Figure 3. LRRK2 and AAK1 play an independent and tissue-specific role on phosphorylation of AP2M1.

(A and B) In vitro kinase assays assessing the amount of ³²P-ATP incorporated by AP2M1 upon equimolar amounts of GST-tagged LRRK2 G2019S (GS) (970–2527aa) and GST-tagged AAK1 (1–501aa). Note that because GST-AAK1 (1–501aa) fragment and GST-AP2M1 have similar molecular weights, the signal in the lane of GST-AAK1 with AP2M1 includes both AAK1 autophosphorylation and AP2M1 phosphorylation by AAK1; thus, to calculate AP2M1 phosphorylation by AAK1, the intensity was subtracted by the AAK1 autophosphorylation signal. (C and D) Immunoblotting analysis of endogenous AP2M1 phosphorylation at Thr¹⁵⁶ upon LRRK2 inhibition with MLi-2 and AAK1 inhibition with LP-935509 (0.1 and 1 μM, respectively, for 2 hours). Immunoblotting was performed with antibodies to pThr¹⁵⁶-AP2M1 and pSer⁹³⁵-LRRK2. (E and F) Immunoblotting analysis of endogenous AP2M1 Thr¹⁵⁶ phosphorylation in LRRK2 WT and KO SH-SY5Y cells with overexpression of AAK1. (G and H) Immunoblotting analysis of endogenous AP2M1 Thr¹⁵⁶ phosphorylation in LRRK2 WT or KO SH-SY5Y cells upon transfection of AAK1 shRNA. (I and J) Immunoblotting analysis of endogenous AP2M1 Thr¹⁵⁶ phosphorylation in brain tissue and thyroid gland lysates from LRRK2 WT or KO mice. All quantification data in (A to J) are mean ± SEM from n=5 independent experiments; each experiment in (I and J) consisted of 3 WT and 3 KO mice. Blots were quantified by ImageJ software. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student’s t tests for two comparisons or one-way ANOVA followed by a Tukey’s post hoc test for multiple comparisons.