Identification of the phosphorylated protein bands 1 and 2 as MAP1A and MAP2.

<p>(<b><i>A</i></b>) <i>MAP1A and MAP2 in the phosphorylated CCVs and MTs</i>. CCVs (20 µg) and purified MTs (5 µg) were incubated with [γ-³²P]-ATP with (+) or without (−) GST-Dyrk1A⁴⁹⁷ as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034845#pone-0034845-g001" target="_blank">Fig. 1</a>, followed by SDS-PAGE without ultracentrifugation. Approximately 9 µg and 2 µg of CCVs and MTs, respectively, were applied per lanes. After transferring proteins, each lane of the PVDF membranes was cut into two strips for immunostaining either with anti-MAP1A (<i>a</i>) or anti-MAP2 (<i>b</i>) antibody, or for Coomassie Blue staining (<i>c</i>). The strips were reassembled (<i>WB/CB</i>) and subjected to autoradiography (<i>³²P</i>). (<i>n = 2</i>). (<b><i>B</i></b>) <i>Coomassie Blue-staining of the CCV and MT preparations</i>. Ten and five µg of CCVs and MTs, respectively, were applied per lane. (<b><i>C</i></b>) <i>Immunoprecipitation of MAP1A and MAP2 from the phosphorylated MTs</i>. MTs (200 µg) were phosphorylated for 1 hr with GST-Dyrk1A⁴⁹⁷ (18 µg) and 0.2 mM [γ-³²P]-ATP in a final volume of 250 µl. After the reaction, the soluble fraction was subjected to immunoprecipitation (<i>IP</i>) by using anti-MAP2 (<i>M2</i>) or anti-MAP1A (<i>M1A</i>) antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034845#s2" target="_blank"><i>MATERIALS AND METHODS</i></a>. A negative control for the immunoprecipitation (−) was obtained without primary antibody. The immunoprecipitates were applied to SDS-PAGE followed by Coomassie Blue staining (<i>CB</i>) and autoradiography (<i>³²P</i>). (<i>n = 1; various preliminary performances carried out to lead the final assay conditions are not included</i>). Scanning of the MAP1A and MAP2 bands from the original material used for immunoprecipitation (<i>Ori</i>) gave the arbitrary units for these proteins as 3306 and 6323, respectively, whereas those for the radioactivity were 6056 and 12582, respectively. (<b><i>D</i></b>) <i>Immunoprecipitation of MAP1A from the extract of the phosphorylated CCVs</i>. CCVs (60 µg) were incubated with GST-Dyrk1A⁴⁹⁷ (7 µg) and 0.2 mM [γ-³²P]-ATP for 1 hr in a final volume of 120 µl. The phosphorylated CCVs were extracted with 0.5 M Tris-HCl, diluted the Tris-HCl concentration, and used for immunoprecipitation with anti-MAP1A antibody (<i>IP</i>). The immunoprecipitates were subjected to blotting (<i>WB</i>) with anti-MAP1A antibody followed by autoradiography (<i>³²P</i>). (<i>n = 2</i>). <i>St</i>, pre-stained standard proteins; <i>M1A</i>, MAP1A; <i>M2</i>, MAP2.</p

Localization of Dyrk1A and clathrin in the primary cultured neurons.

<p>E-18 rat hippocampal neurons were differentiated, fixed with 2% formaldehyde, and immunostained with monoclonal anti-CHC followed by Alexa Fluor 488 conjugated goat anti mouse IgG. After completion of CHC staining, the cells were incubated with monoclonal anti-Dyrk1A antibody (7F3), directly conjugated with Alexa Fluor 568 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034845#s2" target="_blank"><i>MATERIALS AND METHODS</i></a>. (<i>a</i>), single confocal images; (<i>b</i> and <i>c</i>), Z-stack images. (<i>n = 2</i>). Pearson's correlation coefficient for single scanning images was 0.645 as calculated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034845#pone-0034845-g010" target="_blank">Fig. 10</a>.</p

Co-localization of endogenous Dyrk1A and clathrin in the adult mouse brains.

