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, 21 (21), 5611-22

Inducible Gene Deletion Reveals Different Roles for B-Raf and Raf-1 in B-cell Antigen Receptor Signalling

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Inducible Gene Deletion Reveals Different Roles for B-Raf and Raf-1 in B-cell Antigen Receptor Signalling

Tilman Brummer et al. EMBO J.

Abstract

Engagement of the B-cell antigen receptor (BCR) leads to activation of the Raf-MEK-ERK pathway and Raf kinases play an important role in the modulation of ERK activity. B lymphocytes express two Raf isoforms, Raf-1 and B-Raf. Using an inducible deletion system in DT40 cells, the contribution of Raf-1 and B-Raf to BCR signalling was dissected. Loss of Raf-1 has no effect on BCR-mediated ERK activation, whereas B-Raf-deficient DT40 cells display a reduced basal ERK activity as well as a shortened BCR-mediated ERK activation. The Raf-1/B-Raf double deficient DT40 cells show an almost complete block both in ERK activation and in the induction of the immediate early gene products c-Fos and Egr-1. In contrast, BCR-mediated activation of nuclear factor of activated T cells (NFAT) relies predominantly on B-Raf. Furthermore, complementation of Raf-1/B-Raf double deficient cells with various Raf mutants demonstrates a requirement for Ras-GTP binding in BCR-mediated activation of both Raf isoforms and also reveals the important role of the S259 residue for the regulation of Raf-1. Our study shows that BCR-mediated ERK activation involves a cooperation of both B-Raf and Raf-1, which are activated specifically in a temporally distinct manner.

