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. 2016 May;137(4):518-27.
doi: 10.1111/jnc.13571. Epub 2016 Mar 1.

Multisite tyrosine phosphorylation of the N-terminus of Mint1/X11α by Src kinase regulates the trafficking of amyloid precursor protein

Christopher J R Dunning  1 , Hannah L Black  1 , Katie L Andrews  1 , Elizabeth C Davenport  1 , Michael Conboy  1 , Sangeeta Chawla  1 , Adam A Dowle  1 , David Ashford  1 , Jerry R Thomas  1 , Gareth J O Evans  1
Affiliations

Multisite tyrosine phosphorylation of the N-terminus of Mint1/X11α by Src kinase regulates the trafficking of amyloid precursor protein

Christopher J R Dunning et al. J Neurochem. 2016 May.

Abstract

Mint/X11 is one of the four neuronal trafficking adaptors that interact with amyloid precursor protein (APP) and are linked with its cleavage to generate β-amyloid peptide, a key player in the pathology of Alzheimer's disease. How APP switches between adaptors at different stages of the secretory pathway is poorly understood. Here, we show that tyrosine phosphorylation of Mint1 regulates the destination of APP. A canonical SH2-binding motif ((202) YEEI) was identified in the N-terminus of Mint1 that is phosphorylated on tyrosine by C-Src and recruits the active kinase for sequential phosphorylation of further tyrosines (Y191 and Y187). A single Y202F mutation in the Mint1 N-terminus inhibits C-Src binding and tyrosine phosphorylation. Previous studies observed that co-expression of wild-type Mint1 and APP causes accumulation of APP in the trans-Golgi. Unphosphorylatable Mint1 (Y202F) or pharmacological inhibition of Src reduced the accumulation of APP in the trans-Golgi of heterologous cells. A similar result was observed in cultured rat hippocampal neurons where Mint1(Y202F) permitted the trafficking of APP to more distal neurites than the wild-type protein. These data underline the importance of the tyrosine phosphorylation of Mint1 as a critical switch for determining the destination of APP. The regulation of amyloid precursor protein (APP) trafficking is poorly understood. We have discovered that the APP adapter, Mint1, is phosphorylated by C-Src kinase. Mint1 causes APP accumulation in the trans-Golgi network, whereas inhibition of Src or mutation of Mint1-Y202 permits APP recycling. The phosphorylation status of Mint1 could impact on the pathological trafficking of APP in Alzheimer's disease.

Keywords: Mint1; Src; amyloid precursor protein; intracellular trafficking; protein phosphorylation; tyrosine kinase.