<p>Forty micrometer sections of the mouse brain tissues were incubated with polyclonal anti-Dyrk1A (red, <i>a</i>, <i>d</i>, and <i>g</i>) and monoclonal anti-CHC (green, <i>b</i>, <i>e</i>, and <i>h</i>) antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034845#s2" target="_blank"><i>MATERIALS AND METHODS</i></a>. Presented are Z-stack images collected from the subiculum (<i>a–c</i>), the medio-posterior thalamic nucleus (<i>d–f</i>), and individual confocal images from the cerebellar Purkinje cell layer (<i>g–i</i>). Structures showing co-localization of Dyrk1A and CHC are visible on merged images (yellow in <i>c, f, i</i>). The dotted squares shown in <i>a–i</i> are enlarged in the upper right corners. Scale bar = 10 µm. Panels (<i>j–m</i>) show three consecutive scanning images from subiculum. Pearson's correlation coefficients for three consecutive scanning images from subiculum and the medio-posterior thalamic nucleus were calculated as 0.439 and 0.499, respectively, by using NIH Image J with the JACoP plug-in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034845#pone.0034845-Bolte1" target="_blank">[65]</a>.</p

Phosphorylation of the membrane-unbound adaptor proteins.

<p><i>(</i><b><i>A</i></b><i>) Immunoprecipitation of AP180 from the phosphorylated PNP extract</i>. The Tris-HCl extract from PNP (diluted, 300 µl) prepared as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034845#s2" target="_blank"><i>MATERIALS AND METHODS</i></a> was phosphorylated for 1 hr in a mixture containing 0.2 mM [γ-³²P]-ATP, 9 µg Dyrk1A, 5 mM MgCl₂, 0.12 M NaCl, 0.1 mM EGTA, and 0.2 mM DTT. After the reaction, the mixture was subjected to immunoprecipitation by using anti-AP180 antibody as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034845#pone-0034845-g003" target="_blank">Fig. 3<i>B</i></a>. (<i>n = 2</i>). The immunoprecipitate (<i>IP</i>) and an aliquot of the original phosphorylation mixture (<i>Ori</i>) were subjected to immunoblotting (<i>WB</i>) and autoradiography (<i>³²P</i>). (<b><i>B</i></b>) <i>Phosphorylation of the immunoprecipitated AP180</i>. The PNP extract was first subjected to immunoprecipitation with (+) and without (−) anti-AP180 antibody. The pellets of Protein A/G resins were washed three times with PBS-T and once with kinase buffer, and suspended in a small volume of kinase buffer (20 µl). Phosphorylation reaction was carried out in the presence of 0.2 mM [γ-³²P]-ATP and Dyrk1A (2 µg) in a final liquid volume of 25 µl. Aliquots of the reaction mixtures were subjected to SDS-PAGE followed by Coomassie-Blue staining (<i>CB</i>) and autoradiography (<i>³²P</i>). <i>Arrow</i>, AP180. (<i>n = 1</i>). (<b><i>C, D</i></b>) <i>Immunoprecipitation of α- and β-adaptins after phosphorylation reaction</i>. The PNP extract (<b><i>C</i></b>) and cytosol (<b><i>D</i></b>) were incubated with Dyrk1A and [³²P]-ATP and subjected to immunoprecipitation with (<i>IP</i>) and without (−) corresponding antibodies. The immunoprecipitates and the aliquots of original reaction mixtures (<i>Ori</i>) were subjected to immunoblotting and autoradiography. (<i>n = 3</i>). (<b><i>E</i></b>) <i>Incubation of the immunoprecipitated adaptins with Dyrk1A</i>. Cytosol and the PNP extract were first subjected to immunoprecipitation without (−) and with (+) anti-α-adaptin antibody. The resultant precipitates were incubated with Dyrk1A and [γ-³²P]-ATP followed by immunoblotting (<i>WB</i>) and autoradiography (<i>³²P</i>) as described in (<i>B</i>). (<i>n = 1, various preliminary performances carried out to lead the final assay conditions are not included</i>). α, α-adaptin; β, β-adaptin; PNP-Ext, PNP extract; arrowheads, α-adaptin; <i>arrow</i>, β-adaptin; *, autophosphorylated Dyrk1A.</p

Dissociation of the phosphorylated proteins from CCVs.

