Our data reveal the existence of a Ca2+
-dependent pro-apoptotic pathway to Erk activation, which mediates a critical checkpoint during B cell development. We therefore propose a model where Ca2+
and DAG can direct Erk to distinct functional outcomes during B cell development. Because DAG and IP3
are produced at equal molar concentrations downstream of PLCγ-mediated hydrolysis of PtdIns(4,5)P2
, DAG production is inextricably linked to Ins(1,3,4)P3
-induced SOCE. However, the amplitude and duration of DAG production and SOCE largely depends on proteins that amplify or negatively regulate these second messengers. For example, higher amounts of Stim1 protein (which we observed in bone marrow transitional cells) may contribute to amplify SOCE. By contrast, lower DAG production, which have been previously reported in immature B cells10
, may be the result of rapid DAG turn-over mediated by DAG kinases, DAG acyltransferases or DAG lipases, all of which metabolize DAG into different products. Data from the IMMGEN website show that mRNA abundance for some of these proteins, such as DGKδ, DGKτ, DGKη, DGLβ or DGAT2L6, change during B cell development in ways that may affect DAG metabolism. In addition, we have found that considerable changes in expression of proteins that mediate the activation of Erk pathways during development also contribute to their amplification, as demonstrated by the robust increases of RasGRP1 and PKCδ expression in transitional bone marrow B cells relative to other B cell subsets. These changes in expression are likely critical to setting the appropriate threshold of antigenic signal strength that results in negative selection.
The involvement of PKCδ in this pro-apoptotic Erk pathway is intriguing and was not expected. PKCδ has been implicated in apoptosis of multiple cell types43, 44
, but the potential involvement of pro-apoptotic Ras–Erk signaling as an effector pathway downstream of PKCδ has not been explored. Therefore, our data implicating PKCδ in antigen-induced pro-apoptotic Ras–Erk signaling establish a new paradigm that could be tested in other systems where PKCδ is important to mediate apoptosis. It has been clearly demonstrated that Prkcd−/−
mice display a defect in peripheral B cell homeostasis which increases the lifespan of B cells and is independent of selection events21, 42
. However, our data involving PKCδ in pro-apoptotic Erk signaling during negative selection now suggest that loss of PKCδ may also result in previously unrecognized alterations to the B cell repertoire that contribute to the disease pathology of the mice. Interestingly, PKCδ has been reported to be dispensable in a transgenic model of negative selection mediated by expression of membrane-bound hen egg lysozyme (HEL) in MD4 mice, which express IgHEL-specific B cells21
, implying that this may not be the only pathway mediating B cell negative selection. However, membrane-bound HEL in that model provides an exceedingly strong antigenic stimulus, in particular to a high affinity BCR transgene, which may override mechanisms that establish selection thresholds for many antigens in normal cells, thus leading to B cell deletion at the earliest bone marrow immature stage45, 46
. Because the onset of PKCδ-mediated, Ca2+
-dependent Erk activation occurs in IgMhi
bone marrow transitional B cells, it is not surprising that the mHEL system does not reveal the role of PKCδ in negative selection.
Ras and Erk activation have been intensively studied in lymphocytes, and have mostly been thought of as DAG-dependent and Ca2+
-independent. Our findings demonstrate that more than one pathway leading to Erk activation exists in B lymphocytes, and here we show that a Ca2+
-dependent Erk activation pathway directs the functional outcome of the stimulus towards an apoptotic fate in developing B cells. Because RasGRP1 is prominently and exclusively expressed in B cell subsets where this pathway is active, we focused on defining the biochemical mechanisms by which Ca2+
may influence its function. We have thus identified S332 on RasGRP1 as a potential phosphorylation site that is a likely PKCδ target and which was clearly required for Ca2+
-dependent Erk activation. Unexpectedly, this site is located within the CDC25 domain of RasGRP1, the domain that actually mediates the interaction with Ras and consequently the nucleotide exchange function of the protein. Because of this, a phosphorylation event that preserves nucleotide exchange function (as is the case with S332) is unlikely to trigger structural reorganization of the protein. Based on homology modeling with the CDC25 of RasGRF141, 47
, we predict that this site lies in the “flap2” region of the CDC25 domain, which may have a role in positioning the catalytic helical hairpin of the exchange factor for catalysis. This residue is strikingly close to the Ras binding site and, because of the increase in negative charge associated with phosphorylation, electrostatic interactions may influence the choice of Ras isoform that interacts with RasGRP1. This interesting idea that may help explain how distinct Ras protein signaling pathways that rely on Erk achieve specificity.
The Ras–Erk pathway is remarkably promiscuous in terms of potential substrates, and it has been suggested that specificity is achieved through compartmentalization mediated by scaffolds that constrain active Erk to signaling complexes where they can be activated by appropriate stimuli and simultaneously gain access to appropriate substrates48–50
. As mentioned above, phosphorylation of S332 on RasGRP1 could potentially contribute to such specificity. However, although it is clear that phosphorylation of S332 RasGRP1 is essential for SOCE to induce activation of this Erk pathway, the results with the phosphomimetic mutant suggest that this event is also not sufficient to activate it, and that other Ca2+
-dependent events may be required. Because PKCδ is not thought to bind Ca2+
directly, it is possible that Ca2+
binding to RasGRP via the EF hands contributes to promote RasGRP activity, possibly by facilitating the interaction with PKCδ. Moreover, at this point we cannot exclude the possibility that other Ca2+
-responsive proteins may play a role within this signaling complex, perhaps by further modifying PKCδ and/or RasGRP and potentiating their activity. Concentration gradients of DAG and Ca2+
are also known to affect localization of signaling proteins, and may contribute to directing distinct Erk pathways to unique compartments within the cell. Interestingly, recent results in thymocytes have implicated differential localization of MAPK signals in setting the thresholds of positive and negative selection51
. These findings, combined with our data, suggest that Ca2+
and DAG may control selection by directing the assembly and localization of different MAPK signaling complexes.