Phosphorylation by Dyrk1A of Clathrin Coated Vesicle-Associated Proteins: Identification of the Substrate Proteins and the Effects of Phosphorylation
Figure 4
Release of MAP1A and MAP2 into the soluble fraction after phosphorylation.
(A) Release of MAP1A. CCVs (20 µg) were phosphorylated and ultracentrifuged as described in Fig. 1 except for using 2 mM cold ATP and with or without GST-Dyrk1A497. The resultant supernatants were blotted with anti-MAP1A antibody (M1A). (n = 3). (B) Phosphorylation-dependent dissociation of MAP1A and MAP2. CCVs were incubated as in (A) in duplicate tubes containing ATP and either wild type (wt) or double mutant (DF) GST-Dyrk1A497. After adding EDTA, one set of tubes was kept on ice, whereas the other set was ultracentrifuged to collect the soluble fractions. Aliquots of whole mixtures (W) and the supernatants (S) were subjected to immunoblotting. Both MAP1A (M1A) and MAP2 (M2) were identified in the same lane by blotting the PVDF membrane first with anti-MAP2 then with anti-MAP1A antibodies. (n = 1, various preliminary performances carried out to lead the final assay conditions are not included).