<p>Rat brain CCVs (20 µg) were incubated with 0.1 mM [γ-³²P]-ATP with or without GST-Dyrk1A⁴⁹⁷ (3.7 µg) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034845#s2" target="_blank"><i>MATERIALS AND METHODS</i></a>. After mixing with EDTA and phosphatase inhibitors, the samples were transferred on ice and ultracentrifuged at 70,000 rpm for 15 min using a Beckman TLA-100 rotor. The supernatants (<i>S</i>) were collected, and the precipitates (<i>P</i>) were suspended in the original volume of the kinase buffer containing protease- and phosphatase- inhibitors. Each sample was subjected to SDS-PAGE followed by Coomassie Blue staining (<i>CB</i>) and autoradiography (<i>³²P</i>). Half of each SDS sample was applied per lane. The numbers in the right panel refer to the phosphorylated protein bands 1–5. Dyrk1A⁴⁹⁷ was used in most of our experiments, because this truncated form 1) is highly purified in contrast to the full-length protein, which always contains kinase bands degraded to various extents <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034845#pone.0034845-Murakami2" target="_blank">[20]</a>, and 2) exhibits the similar kinase activity as the full-length protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034845#pone.0034845-Adayev2" target="_blank">[21]</a>. *, denotes clathrin heavy chain (CHC). (<i>n = 3; the assay was repeated three times by using different CCV preparations giving similar results</i>.)</p

Ratios of adaptin subunits recovered in cytosol to the precipitated fractions from cultured cells.

<p>Cytosol (<i>Sup</i>) and precipitated (<i>Ppt</i>) fractions from CHO and PC12 cells were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034845#s2" target="_blank"><i>MATERIALS AND METHODS</i></a>. SDS-PAGE was carried out by applying equal volumes of the soluble and insoluble fractions in each lane side-by-side. Immunoblotting was performed by using corresponding antibodies derived from mouse and rabbit. The first blots with mouse and rabbit antibodies were stripped (stripping buffer, PIERCE) and re-blotted with rabbit and mouse antibodies, respectively, for detecting other adaptin subunits in the same membranes. The antibodies used were mouse monoclonal antibodies against anti-α and γ-adaptins, rabbit monoclonal anti-β-adaptin antibody, and rabbit polyclonal anti-μ adaptin antibody. Each adaptin band in the <i>Sup</i> and <i>Ppt</i> was scanned, and the <i>Sup</i> to <i>Ppt</i> ratio was calculated. Four independent samples per each cell type were shown.</p

Effect of phosphorylation on dissociation of adaptor proteins in the presence of Mg²⁺.

<p><i>(</i><b><i>A</i></b><i>) Effect of Mg²⁺ on AP180 dissociation</i>. CCVs (3 µg) were incubated with Dyrk1A and cold ATP as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034845#pone-0034845-g005" target="_blank">Fig. 5<i>A</i></a>, but with modified conditions including 75 mM HEPES (pH 7.0), 1 mg/ml BSA, and a mixture of phosphatase-inhibitors. After the reaction, the tubes were mixed with either 10 mM EDTA (<i>EDTA</i>) or with H₂O (<i>Mg²⁺</i>) and ultracentrifuged. The resultant supernatant fractions were blotted with anti-AP180 antibody. (<i>n = 3</i>). Duplicated samples were shown. <i>(</i><b><i>B</i></b><i>) Effect of phosphorylation on adaptor protein dissociation.</i> CCVs were incubated as in <i>(A)</i> with ± Dyrk1A and ± ATP as indicated in the panel. The reaction mixtures were ultracentrifuged without adding EDTA. The same volumes of the supernatants and whole reaction mixtures (<i>W</i>) were immunoblotted using the indicated antibodies. Duplicated samples were shown. (<i>n = 1, various preliminary performances carried out to lead the final assay conditions are not included</i>).</p

Proposed Dyrk1A functions in regulating synaptic vesicle endocytosis.

<p>Dyrk1A phosphorylated AP180 in cytosol. The phosphorylated AP180 may decrease its binding affinity to the AP2 complex; we hypothesize here that the decrease in such binding affinity would reduce recruitment of clathrin at the <i>nucleation</i> and <i>invagination</i> sites. Phosphorylation of dynamin 1, amphiphysin 1, and synaptojanin 1 at their PRD reduces the interaction between the PRDs and the SH3 domains of amphiphysin and endophilin. This likely slows down the <i>invagination</i> and <i>fission</i> steps of synaptic vesicle formation. Once endocytosed, the vesicle-associated proteins are quickly removed from the membranes (<i>uncoating</i>). The Dyrk1A-mediated phosphorylation releases first AP180 and β-adaptin from the vesicle membranes, while both α- and μ-adaptin subunits remain bound with the membranes. Additional factor(s) are required to release the membrane-bound subunits. Clathrin release from the vesicles is independent from Dyrk1A. We speculate that the adaptin subunits released in cytoplasm may reassemble into the AP2 complex after dephosphorylation.</p

Relative kinase concentrations in the CCV fraction.