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Figures

Figure 1
Figure 1
The Mint1 N‐terminus is phosphorylated sequentially by C‐Src. (a) Recombinant GST fusions of the N‐terminal domains of Mint1(1‐314), Mint2(1‐341) and Mint3(1‐184) were subjected to an in vitro kinase assay with C‐Src. Tyrosine phosphorylation was detected by immunoblotting (top panel) and protein loading was confirmed by Coomassie staining [bottom panel: asterisks indicate Mint proteins; solid arrowhead = bovine serum albumin (BSA); hollow arrowhead = C‐Src]. Lower molecular weight proteolytic fragments of Mints 1 and 2 were also phosphorylated. (b) Twenty micrograms of phosphorylated Mint1(1‐314) was separated by 2D gel electrophoresis and stained with Coomassie (asterisk, top panel; + = BSA and # = dnaK). Phosphorylation was confirmed by immunoblotting (asterisk, bottom panel). A schematic of the Mint1 spot indicates how it was sampled at four sites by mass spectrometry. (c) Sequences and masses of the predominant phosphopeptides identified in spots 1–4. Phosphorylated tyrosine residues are highlighted in grey. (d) Schematic of the domain structure of Mint1 indicating the location of the three tyrosine residues sequentially phosphorylated by C‐Src.
Figure 2
Figure 2
Mint1‐Y202 is phosphorylated in cells and recruits C‐Src kinase. (a) Recombinant GST Mint1(1‐431) protein and a Y202F mutant were subjected to the same phosphorylation assay described in Fig. 1a. The samples were processed for tyrosine phosphorylation (PY; top panel) and protein loading (Coomassie; bottom panel). (b) COS‐7 cells transfected with either wild‐type or mutant (Y202F) myc‐Mint1 N‐terminus (1‐431) were treated with or without NaVO 4 3− prior to immunoprecipitation with anti‐myc antibodies. Immunoprecipitated Mint1 (top panel) and tyrosine phosphorylated Mint1 (bottom panel) were detected by immunoblotting. (c) COS‐7 cells transfected with C‐Src‐FLAG and either wild‐type or mutant (Y202F) Mint1‐HA N‐terminus (1‐431) were treated with or without NaVO 4 3− prior to immunoprecipitation with anti‐HA antibodies. Input (bottom panel) or immunoprecipitated (top panel) Mint1, total C‐Src‐FLAG and active C‐Src phosphorylated on Y416 were detected by immunoblotting. All gels and immunoblots are representative of three independent experiments.
Figure 3
Figure 3
Tyrosine phosphorylation of Mint1 does not regulate Munc18 binding. GST or GST‐Mint1(1‐314) were incubated in duplicate in the presence or absence of C‐Src kinase prior to incubation with glutathione resin and bacterial lysate containing recombinant His‐Munc18. Following washing, Munc18 present in the bound fraction or in 0.5% of the input lysate was detected by immunoblotting (top panel). Phosphorylation of Mint1 was confirmed by anti‐phosphotyrosine (middle panel). Protein levels were determined by Coomassie staining of the PVDF membrane (bottom panel). The immunoblots are representative of three independent experiments.
Figure 4
Figure 4
Mint1‐Y202 regulates amyloid precursor protein (APP) metabolism. (a) COS‐7 cells transfected in triplicate with APP‐FLAG and empty vector or myc‐Mint1‐WT or ‐Y202F were processed for immunoblotting with anti‐FLAG and anti‐myc antibodies. (b) APP immunoreactivity was quantified by densitometry and normalised to APP + Mint1‐WT. Data are plotted as mean ± SEM, n = 3 separate experiments, each performed in triplicate. **p < 0.01, ***p < 0.001. (c) COS‐7 cells transfected as described in (a) were subjected to immunocytochemistry with anti‐FLAG (APP) and anti‐myc (Mint1) antibodies and the number of FLAG reactive puncta/cell was counted using ImageJ software in myc positive cells. Data are from ≥ 20 cells per condition, ***p < 0.001. (d) Representative images are shown of COS‐7 cells transfected with APP‐FLAG and CFP or CFP‐Mint1‐WT or ‐Y202F that were stained with anti‐FLAG (red) and anti‐TGN‐46 (green) antibodies. Merged images are of the red and green channels only. Scale bar = 10 μm.
Figure 5
Figure 5
Regulation of amyloid precursor protein (APP) trafficking by Mint1 is Src dependent. COS‐7 cells were transfected with APP‐FLAG and empty myc vector (pMH) or myc‐Mint1‐WT or ‐Y202F and then incubated in the presence of 5 μM PP2 or its inactive analogue, PP3, for 48 h prior to immunocytochemistry with anti‐FLAG (APP; red) and anti‐myc (Mint1; green) antibodies. Representative images are shown of merged red and green channels except for APP alone which was co‐transfected with an empty vector. Scale bar = 10 μm.
Figure 6
Figure 6
Amyloid precursor protein (APP) recycling is Mint1‐Y202 dependent. Tetracycline‐inducible APP‐FLAG Flp‐in HeLa cells were transfected with CFP, CFP‐Mint1‐WT or ‐Y202F and then induced to express APP‐FLAG for 3, 6, 12 or 24 h prior to processing for immunofluorescence with anti‐FLAG or anti‐CFP antibodies. (a) Top panels show representative grey scale images of cells with APP‐FLAG immunoreactivity predominantly cytoplasmic, perinuclear or distributed between the two (cytoplasmic and perinuclear). Cells from each time point were then classified into one of the three phenotypes according to the localisation of APP‐FLAG puncta. Data are plotted as percentages, n ≥ 30 cells for each condition. (b) The number of APP‐FLAG reactive puncta in each CFP positive cell was quantified using ImageJ software. Data are plotted as mean ± SEM, from ≥ 30 cells per condition. Pair‐wise chi‐squared statistical analysis was carried out for each condition, with Bonferroni correction. ***p < 0.0001, comparing all conditions at 24 h.
Figure 7
Figure 7
Mint1 is phosphorylated on Y202 in cultured neurons. (a) A phosphospecific antibody (anti‐Mint‐PY202) raised to a peptide containing phospho‐Y202 was tested for specificity on lysates of recombinant N‐terminal Mint1 expressed in TKX bacteria (left panel) and full length Mint1‐WT and ‐Y202F phosphorylated in vitro by C‐Src (right panel). The lower band of ~ 90 kDa in the right panels represents a proteolytic fragment of full length Mint1. (b) Lysates from cortical neurons incubated in the presence or absence of 100 μM sodium pervanadate and/or 10 μM PP2 were processed for immunoblotting with anti‐Mint1‐pY202, anti‐Src‐pY416 (active Src) or anti‐βIII‐tubulin.
Figure 8
Figure 8
Mint1‐Y202 controls neuronal trafficking of amyloid precursor protein (APP). (a) Representative fluorescence images of cultured hippocampal neurons transfected with EGFP, myc‐Mint‐WT or myc‐Mint‐Y202F (top panels, grey scale) and APP‐FLAG (middle panels, grey scale). Bottom panels show merge of red and green channels. The extent of APP trafficking was quantified by calculating the neurite length (b) or neurite number (c) occupied by APP staining as a percentage of that occupied by EGFP or Mint staining. Data are mean ± SEM, n = 3 individual experiments (10–15 neurons analysed/condition/experiment). *p < 0.05 compared to EGFP or Mint‐Y202F. Scale bar = 20 μm.
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