Previous studies have identified apoptosis and cell cycle arrest as mechanisms of BCR-induced growth arrest in BCL1
.3B3 cells [19
]. The studies presented here have demonstrated that stimulation of the BCR resulted in the rapid and transient activation of the non-receptor tyrosine kinase, Syk. Previous studies have implicated Syk in BCR-induced apoptosis in other cell lines [37
]. To examine the putative role of Syk in BCR-induced apoptosis and cell cycle arrest in BCL1
.3B3, we attempted to alter Syk activity through the expression of mutant forms of Syk. When the (K395A/3F)Syk mutant was co-expressed with endogenous Syk in BCL1
.3B3 cells, a constitutive Syk activation phenotype was observed. The presence of elevated Syk activity allowed us to evaluate the role of Syk activation on the growth and viability of BCL1
.3B3, and on other biochemical changes associated with BCR signaling. To our surprise, no evidence for downstream signaling was found in cells with high levels of Syk kinase activity.
Two novel observations were obtained from these studies. First, expression of the (K395A/3F)Syk molecule in BCL1.3B3 results in constitutive activation of endogenous Syk. Since (K395A/3F)Syk was enzymatically inactive when transfected into Syk-deficient DT40 cells, the observed constitutive kinase activity must be derived from endogenous Syk and not from the (K395A/3F)Syk protein. Mutant molecules that inhibit the activation or function of the wildtype counterpart in the same cell have been defined as dominant-negative molecules. Since (K395A/3F)Syk has an activating effect on endogenous wildtype Syk, we propose that (K395A/3F)Syk is a dominant-positive molecule.
It is likely that the replacement of the three carboxy-terminal tyrosine residues with phenylalanine is necessary for the dominant-positive phenotype. Zeitlmann et al. found that transfection of a mutant Syk expression construct carrying only the 3F alteration into a T cell lines could overcome the need for TCR occupancy for IL-2 expression in PMA-treated cells [62
]. However, since the mutant Syk used retained kinase activity, these studies could not distinguish between a gain-of-function effect versus a dominant-positive effect as found here.
The mechanism by which the (K395A/3F)Syk molecule activates endogenous Syk is unknown. One hypothesis is that a negative regulator of Syk is bound and sequestered by the (K395A/3F)Syk molecule. By sequestering this negative regulator, expression of the mutant Syk molecules gives rise to the activation of the endogenous wildtype protein in the absence of specific stimulation, leading to a constitutively active Syk phenotype. Several negative regulators of Syk have been reported. BCR-induced interaction between Cbl and Syk increases the susceptibility of Syk to proteolysis [55
], possibly through enhancement of ubiquitination [56
]. Expression of a dominant-negative SHP-1 phosphatase in the mature murine B cell line K46 resulted in an increase in BCR-induced Syk kinase activity as measured by C3b phosphorylation in an ICKA and enhanced BCR-induced Ca2+
]. It remains to be determined if interference with any of these known regulatory proteins is responsible for the dominant-positive effect.
The second novel observation, that Syk kinase enzymatic activity was insufficient for signal transduction, suggests that multiple biochemical processes may be required for the Syk-dependent step of signal transduction. Presently, it is unclear what biochemical changes, other than enzymatic activation, are required. However, in light of the present model of signaling through the BCR, perhaps appropriate subcellular localization to the BCR protein complex and the ITAMs of CD79b (Igβ) is also required. In support of this hypothesis, Syk did not co-precipitate with IgM in (K395A/3F)Syk-containing cells, without stimulation (data not shown). This is not surprising since the ITAMs in CD79, which are proposed to be docking sites for Syk, are unlikely to be phosphorylated in the absence of BCR stimulation since phosphorylation of these residues likely requires activation of Src family tyrosine kinases expressed in B cells. Therefore, despite constitutive Syk kinase activity, the ITAMs in the dominant-positive-expressing cells remain hypophosphorylated because kinases that are upstream of Syk and/or are Syk independent are not active in the absence of BCR crosslinking.
This result implies that in addition to enzymatic activation, localization of Syk to the BCR might be required for functional signal transduction. There is precedence for this model. Mutation of tyrosine 130 in Syk to glutamic acid had two effects. First, the Y130E mutation resulted in constitutive activity of Syk. Second, molecules with the Y130E mutations did not co-immunoprecipitate with the BCR [58
]. A second example involves an in vitro
model of histamine release in the Syk-deficient RBL-2H3 cell line [59
]. When tyrosines 519 and 520 were mutated and introduced into Syk-deficient RBL-2H3 cells, these Syk molecules were capable of phosphorylation of c3b substrate in an ICKA. However, despite this activity, there was no high affinity IgE receptor (FcεRI)-induced degranulation. Further analysis demonstrated neither increases in phosphotyrosine nor phosphorylation of PLCγ2.
Alternatively, the absence of signal transduction in the presence of Syk activity may be related to its ability to interact with its downstream targets. Within Syk there are two phosphotyrosine-binding SH2 domains, allowing for protein-protein interactions. In addition, phosphotyrosine residues in Syk would provide additional sites for protein-protein interactions. Perhaps, further modification is necessary to increase its affinity for downstream binding partners, e.g. BLNK or PLCγ2.
The absence of signaling in the presence of constitutive enzymatic activity described for Syk in different systems implies that execution of the Syk-dependent step in this signaling cascade is a multi-step process. Multiple requirements for progression through this step in the signal transduction pathway may serve to integrate the cellular response to multiple environmental cues. In this case, the ultimate cellular response to the activation of the BCR signaling pathway is cell death. Each of these required steps may provide a regulatory site where other signal transduction pathways could intersect the default response to antigenic stimulation giving rise to an altered cellular outcome.