Journal: Scientific Reports
Article Title: Development of a novel AAK1 inhibitor via Kinobeads-based screening
doi: 10.1038/s41598-024-57051-9
Figure Lengend Snippet: Identification and characterization of AAK1 as a TIM-063–interactant. ( a ) Protocol for identification of TIM-063–interactants using TIM-063–immobilized sepharose (TIM-127-sepharose ) combined with mass spectrometry analysis. Mouse cerebrum extracts were applied onto TIM-127-sepharose, and after extensive washing of resin, TIM-063–interactants were eluted by the addition of 100 µM TIM-063 containing buffer, followed by identification via mass spectrometry analysis as described in the “ ”. ( b ) Eluates from TIM-127-sepharose (TIM-127–S) or a control sepharose without the inhibitor (Control–S) as described in ( a ) were analyzed by immunoblotting with either anti-CaMKKβ/2 antibody ( upper panel ), anti-AAK1 antibody ( middle panel ), or anti-ERK1/2 antibody ( bottom panel ) together with mouse cerebrum extracts (Input). ( c ) Extracts from COS-7 cells expressing His-tagged AAK1 wild type (1–863) or His-tagged AAK1 catalytic domain (25–396) were incubated with either TIM-127-sepharose or control sepharose, and after extensive washing of resin, TIM-063–interactants were eluted by the addition of 100 µM TIM-063–containing buffer, followed by immunoblot analysis with an anti-AAK1 antibody as described in the “ ”. An arrow and asterisk indicate His-tagged AAK1 wild type (1–863) and His-tagged AAK1 catalytic domain (25–396), respectively. ( d ) Phosphorylation of GST-AP2µ2 (145–162) at Thr156 by the His-tagged AAK1 catalytic domain (25–396) for various time periods (0–120 min) was detected by immunoblot analysis with an anti-phospho-AP2µ2 (pThr156) antibody ( insert ), quantitated by densitometric scanning of the immunoreactive bands, and expressed as a percentage of the value at 120 min of the reaction. An asterisk indicates phospho-Thr156 in GST-AP2µ2 (145–162). ( e ) Inhibition of AAK1 activity by TIM-063 in a dose-dependent manner. Phosphorylation of GST-AP2µ2 (145–162) by His-tagged AAK1 catalytic domain (25–396) was measured in the presence or absence of various concentrations of TIM-063 at 30 °C for 20 min using 100 µM [γ- 32 P]ATP as described in the “ ”. AAK1 activities are expressed as a percentage of the average value in the absence of the compound. Results represent duplicate experiments. Chemical structure of TIM-063 is indicated ( insert ). Molecular mass markers (kDa) are indicated in the left lanes of immunoblot panels.
Article Snippet: Anti-phospho-AP2µ2(AP2M1) at Thr156 (D4F3, 7399S) and anti-His-tag (66005-1-Ig) antibodies were obtained from Cell Signaling Technology (Danvers, MA) and Proteintech (Rosemont, IL), respectively.
Techniques: Mass Spectrometry, Western Blot, Expressing, Incubation, Inhibition, Activity Assay
Cell Signaling Technology Inc is a verified supplier 
Cell Signaling Technology Inc manufactures this product 
![Identification and characterization of AAK1 as a TIM-063–interactant. ( a ) Protocol for identification of TIM-063–interactants using TIM-063–immobilized sepharose (TIM-127-sepharose ) combined with mass spectrometry analysis. Mouse cerebrum extracts were applied onto TIM-127-sepharose, and after extensive washing of resin, TIM-063–interactants were eluted by the addition of 100 µM TIM-063 containing buffer, followed by identification via mass spectrometry analysis as described in the “ ”. ( b ) Eluates from TIM-127-sepharose (TIM-127–S) or a control sepharose without the inhibitor (Control–S) as described in ( a ) were analyzed by immunoblotting with either anti-CaMKKβ/2 antibody ( upper panel ), anti-AAK1 antibody ( middle panel ), or anti-ERK1/2 antibody ( bottom panel ) together with mouse cerebrum extracts (Input). ( c ) Extracts from COS-7 cells expressing His-tagged AAK1 wild type (1–863) or His-tagged AAK1 catalytic domain (25–396) were incubated with either TIM-127-sepharose or control sepharose, and after extensive washing of resin, TIM-063–interactants were eluted by the addition of 100 µM TIM-063–containing buffer, followed by immunoblot analysis with an anti-AAK1 antibody as described in the “ ”. An arrow and asterisk indicate His-tagged AAK1 wild type (1–863) and His-tagged AAK1 catalytic domain (25–396), respectively. ( d ) Phosphorylation of <t>GST-AP2µ2</t> (145–162) at <t>Thr156</t> by the His-tagged AAK1 catalytic domain (25–396) for various time periods (0–120 min) was detected by immunoblot analysis with an anti-phospho-AP2µ2 (pThr156) antibody ( insert ), quantitated by densitometric scanning of the immunoreactive bands, and expressed as a percentage of the value at 120 min of the reaction. An asterisk indicates phospho-Thr156 in GST-AP2µ2 (145–162). ( e ) Inhibition of AAK1 activity by TIM-063 in a dose-dependent manner. Phosphorylation of GST-AP2µ2 (145–162) by His-tagged AAK1 catalytic domain (25–396) was measured in the presence or absence of various concentrations of TIM-063 at 30 °C for 20 min using 100 µM [γ- 32 P]ATP as described in the “ ”. AAK1 activities are expressed as a percentage of the average value in the absence of the compound. Results represent duplicate experiments. Chemical structure of TIM-063 is indicated ( insert ). Molecular mass markers (kDa) are indicated in the left lanes of immunoblot panels.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4696/pmc10954696/pmc10954696__41598_2024_57051_Fig1_HTML.jpg)
