B cells deficient for Btk or expressing a mutant form of the Btk protein are characterized by drastically reduced life-span in vitro and in vivo (12
). Similar properties are characteristic for PKCβ-deficient B cells. Incubation of ex vivo isolated splenic PKCβ-deficient B cells in vitro in the absence of exogenously added agonists of B cell survival is accompanied by a rapid decline of cell viability ( A). Similar to B cells with impaired Btk function, the PKCβ-deficient B cells are characterized by drastically decreased proliferative responses to the antibody-mediated cross-linking of BCR, or to the polyclonal activation by anti-CD38 or anti-RP105 antibodies ( B). Addition of the general B cell survival factor IL-4 (20
) to the culture medium promotes B cell survival ( C) and increases the fraction of dividing cells ( B). These data suggest that accelerated death of PKCβ-deficient B cells in vitro is likely causing defective proliferative responses of PKCβ-deficient B cells to various stimuli in vitro.
Figure 1. Impaired survival and proliferation of PKCβ-deficient B cells in vitro. (A) Accelerated cell death of PKCβ−/− B cells cultured without stimulation. Percentage of cell viability of purified splenic B cells is plotted for (more ...)
Poor survival of PKCβ-deficient B cells in vitro correlates with their inability to up-regulate expression of the anti-apoptotic protein Bcl-xL upon stimulation with anti-IgM () . Incubation of wild-type splenic B cells with anti-IgM results in progressive increase in the expression levels of the Bcl-xL (). Similar treatment of the PKCβ-deficient B cells has a negative impact on the Bcl-xL expression level (). The inability of the surface expressed BCR to promote the expression of the anti-apoptotic Bcl-xL and Bcl-2 proteins likely causes the rapid death of the PKCβ-deficient B cells.
Figure 2. Reduced expression of Bcl-xL and Bcl-2 after IgM cross-linking on B cells of PKCβ−/− compared with control mice. Splenic B cells were isolated from PKCβ−/− and PKCβ+/+ mice and incubated with 10 (more ...)
Expression of Bcl-xL in B cells is dependent on the activity of the NF-κB transcription factor (6
), thus suggesting a possible involvement of PKCβ in NF-κB regulation. Members of the NF-κB/Rel family of proteins include RelA, c-Rel, RelB p50/NF-κB1, and p52/NF-κB2 (7
). Deficiency in PKCβ does not affect expression levels of RelA, c-Rel, and RelB in splenic B cells ( A). NF-κB2 is synthesized as a large precursor (p100) that requires proteolytic processing to produce p52 (7
). The relative amount of unprocessed p100 NF-κB2 precursors is increased in PKCβ-deficient B cells compared with control B cells ( A).
Figure 3. Expression of Rel family proteins and IKKα/IKKβ/IKKγ complex in splenic B cells of PKCβ−/− (−/−) and PKCβ+/+ (+/+) mice. (A) The amount of RelA, RelB, and c-Rel (left panel) as well (more ...)
Defective NF-κB2 processing has been previously found in B cells deficient for IKKα or expressing a catalytically inactive form of IKKα, which is a component of the tri-member IκB kinase complex IKKα/IKKβ/IKKγ (23
). To test whether PKCβ controls NF-κB2 processing through the regulation of IKKα expression or activation we have analyzed the expression and activation state of IKKs in PKCβ-deficient splenic B cells. Both IKKα and IKKβ, as well as the regulatory component of the IκB kinase complex, IKKγ, are expressed in PKCβ-deficient B cells at wild-type levels ( B, WCL lanes). Immunoprecipitation of IKKα or IKKβ from PKCβ-deficient and control B cell lysates results in coprecipitation of IKKβ/IKKγ or IKKα/IKKγ, respectively ( B, and data not shown). These data suggest that expression of IKKα and formation of the IκB kinase complex is not controlled by PKCβ in B cells.
