long-lived mature B cells (45). At the same time the B cells are screened for tolerance against autoantigens. Initially, the pro-B/preB cells undergo rearrangement in their Heavy-chain gene loci. Those that contain the proper in frame gene rearrangement undergo Light-chain gene rearrangement to form immature B cells. After further secondary Light-chain gene rearrangement and selection against autoantigens, immature B cells exit the bone marrow to become mature B cells.

To examine the biological relevance of the Src protein tyrosine kinases in lymphocyte signaling and maturation, mice expressing mutations in a Src kinase or a deficiency in Src kinases are analysed. In mice deficient in Lck, thymocytes are generally blocked at the DP stage, but a small number of single positives are found (46). However, those that do develop exhibit only partial signaling in response to TCR stimulation. In contrast, thymocytes from mice deficient in Fyn are able to mature but SP thymocytes are hyporesponsive to TCR ligation (47,48). Thus unlike Lck, Fyn contributes to TCR signaling but is not critical for thymopoiesis. For mice deficient in both Lck and Fyn, thymocyte development is blocked at the DN stage and no mature alphabeta T cells are observed in the peripheral lymphoid organs (49,50). However, there are normal numbers of natural killer cells which have normal cytolytic activity (50). In all, these results point to a possible redundancy effect of Fyn for Lck in T cell development.

Expression of a gain of function Fyn (Y528F) transgene in lck^-/- mice restores DP thymocyte development and enhances the DP to SP transition of thymocytes, further supporting a redundancy between Lck and Fyn (49). However, the Fyn transgene only marginally affects RAG1^-/- mice whereas expression of a constitutively active Lck restores normal DP thymocyte development (49,51). In addition, a dominant negative Fyn does not affect T cell development while a dominant negative Lck abrogates DP thymocyte development (52-54). Taken together, these results show that Fyn and Lck do not have identical functions. Nonetheless, Fyn can transduce signals required for positive selection of DP thymocytes and can subserve Lck in some aspects of T cell development.

It is apparent that Lck is required for positive selection of thymocytes. In support of this, a catalytically inactive Lck expressed in DP thymocytes unambiguously blocks positive selection (55). However, other defects present in DP thymocytes deficient in Lck can also influence positive selection. It is noted that thymocytes deficient in Lck are blocked at the DP stage and have decreased CD4 dependent signaling but relatively unaffected TCR signaling (41). In addition, these thymocytes display decreased CD5 expression and increased TCR expression. CD5, an accessory signaling molecule, is important since it negatively regulates TCR and BCR signaling, and is required for the positive selection of thymocytes (56,57). Similarly, CD4/TCR co-aggregation is essential for ZAP-70 activation in DP thymocytes (58). Hence, altered CD5 or TCR expression can affect the positive selection of DP thymocytes deficient in Lck. Nevertheless expression of the Fyn transgene (Y528F) normalizes CD5 and TCR expression (49).

Lyn is critical for BCR signal transduction and this is supported by the findings from mice deficient in Lyn (59). These mice exhibit decreased numbers of B cells, which may result from a failure in B cell expansion. It is noteworthy that the lymph nodes of mice deficient in Lyn have deformed germinal centers. B cell function is also impaired as shown by their poor response to lipopolysaccharide stimulation. However, their response to CD40 stimulation is normal. In addition, these mice exhibit elevated levels of serum IgM due to increased numbers of plasma cells producing IgM, circulating autoreactive antibodies and symptoms characteristic of an autoimmune disease. Therefore, these results demonstrate that Lyn is vital for proficient B cell signaling and establish Lyn dependence in B cell selection.

ZAP-70 and Syk are also important for lymphocyte development. A portion of humans or mice with severe combined immunodeficiency (SCID), display a defective ZAP-70 or a deficiency in ZAP-70 (60-62). The normal number of CD4⁺ cells are present but they are non-functional. No CD8⁺ thymocytes are present in the periphery. Mice deficient in ZAP-70 have a thymocyte block at CD4⁺ CD8⁺ TCR^low stage but can be rescued with human ZAP-70 (63). Deficient mice exhibit elevated numbers of normal DP thymocytes. Mice deficient in Syk exhibit normal thymopoiesis (64,65). Therefore ZAP-70 but not Syk is vital for thymocyte development. However, mice deficient in Syk have decreased numbers of mature B cells and signaling through the BCR is impaired (64-66). Furthermore, they display impaired gammadelta T cell development (67).

ZAP-70 and Syk have similar structural and functional properties, which could imply a redundancy between these two kinases. However the different expression patterns of ZAP-70 and Syk may affect their roles (68). ZAP-70 is not expressed in peripheral B cells but is restricted to T cells, natural killer cells and thymocytes. However, Syk is expressed in thymocytes and predominantly in peripheral B cells but down regulated in peripheral T cells. Recent findings attempt to address the functional overlap between ZAP-70 and Syk. In B cells deficient in Syk, the BCR is non-functional. Nonetheless, expression of ZAP-70 in these cells reconstitutes BCR signaling (69). Functionally competent SH2 and catalytic domains of ZAP-70 are essential for full BCR activity. ZAP-70, like Syk, binds to the phosphorylated Igalpha and Igbeta subunits, with affinities similar to their interactions with the CD3eta subunit. Therefore under these conditions, ZAP-70 can substitute for Syk in its role in BCR signal transduction.