Figure 8. Phosphorylation of p27^Kip1 and Cyclin D1 by DYRK1A in vitro and in cells. (A) In vitro kinase assay. Kinase assay with GFP-DYRK1A and GFP-DYRK1B immunoprecipitated from HeLa cells and subjected to a kinase assay with recombinant GST-p27^Kip1. The phosphorylation reaction was started by ATP addition (+ ATP). No ATP addition served as negative control. (B) In vitro kinase assay with bacterial GST fusion proteins of DYRK1A or kinase-deficient DYRK1A-K188R and p27^Kip1. Aliquots of the kinase reaction were taken at the indicated time points. Control lanes (c) were loaded only with GST-p27^Kip1. The majority of GST-DYRK1A is isolated from E. coli as a catalytically active C-terminally truncated product (marked by an asterisk).⁷⁹ (C) Phosphorylation of p27^Kip1 by DYRK1A in HeLa cells. HeLa cells were transiently transfected with expression plasmids for p27^Kip1, GFP-DYRK1A, and GFP-HIPK2 as indicated. To analyze p27^Kip1 phosphorylation by endogenous DYRK1A, cells were treated with 1 µM of the DYRK1A inhibitor AnnH31 for 5 h before cell lysis. Total protein extracts then were analyzed for p27^Kip1 phosphorylation by western blotting with the indicated antibodies. Relative phosphorylation of Ser10 is indicated below the bottom panel. GFP-HIPK2 was not resolved on this gel due to its large size. One representative blot from n = 3 each. (D) DYRK1A induces Cyclin D1 Thr286 phosphorylation independently of GSK3β. SH-SY5Y cells were treated with 0.5 µg/ml doxycycline (+dox) to induce DYRK1A overexpression. After 5 h, cells were additionally treated with the DYRK1A inhibitor leucettine L41 (1 µM) or the GSK3β inhibitor CHIR99021 (3 µM) for further 24 h before total cellular protein was analyzed by western blotting with the indicated antibodies. Stabilization of β catenin due to reduced GSK3β-mediated phosphorylation was used to validate GSK3β inhibition. The vertical line indicates where an irrelevant lane was deleted from the final image. Column diagrams show the densitometric quantification of Cyclin D1 Thr286 phosphorylation and total β catenin protein levels from 3 independent experiments.