Abl kinase-mediated FUS Tyr526 phosphorylation alters nucleocytoplasmic FUS localization in FTLD-FUS

Abstract

Nuclear to cytoplasmic mislocalization and aggregation of multiple RNA-binding proteins (RBPs), including FUS, are the main neuropathological features of the majority of cases of amyotrophic lateral sclerosis (ALS) and frontotemporal lobular degeneration (FTLD). In ALS-FUS, these aggregates arise from disease-associated mutations in FUS, whereas in FTLD-FUS, the cytoplasmic inclusions do not contain mutant FUS, suggesting different molecular mechanisms of FUS pathogenesis in FTLD that remain to be investigated. We have previously shown that phosphorylation of the C-terminal Tyr526 of FUS results in increased cytoplasmic retention of FUS due to impaired binding to the nuclear import receptor TNPO1. Inspired by the above notions, in the current study we developed a novel antibody against the C-terminally phosphorylated Tyr526 FUS (FUSp-Y526) that is specifically capable of recognizing phosphorylated cytoplasmic FUS, which is poorly recognized by other commercially available FUS antibodies. Using this FUSp-Y526 antibody, we demonstrated a FUS phosphorylation-specific effect on the cytoplasmic distribution of soluble and insoluble FUSp-Y526 in various cells and confirmed the involvement of the Src kinase family in Tyr526 FUS phosphorylation. In addition, we found that FUSp-Y526 expression pattern correlates with active pSrc/pAbl kinases in specific brain regions of mice, indicating preferential involvement of cAbl in the cytoplasmic mislocalization of FUSp-Y526 in cortical neurons. Finally, the pattern of immunoreactivity of active cAbl kinase and FUSp-Y526 revealed altered cytoplasmic distribution of FUSp-Y526 in cortical neurons of post-mortem frontal cortex tissue from FTLD patients compared with controls. The overlap of FUSp-Y526 and FUS signals was found preferentially in small diffuse inclusions and was absent in mature aggregates, suggesting possible involvement of FUSp-Y526 in the formation of early toxic FUS aggregates in the cytoplasm that are largely undetected by commercially available FUS antibodies. Given the overlapping patterns of cAbl activity and FUSp-Y526 distribution in cortical neurons, and cAbl induced sequestration of FUSp-Y526 into G3BP1 positive granules in stressed cells, we propose that cAbl kinase is actively involved in mediating cytoplasmic mislocalization and promoting toxic aggregation of wild-type FUS in the brains of FTLD patients, as a novel putative underlying mechanism of FTLD-FUS pathophysiology and progression.

Keywords: FTLD; FUS; c-Abl; c-Src; cytoplasmic aggregates; phosphorylation.

Figure 1

Members of src family kinases (c-src, c-fyn, c-abl) phosphorylate the c-terminal Tyr526 of FUS. (A) Web prediction programmes NetPhos (top), DisPhos (middle), and BioCuckoo (bottom) were run to search for Tyr526 phosphorylating kinase candidates and kinases (c-Src, c-Fyn, and c-Abl) of Src-family (SFK) revealed as most probable candidates. (B) The phosphorylated target protein of all three SFK was detected with FUS^p-Y526 Ab (left) at the shifted position compared to the FUS band detected with NB100-565 (middle), and GAPDH (right). Dasatinib, an SFK inhibitor, inhibited the activity of expressed wild-type kinases (C) c-Src, (D) c-Fyn, and (E) c-Abl and decreased the signal of FUS^p-Y526 in cell lysates in a concentration-dependent manner.

Figure 2

The specificity of FUS^p-Y526 antibody was confirmed by in vitro phosphatase assay and FUS knockdown studies in HEK293T cells. (A) The C-terminal (His₆-GFP-FUS⁴⁹⁰–^526Y, His₆-GFP-FUS^490-Y526P) fragments of FUS containing either Tyr or Phe as the last residue were expressed together with constitutively active kinases in HEK293T cells, and the lysates were immunoblotted against FUS^p-Y526, FUS, GFP, and GAPDH. (B) His-tag pull-down of His₆-GFP-FUS⁴⁹⁰–^526Y from NT, kinase-transfected and kinase/His₆-GFP-FUS⁴⁹⁰–^526Y co-transfected cell lysates, immunoblotted against FUS^p-Y526 (left) and FUS (right). (C) The lysates of non-transfected and c-Src, c-Fyn, and c-Abl transfected HEK293T cells and (D) the lysates of non-transfected, His₆-GFP-FUS^490–526Y and kinase co-transfected HEK293 cells were subjected to calf intestinal phosphatase activity and analysed by western blot for the FUS^p-Y526, which decreased in the phosphatase treated samples. (E) Knockdown of FUS by siRNA was performed in HEK293T cells and then transfected with either c-Src, c-Fyn, or c-Abl to induce Tyr526 FUS phosphorylation. Western blots with anti- FUS^p-Y526, FUS, c-Abl, c-Src, and c-Fyn antibodies showed the specificity of the FUS^p-Y526 antibody.