Figures

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Fig. 1. The course of the phosphorylation of Raf-1 and B-Raf following BCR engagement is different. (A) Western blot analysis of DT40 cells stimulated with anti-IgM Ab (M4). Detection of tyrosine-phosphorylated proteins (pY) indicates successful stimulation. This result is representative of at least five independent experiments. (B) DT40 and Ramos B cells were stimulated with anti-IgM Abs as indicated, and Raf proteins were purified using anti-B-Raf H-145 or anti-Raf-1 Abs, respectively. The immunocomplexes were subjected to western blot analysis. Phosphorylation at S445 (B-Raf) and S338 (Raf-1) was detected by the anti-pS338 Ab. This result is representative of at least four (DT40) or two (Ramos) independent experiments.
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Fig. 2. Conditional targeting of the chicken raf-1 locus. (A) Map of the wild-type raf-1 locus (wt) with exons 2–5 (black boxes). The targeting vector pTraf-1flE3 contains a loxP site and a loxP-FRT-neo-FRT cassette inserted into the SnabI and EcoNI sites, respectively. LoxP and FRT sites are indicated by black and white triangles, respectively. (B) Map of the modified raf-1 alleles. The flE3Δneo allele respresents the first targeted allele after excision of the neoR gene by FLP-e expression. The flE3neo allele is the result of the second round of transfection containing the neoR gene. Following 4-HT treatment, MCM excises E3 from the alleles flE3Δneo and flE3neo, thereby generating the ΔE3Δneo and ΔE3neo alleles, respectively. (C) MCM-mediated recombination in DT40MCM/raf-1flE3 cells was examined by Southern blot analysis. HincII-digested genomic DNA derived from two clones, which were either exposed to 4-HT for 24 h (+) or left untreated (–), was detected by the indicated probes. MCM-mediated recombination results in excision of genomic sequences around exon 3 and concomitant loss of a HincII site as indicated by the increased fragment size. The polymorphism (by the neoR cassette) of the modified raf-1 loci in these clones was used here to demonstrate the efficient and autonomous recombination of both alleles. (D) Western blot analysis of Raf-1 expression in total cellular lysates after induction with 4-HT.
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Fig. 3. Conditional targeting of the chicken B-raf locus. (A) Map of the wild-type B-raf locus with exons 4–7 (black boxes). The targeting vector pTB- rafflE6 contains a loxP site and a loxP-FRT-neo-FRT cassette inserted into the EcoRV and PacI sites, respectively. LoxP and FRT sites are indicated by black and white triangles, respectively. After removal of the FRT-flanked neoR by transient FLP-e expression, the same construct was used for targeting of the second allele. (B) Map of the modified B-raf allele after homologous and FLP-e-mediated recombination. (C) MCM-mediated recombination was confirmed by Southern blot analysis. VspI-digested genomic DNA fragments derived from either parental DT40MCM or DT40MCM/B-rafflE6 cells [either exposed to 4-HT (+) for 24 h or left untreated (–)] were detected with the E6/E7 probe. Homologous recombination of both alleles (lane 3) is indicated by the increased size of the fragment. MCM-mediated recombination results in excision of exon 6 indicated by the decreased fragment size (lane 4). (D) Western blot analysis using anti-B-Raf H-145 Abs. DT40MCM/ B-rafflE6 cells were harvested at 5 days post-induction. The aberrant B-Raf (B-Raf*) and the N-terminal peptide (N-term.) are only detected after MCM-mediated recombination.
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Fig. 4. Loss of Raf-1 does not affect the kinetics of BCR-mediated ERK activation. Populations of DT40MCM/raf-1flE3 cells, which were either left untreated (–) or exposed to 4-HT for 24 h (+), were stimulated with M4 at 8 days post-induction. Total cellular lysates were subjected to western blot analysis. This result is representative of at least seven independent, comparable experiments.
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Fig. 5. The early and late phases of BCR-mediated ERK activation are impaired in B-Raf-deficient DT40 cells. (A) Western blot analysis of B-Raf-positive and -negative populations of DT40MCM/B-rafflE6 cells, which were conducted as described in Figure 4. This result is representative of three independent experiments with an identical procedure and several other experiments with a similar design. The apparent decrease in the level of ERK2 is caused by sequential detection of phospho-ERK followed by detection with anti-ERK2 antibody on the same western blot membrane. (B) The ERK phosphorylation kinetics of the B-Raf-positive and -negative populations from two independent clones were quantified by Lumiimager analysis. The values represent the fold activation over the dual ERK phosphorylation measured in unstimulated B-Raf-positive cells. This figure shows two representative results obtained from the analysis of several clones.
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Fig. 6. Severe block in BCR-mediated ERK activation in Raf-1/B-Raf double deficient DT40 cells. Populations of DT40MCM/raf-1flE3/ B-rafflE6 cells were harvested at 5 days after induction of MCM-mediated deletion (+) and stimulated with M4. Total cellular lysates were subjected to western blot analysis. This result is representative of two experiments with an identical procedure (two independent clones) and at least five other experiments with a similar design. The apparent decrease in the level of ERK2 is caused by sequential detection of phospho-ERK followed by detection with anti-ERK2 antibody on the same western blot membrane.
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Fig. 7. The significance of Ras–Raf interaction for BCR-mediated ERK activation. Western blot analysis of total cellular lysates. (A) DT40MCM/ raf-1flE3/B-rafflE6 cells were treated with 4-HT to delete both raf-1 and B-raf, and transfected with expression vectors for either wild-type Raf-1 (R), Raf-1 R89L (L) or Raf-1 S259D (S). These cell lines were stimulated with M4 together with parental, Raf-deficient 4-HT-treated DT40MCM/raf-1flE3/B-rafflE6 cells (Δ) and their non-4-HT-treated, Raf-positive sister population (W) as indicated. (B) After the deletion of both raf genes, DT40MCM/ raf-1flE3/B-rafflE6 cells were transfected with expression vectors for either wild-type HA-tagged B-Raf (R) or HA-B-Raf R188L (L). These cell lines were stimulated with M4 together with parental, Raf-deficient cells (Δ) and their non-4-HT-treated, Raf-positive counterpart (W) as indicated. Detection of BAP37 serves a loading control. (C) Detection of S445 phosphorylation in HA-B-Raf and HA-B-Raf R188L by anti-pS338 Ab. DT40MCM/raf-1flE3/B-rafflE6 cells were transfected with expression vectors for HA-B-Raf or HA-B-Raf R188L and then treated with 4-HT. Nine days after induction, the cells were stimulated with M4 for the indicated times and HA-B-Raf proteins were purified as in (B). For each panel, representative results of at least three independent, comparable experiments are shown.
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Fig. 8. BCR-mediated induction of the transcription factors NFAT, c-Fos and Egr-1 is regulated by both Raf kinases. (A) DT40 lines allowing inducible deletion of either raf-1, B-raf or both raf genes were exposed to 4-HT for 24 h (white bars) or left untreated (grey bars) and cultivated for an additional 4 days. The cells were then transfected with the NFAT reporter plasmid. Cells were stimulated with 10 µg of M4 for 6 h. (B) Raf-1/B-Raf double-deficient DT40 cells were transfected with the NFAT reporter plasmid and 10 µg of the expression vector pFlu/B-raf or the empty vector pFlu as indicated. As wild-type reference, uninduced DT40MCM/raf-1flE3/B-rafflE6 cells were also included. The DT40 lines were treated as described in (A) and stimulated with M4 hybridoma supernatant (a stimulus equivalent to 5–10 µg M4/ml) for 6 h. The mean of the standardized luciferase activity derived from three independent, simultaneously performed transfections is shown. Standard deviation is indicated by an error bar. For each panel, representative results of at least three independent experiments are shown in both (A) and (B). (C) Western blot analysis of BCR-mediated synthesis of c-Fos and Egr-1. The DT40 lines were treated as described in (A), stimulated with M4 hybridoma supernatant (a stimulus equivalent to 5–10 µg M4/ml) for 1 h and lysed in RIPA buffer. This result is representative of three independent experiments.
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Fig. 9. A new model for BCR-mediated ERK activation. (A) Kinetic model of the different requirements for B-Raf and Raf-1 in BCR-mediated ERK activation as defined by the phenotypes of B-Raf or Raf-1 single deficient DT40 cells. The x-axis is not drawn to scale. The immediate early (<2.5 min) and late (>30 min) phases of ERK activation as well as the basal ERK activity are dependent on B-Raf (grey areas). In contrast, Raf-1 and B-Raf cooperate during the intermediate phase of BCR-mediated ERK activation, which is characterized by maximum ERK activity (white area). Raf-1 can partially compensate B-Raf deficiency in the intermediate phase and this genetically defined time window for the contribution of Raf-1 to BCR-mediated ERK activation correlates with the kinetics of S338 phosphorylation (Figure 1B). (B) Basal ERK activity is maintained in DT40 cells by B-Raf. Phosphorylation of B-Raf at S445 by an unknown kinase primes B-Raf for activation prior to its interaction with Ras-GTP. (C) Upon BCR engagement, more primed B-Raf molecules are recruited and activated at the plasma membrane by increasing levels of Ras-GTP and other activators (Chong et al., 2001). This pool of BCR-activated B-Raf increases ERK activity, leading to its rapid feedback phosphorylation by activated ERK. With persistent BCR engagement, Raf-1 is also recruited to the plasma membrane by Ras-GTP and subsequently is activated by dephosphorylation of S259 followed by phosphorylation of its N-region and other events. This pool of BCR-activated Raf-1 contributes to intermediate ERK activation leading to its own feedback phosphorylation, which is proposed as a negative feedback loop involved in its detachment from the plasma membrane (Wartmann et al., 1997).

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