Figure 4

Maximal activity of HIPKs depends on the activation loop tyrosine. Wild type GFP-HIPK fusion proteins and the respective Tyr→Phe mutants were immunoprecipitated from HeLa cells and subjected to kinase assays with recombinant GST-p27^Kip1 (A), myelin basic protein (B) or DYRKtide (C). GFP served as background control. A, Phosphorylation of p27^Kip1 at Ser10 was detected by immunoblotting with a phosphorylation-specific antibody. For quantitative evaluation, pSer10 immunoreactivity was normalised to GFP immunoreactivity, which reflects the amount of kinase in the reaction. The blots illustrate a representative experiment, and the relative catalytic activities as determined from 3–4 assays are shown below the panels (means ± SD). One-sample t test: *, p < 0.05; **, p < 0.01. B and C, Phosphorylation of MBP and DYRKtide was measured in triplicate as incorporation of ³²P. Background values from the GFP control samples were subtracted and activities were normalised to the amount of kinase in the reaction as determined by GFP immunoreactivity. Column diagrams illustrate catalytic activities relative to HIPK2 (WT). The results were replicated in independent experiments, except for a missing value of HIPK1.