Figure 3

Kinases affect localization and aggregation of non-phosphorylated and phosphorylated Y526 FUS. HEK293T cells were transfected with (A) constitutively active c-Src, c-Fyn, and c-Abl expression plasmids and co-transfected with (B) constitutively active kinases and a GFP tagged full-length FUS (green). (C and D) Quantitative assessment of nuclear and cytoplasmic FUS (magenta) and FUS^p-Y526 (red) signal was performed using ImageJ and the ratio of nuclear to cytoplasmic FUS and FUS^p-Y526 signals in positively transfected cells was determined alongside their relative cytoplasmic abundance. (E) The culture of mouse primary cortical neurons was established, and neurons were transfected with active kinases. Cell nuclei were counterstained with DAPI (blue). One-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars = 20 μm in A and B; 15 μm in E. ROI = region of interest.

Figure 4

FUS^p-Y526 is detected in vivo in mouse brains. Immunocytochemical analyses performed on transverse frozen-fixed and paraffin-embedded tissue sections of mouse brain showed sparse detection of FUS^p-Y526-positive cells (green) throughout brain tissue, in (A) astrocytes co-stained for GFAP (red), (B) Pyramidal neurons of the cerebral cortex co-stained for A2B5 (red), SMI33 and NeuN proteins (red), and (C) Neurons in the cerebellum co-stained for SMI-33. (D) No co-localization of FUS^p-Y526 and GFAP was observed in the neurons or in (E) brainstem. The neuronal nuclei were counterstained with DAPI. NeuN/FUS^p-Y526 = paraffin sections; others = frozen-fixed sections. Scale bar = 15 μm.

Figure 5

In vivo FUS^p-Y526 signal overlaps with the expression of active src family kinases and appears brain region-specific. Frozen-fixed and paraffin sections of brain from wild-type mice were co-immunostained for active phosphorylated pSrc, pFyn, pAbl and FUS^p-Y526, and co-localization of signals was observed in neurons of (A) hippocampus, (B) brainstem, and (C) frontal cortex. Frozen-fixed sections = pFyn/FUS^p-Y526; paraffin sections = pAbl/pSrc/FUS^p-Y526. Scale bar = 15 μm.

Figure 6

FUS^p-Y526 shows altered nucleocytoplasmic localization in cortical neurons in FTLD-FUS patients. (A) DAB immunolabelling using FUS^p-Y526 antibody in a representative control section showing diffuse nuclear immunoreactivity, whereas in FTLD-FUS cortical neurons (right) showing diffuse nuclear as well as cytoplasmic localization. FUS^p-Y526 antibody also recognizes smaller atypical granular (black arrows) and small globular (white arrow) aggregates. (B) Co-immunolabelling of FUS^p-Y526 and FUS in the cortical neurons of the control and FTLD-FUS patients, (C) showing the FUS and FUS^{p-Y526 signals} to only partially overlap in large cytoplasmic inclusions (white arrow). (D) Quantification of localization of the fluorescent FUS^p-Y526 immunolabelling (Nu = nuclear; Nu & Cyto = nuclear and cytoplasmic; Cyto = cytoplasmic only) by counting neurons in the frontal cortex of control and FTLD-FUS post-mortem brain tissue sections. ImageJ analyses of (E) FUS^p-Y526 and (F) FUS nuclear/cytoplasmic ratio in counted neurons. Statistical analyses were performed using ANOVA. Control n = 4, FTLD-FUS = 4. *P < 0.05, **P < 0.01. Paraffin sections, scale bar = 18 μm.

Figure 7

Activity of pAbl increases in the nuclei of FTLD-FUS frontal cortex neurons. (A) Co-immunolabelling using FUS^p-Y526 and pAbl antibody in the cortical neurons of control and FTLD-FUS patients. (B) Counting the frontal cortex neurons with pAbl signal localization in nucleus only (Nu), nucleus and cytoplasm (Nu & Cyto), or in the cytoplasm only (Cyto) in post-mortem tissue sections of control and FTLD-FUS. ImageJ analyses of (C) FUS^p-Y526 and (D) pAbl nuclear/cytoplasmic ratio in counted cortical neurons. Statistical analyses were performed using ANOVA. *P < 0.05, **P < 0.01. Paraffin sections, scale bar = 20 μm.

Figure 8

Activity of pAbl induces FUS^p-Y526 sequestration into G3BP1 positive granules in stressed sh-SY5Y. The FlpIn SH-SY5Y-TR-FRT-mScarlet-G3BP1-Myc cells generated by us were induced by doxycycline and transfected with constitutively active c-Abl kinase. Twenty hours post-transfection they were exposed to 50 μM sodium arsenite (As) for 1.5 h and processed for FUS^p-Y526 (green) and TDP-43 (magenta) immunolabelling, which revealed co-localization of FUS^p-Y526 and mScarlet-G3BP1 granules (red) only in stressed c-Abl transfected cells. The cell nuclei were counterstained with DAPI. Scale bar = 20 μm.