Figure S1 - Phosphorylation by Dyrk1A of Clathrin Coated Vesicle-Associated Proteins: Identification of the Substrate Proteins and the Effects of Phosphorylation

Phosphorylation of the CCV-Associated Proteins by Full-Length and Truncated Dyrk1A. Four different CCV preparations (a–d) were incubated with full-length Dyrk1A (DyrkFL), GST-full-length Dyrk1A (GST-DyrkFL), or GST-truncated Dyrk1A (GST-Dyrk497) under the conditions as described in Fig. 1. The ultracentrifugation step was omitted, and the reaction mixtures were directly subjected to SDS-PAGE followed by autoradiography. Two distinct kinase preparations (#1 and #2) were used for each DyrkFL and GST-DyrkFL. (A) CCV (preparation a, 12 µg/assay) was incubated with 1.25 µg each of DyrkFL (#1) and GST-Dyrk497. The reaction mixtures were subjected to SDS-PAGE using 7% acrylamide-0.128% bis-acrylamide gels as in Fig. 1. In this panel, the two lanes with DyrkFL were exposed twice as long as those with GST-Dyrk497. Under our SDS-PAGE conditions, DyrkFL migrated with an apparent molecular weight (∼115 kDa) greater than the calculated value of DyrkFL (87 kDa) for both endogenous (expressed in rat brain) and recombinant kinases. (B) Two CCV preparations (b and c; 7 and 14 µg, respectively) were phosphorylated by 0.5 µg each of GST-DyrkFL (#1) and GST-Dyrk497. To avoid co-migration of the autophosphorylated Dyrk1A band and band 4, SDS-PAGE was carried out using 7% acrylamide-0.22% bis-acrylamide gels. Autoradiogram is shown. All lanes have the same exposure time. The phosphorylated bands 1+2, 3, and 4 were scanned, and the relative ratios of the 32P-labeled bands 1+2 to bands 3 and 4 were calculated for each CCV preparation (b & c) phosphorylated with either truncated or full-length Dyrk1A; the ratios were 1∶0.69∶0.75 and 1∶0.73∶0.78 for CCV-b with truncated and full-length Dyrk1A, respectively. Similarly, the ratios were 1∶0.70∶0.54 and 1∶0.80∶0.61 for CCV-c with truncated and full-length Dyrk1A, respectively. (C) CCVs (preparation d) were incubated with DyrkFL (#2), GST-DyrkFL (#2), or GST-Dyrk497, and subjected to SDS-PAGE as described in (B). In the panel, the CCV lanes with DyrkFL were exposed twice as long than those by GST-DyrkFL and GST-Dyrk497. The phosphorylation patterns of bands 1–4 were very similar with all three kinase preparations. Relative ratios of phosphorylated bands 1+2 to bands 3 and 4 were 1∶0.74∶0.53, 1∶0.84∶0.68, and 1∶0.71∶0.49 for phosphorylation with DyrkFL, GST-DyrkFL, and GST-Dyrk497, respectively. Thus, full-length and the truncated forms of Dyrk1A recognize the CCV-associated substrates with a similar manner. For the CCVs with GST-DyrkFL, the ratios were not normalized for the autophosphorylated kinase band. Note that full-length kinases showed much higher autophosphorylation levels than that of GST-Dyrk497. This is because the C-terminal domain contains multiple autophosphorylation sites and truncated kinase should be less autophosphorylated than the full-length kinase. Reduction in the autophosphorylation levels when incubated with the substrates may indicate that the autophosphorylation of Dyrk1A is in intermolecular, not intramolecular, event. *, autophosphorylated GST-Dyrk497.
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