<p>Rat brain fractions S1, P2, P3 were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034845#s2" target="_blank"><i>MATERIALS AND METHODS</i></a>. These fractions together with CCVs were subjected to SDS-PAGE followed by immunoblotting using 8D9 (<i>Dyrk1A</i>). Fifteen µg of protein were applied per each lane. After stripping, the same membrane was re-blotted with anti-CHC antibody. (<i>n = 1</i>, <i>various preliminary performances carried out to lead the final assay conditions are not included</i>).</p

Identification of the phosphorylated protein bands 3 and 4.

<p>(<b><i>A</i></b>) <i>Migration patterns of AP180 and band 3 in SDS-PAGE</i>. After [³²P]-phosphorylation, CCVs were subjected to SDS-PAGE followed by blotting with anti-AP180 antibody (WB), Coomassie-Blue staining (<i>CB</i>), and autoradiography (<i>³²P</i>) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034845#pone-0034845-g002" target="_blank">Fig. 2<i>A</i></a>. (<i>n = 2</i>). Approximately 1.3 µg proteins were applied per lane, and a single lane of the PVDF membrane was cut into two strips. Arrowheads, AP180; asterisk, CHC. Two lower bands in the autoradiogram are band 4 and the autophosphorylated GST-Dyrk1A⁴⁹⁷. (<b><i>B</i></b>) <i>Immunoprecipitation of AP180 from the extract of the phosphorylated CCVs</i>. CCVs were incubated with GST-Dyrk1A⁴⁹⁷ and [γ-³²P]-ATP, extracted with Tris-HCl, and used for immunoprecipitation (<i>IP</i>) using anti-AP180 antibody (anti-SNAP91), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034845#pone-0034845-g002" target="_blank">Fig. 2<i>D</i></a>. The resultant immunoprecipitates were subjected to blotting using anti-AP180 antibody (<i>WB:AP180</i>) followed by autoradiography (<i>³²P</i>). (<i>n = 2</i>). (<i>−</i>) and (<i>+</i>), immunoprecipitation without and with anti-AP180 antibody, respectively; <i>Ori</i>, the CCV extract used for immunoprecipitation. <i>(</i><b><i>C</i></b><i>) Immunoprecipitation of β-adaptin</i>. The CCV extract prepared as in <i>(B)</i> was subjected to immunoprecipitation without (−) or with (+) anti-β-adaptin antibody (<i>IP:β</i>). (<i>n = 2</i>). One lane of the starting extract (<i>Ori</i>) was cut into two strips; one was probed with anti-β-adaptin (<i>strip 1</i>) together with the immunoprecipitates (<i>WB:β</i>), and the other (<i>strip 2</i>) and lane <i>3</i> were incubated with anti-α-adaptin antibody (<i>α</i>). All strips were reassembled for autoradiography (<i>³²P</i>). The lower band in the WB panel is IgG heavy chain. <i>(</i><b><i>D</i></b><i>) Immunoprecipitation of β-adaptin but not α-adaptin by anti-β-adaptin antibody</i>. The membranes containing <i>lane (+)</i> and <i>strip 1</i> from <i>(C)</i> were re-blotted with anti-α-adaptin (<i>WB:β+α</i>) and re-assembled with strip <i>2</i> from (<i>C</i>). The ratios in relative intensities of α- (<i>APα</i>) to β (<i>APβ</i>)-adaptins in the <i>IP:β</i> lane and <i>strip 1</i> shown in here were 1∶40.5 and 1∶2.7, respectively. <i>(</i><b><i>E</i></b><i>) Immunoprecipitation of α-adaptin</i>. After the first immunoprecipitation using anti-β-adaptin antibody <i>(C)</i>, the unbound fraction was incubated with (+) or without (−) anti-α-adaptin antibody (<i>IP:α</i>) for the second immunoprecipitation. The precipitates were subjected to immunoblotting using anti-α-adaptin antibody (<i>WB:α</i>) followed by autoradiography (<i>³²P</i>). (<i>n = 1</i>). <i>(</i><b><i>F</i></b><i>) Co-precipitation of α- and β-adaptins from the extracts of unphosphorylated CCVs</i>. CCVs in two tubes were diluted in kinase buffer, mixed with either H₂O (<i>Mg²⁺</i>) or 10 mM EDTA, and extracted with 0.5 M Tris-HCl for immunoprecipitation with anti-β-adaptin antibody as in <i>(C)</i>. (<i>n = 3</i>). The immunoprecipitates (<i>IP</i>) and the original extracts (<i>Ori</i>) were blotted using antibodies against α- or β-adaptin (<i>WB: α, β</i>).</p