BRAF interactome

BRAF involvement in antigen-dependent BCR signaling in DG75 was further investigated by the analysis of its interactome. BRAF is a serine-threonine kinase, namely a MAPKKK (or MAP3K) and therefore an upstream kinase in the classical MAP-kinase signaling cascade (reviewed in Chang and Karin, 2001 and Dhillon et al., 2007). As BRAF is not described to be mutated in Burkitt’s lymphoma, for example the V600E point mutation leading to its constitutive activation, the kinase is amenable to activity regulation (Davies et al., 2002). As other MAP-kinases, BRAF activation depends amongst other processes on activating and inhibiting of phosphorylation sites and their dephosphorylation (reviewed in Chang and Karin, 2001 and Dhillon et al., 2007). BRAF is therefore expected to interact with its upstream interactors as well as with its downstream targets. The interactome analysis to identify putative BRAF binding partners was performed with a triple SILAC experimental design: light- and medium-labeled DG75 cells were left non-stimulated whereas the BCRs of heavy-labeled cells were stimulated for 5 min via their BCR (Figure 3.10 A). BRAF was purified with a BRAF-specific antibody from the medium and heavy cell lysates (Figure 3.10 A). As a control, the light-labeled lysates were incubated with the beads lacking the antibody. Immunopurified proteins were mixed in equimolar amounts, separated by SDS-PAGE, digested with trypsin and analyzed by LC-MS/MS (Figure 3.10 A). Of note, the DG75 cells used for the BRAF interactome analysis were affected to some extent by metabolic arginine-to-proline conversion as described in 3.1.12 and shown in Figure 3.13 E (Blagoev and Mann, 2006; Lössner et al., 2011;

Ong et al., 2003; Van Hoof et al., 2007). However, the distribution of the normalized protein ratios was normally distributed and specific BRAF enrichment could be observed. Therefore results were used for analysis but might be considered carefully.

Results

Highly specific enrichment of BRAF by immunopurification compared to the control state is demonstrated by high medium-to-light (M/L) and heavy-to-light (H/L) quantitation ratios (right upper quadrant; Figure 3.10 B). As a proof-of-principle, the downstream MAP-kinase of BRAF, MAP2K1 (MEK1) was found to be enriched together with BRAF in both the non-simulated (medium) and the BCR stimulated cells (heavy) (reviewed in Chang and Karin, 2001;

Dhillon et al., 2007). The diagonal distribution of protein H/L and M/L SILAC ratios showed a Pearson’s correlation efficient of 75.9%, indicating correlation in the binding behaviour of BRAF interactors after 5 min of BCR stimulation compared to the non-stimulated state. The majority of proteins (grey squares) cluster around the intersection of the y- and x-axis base lines and presumably present non-specific binders (light grey dashed lines, Figure 3.10 B).

Figure 3.10: BRAF interactome analysis in DG75

(A) For the BRAF interactome analysis, light- and medium-labeled DG75 cells were left non-stimulated whereas the BCRs of heavy-labeled cells were stimulated for 5 min. BRAF was affinity purified from the medium and heavy lysates with a BRAF-specific antibody. As a control, the light-labeled lysates were incubated with the beads lacking the antibody. Immunopurified proteins were mixed in equimolar amounts, separated by SDS-PAGE, digested with trypsin and analysed by LC-MS/MS. (B) The scatter plot shows the distribution of the normalized SILAC heavy-to-light (H/L) ratio of proteins (y-axis) reflecting 5 min of BCR stimulation and the medium-to-light (M/L) ratio (x-axis; both log2) reflecting the non-stimulated ground state.

Interaction partners with ratios ≤ -1.5 and ≥ 1.5 are highlighted in red and gene names are indicated, proteins below the threshold (red dashed lines) are marked in grey. Proteins with a H/L ratio above or below the threshold are highlighted in blue. The vertical and horizontal base lines are shown as light grey dashed line. The Pearson’s correlation coefficient is displayed in the upper right corner of the scatter plot. BRAF is represented two times in the scatter plot due to two different sequence database entries (C) The scatter plot shows the distribution of the intensity (y-axis; log10) against the normalized SILAC ratio H/M (x-axis; log2). The H/M ratio reflects binding after 5 min of BCR stimulation compared to ground state.

Proteins highlighted in green are the most abundant seen in (B). The threshold and color code settings are mentioned in (B).

All detected proteins are listed in supplementary table S6.

Results

The proteins enriched together with BRAF in the ground and the BCR stimulated state in the upper right quadrant: CUX1, TRIM21, KIF3A, DHX8, ARHGEF2, AKAP13 and MAP2K1 could potentially be interaction partners of BRAF (highlighted in red, Figure 3.10 B; supplementary table S6). Potential contaminant proteins were depleted in the medium- and heavy-labeled state i.e. enriched in the light-labeled control experiment and appeared in the left lower quadrant: IGHG1, SPATC1, KRT2, SH3RF3, DCD, IGLC2, CRYAA and SOAT1. Proteins specifically depleted (CD79A and SCCPDH) or enriched (SRPR, RIF1 and histone proteins) only after 5 min of BCR stimulation (heavy label) and not in the non-stimulated (medium label) state are highlighted in blue (Figure 3.10 B; supplementary table S6). No protein was found to be specifically depleted in one state and enriched in the other state (right lower or the left upper quadrant). To further link the experimental BRAF interaction data with the BCR stimulation data in DG75, p-site and protein expression levels of potentially interesting interacting candidates were mapped. For three proteins with the highest SILAC ratios, CUX1, TRIM21 and KIF3A, no regulated p-site could be mapped from the phosphoproteomic dataset and no protein expression change could be detected over the time course of stimulation (supplementary table S6).

To thoroughly visualize BCR stimulation induced partners, the normalized heavy-to-medium (H/M) SILAC ratios which directly compare 5 min of BCR stimulation to the ground state were plotted against the log10 intensity (Figure 3.10 C). The majority of proteins did not show an altered binding behaviour as can be seen by the evenly distribution around the vertical zero line (light grey dashed line) and SILAC ratios between -1 and 1 on a log2 scale (grey squares).

BRAF and its co-purified putative interaction partners (CUX1, TRIM21 and KIF3A) shown in Figure 3.10 B are highlighted in green (Figure 3.10 C). Potential BRAF interaction partners enriched prior to BCR stimulation can be found on the left side of the scatter plot: CD79A/B (signaling module of the BCR), SCCPDH, and S1PR4 whereas putative BRAF interactors after 5 min of BCR engagement can be found on the right side of the scatter plot: HIST1H2BL/AJ, HIST1H4A, HIST2H3A, SH3RF3, SRPR, NDUFS3, and RIF1.