Activation of IKKα and IKKβ is controlled by their phosphorylation in the activation loop of the kinase domain at serine residues 176/180 and 177/181, respectively (24
). Hence, the levels of phospho-IKKα (Ser-180) and phospho-IKKβ (Ser-181) reflect indirectly the fraction of activated IKKs within a cell. In wild-type B cells more phospho-IKKα than phospho-IKKβ can be detected. Incubation of the wild-type B cells with anti-IgM results in a drastic increase of the amount of phospho-IKKβ while the phospho-IKKα levels remain relatively stable in the course of B cell stimulation ( A, top panel). Deficiency in PKCβ has a dramatic impact on the phosphorylation state of IKKα and to a lesser extent on IKKβ. Phospho-IKKα is virtually absent both in nonstimulated and anti-IgM treated PKCβ-deficient B cells ( A, top panel). Moreover, the duration of IgM-mediated phosphorylation of IKKβ is diminished in PKCβ-deficient B cells compared with the wild-type B cells ( A, top panel). Considering the essential role of serine phosphorylation in IKKα and IKKβ activation, these results reveal PKCβ as a key regulatory serine/threonine kinase connecting the BCR and IKK activation.
Figure 4. Impaired activation and NF-κB signaling in PKCβ−/− B cells after BCR cross-linking. Absence of IKKα activation and shortened IKKβ activation in PKCβ−/− B cells. Splenic B cells of (more ...)
In wild-type B cells, the BCR-mediated activation of IKKα is followed by phosphorylation of IκBα. This in turn leads to IκBα degradation and translocation of the Rel proteins to the nucleus. Deficiency in PKCβ and ensuing defective IKKα and IKKβ phosphorylation result in impaired IκBα phosphorylation and degradation ( A, middle panel). Congruently, activation of NF-κB is reduced in PKCβ-deficient B cells ( A, bottom panel).
To test whether impaired NF-κB activation in PKCβ-deficient B cells is specific for BCR-mediated signals, we tested stimulation of PKCβ-deficient B cells through CD40, which in wild-type B cells also leads to NF-κB activation. The analysis of IKKα and IKKβ phosphorylation in PKCβ-deficient B cells stimulated through CD40 reveals a paucity in IKKα phosphorylaytion similar to that observed after BCR engagement ( B, top panel). However, the CD40-mediated phosphorylation and degradation of IκBα, as well as the induction of NF-κB DNA-binding activity, are not impaired in PKCβ-deficient B cells ( B, middle and bottom panel). As a member of the TNF receptor family, CD40-mediated NF-κB activation is thought to require a different set of signaling molecules compared with BCR-mediated NF-κB activation, namely TRAF2, 3, 5, and 6 (25
). Possibly in CD40/TRAF-mediated NF-κB activation IKKβ activation by itself is sufficient, or other kinases, like NIK, can substitute for IKKα (26
). In agreement with the normal induction of Rel family proteins CD40 stimulation alone improves the survival of PKCβ-deficient B cells to wild-type levels (data not shown). This is also consistent with the finding that the CD40-mediated induction of anti-apoptotic A1 is not impaired in IKKα-deficient B cells (27
). In contrast to IgM stimulation, CD40-mediated proliferative responses are only mildly impaired in PKCβ-deficient B cells (13
), which is also seen in IKKα-deficient B cells (27
). In conclusion, the PKCβ-mediated regulation of NF-κB activation is largely specific for BCR-mediated signaling and has only a minor impact on CD40 mediated B cell activation.
The pattern of defective NF-κB activation is very similar in B cells of PKCβ-deficient and Xid mice (9
). As PKCβ can act as a negative regulator of Btk, the similar effect of both mutations on NF-κB activation appears paradoxical. However, increased or decreased Btk-mediated signaling may have the same outcome, as shown by the severe Xid-like phenotype of mice expressing a constitutively active Btk mutant (28
). We think that in addition to its inhibitory role for Btk activation, PKCβ may also act downstream of Btk to mediate the activation of IKKs. It remains to be seen whether PKCβ regulates IKKs through direct phosphorylation. As the activation loop serine residues of IKKα or IKKβ are not part of a PKC consensus phosphorylation site, other serine/threonine residues of IKKα and IKKβ may serve as direct substrates for PKCβ. Interestingly, a low stringency scan for common PKC phosphorylation sites returns several potential sites for IKKα, but none for IKKβ (30
). Alternatively, PKCβ may regulate IKKs indirectly through an intermediate kinase that would be directly controlled by PKCβ. Regardless of the exact mechanism of the PKCβ involvement in IKKs phosphorylation, the data presented reveal PKCβ as a novel component of the NF-κB signaling axis responsible for the survival and activation of B cells after BCR cross-linking.