Azorsa et al. BMC Genomics 2010, 11:25 http://www.biomedcentral.com/1471-2164/11/25 Table 1 Candidate kinases that phosphorylate 12E8 tau Page 4 of 10 Gene Name MARK2 PAK3 PAK2 ADCK5 AKAP13 LOC55971 PLK2 DYRK1A MAK ITK PIM1 RAGE ITPK1 CKB PFKM DGKB SPHK2 Cell Count 453 170 287 280 181 152 335 316 393 285 409 397 399 223 375 440 333 12E8 Tau/Control Average St Dev p value 0.672 0.044 0.006 0.803 0.067 0.036 0.726 0.081 0.028 0.716 0.104 0.042 0.738 0.069 0.023 0.836 0.035 0.014 0.774 0.087 0.046 0.774 0.051 0.017 1.109 0.021 0.012 1.124 0.036 0.027 1.248 0.097 0.047 1.305 0.106 0.038 1.324 0.119 0.042 1.537 0.143 0.023 1.305 0.096 0.032 1.350 0.096 0.024 1.792 0.282 0.040 Average 0.979 1.112 0.986 0.934 0.929 0.982 0.906 0.848 0.919 0.887 0.965 0.960 0.930 1.065 0.888 0.921 1.074 Total Tau St Dev 0.039 0.197 0.101 0.094 0.070 0.019 0.114 0.104 0.037 0.059 0.105 0.088 0.084 0.146 0.112 0.163 0.121 p value 0.451 0.429 0.831 0.351 0.223 0.241 0.289 0.127 0.063 0.079 0.624 0.511 0.285 0.523 0.227 0.488 0.399 12E8 Tau/Total Tau Average St Dev p value 0.687 0.060 0.012 0.730 0.072 0.023 0.741 0.109 0.054 0.765 0.069 0.028 0.797 0.086 0.054 0.852 0.042 0.026 0.856 0.052 0.040 0.860 0.038 0.023 1.209 0.054 0.021 1.273 0.118 0.057 1.297 0.080 0.023 1.361 0.053 0.007 1.424 0.024 0.001 1.454 0.138 0.030 1.476 0.082 0.010 1.489 0.200 0.052 1.664 0.114 0.010 A list of kinases affecting 12E8 tau phosphorylation. Shown are the most significant effectors of 12E8 tau phosphoylration emerging from a high-content siRNA screen of the complete “kinome.” Indicated in the columns from left to right are, the Gene Name, the cell counts (which refers to the number of cells in the well following transfection and is and indicator of potential siRNA toxicity), and then the average fold effect, standard deviation, and p value for 12E8 tau, total tau, and the ratio of 12E8 tau/total tau. For a reference on cell counts, non-siliencing s iRNA control cell counts range from ~330-380, depending on the individual plates in the assay. This tabel contains only those siRNAs that affect pS262 tau/total tau through significant effects on pS262 tau alone. Total tau levels were selected to be insignificantly effected (p > 0.05), although some candidates clearly show a strong trend toward significant reductions in total tau. confirm this hit via secondary measures. Rather, we chose to validate two additional candidates. These were A-kinase anchor protein 13 (AKAP13) and dual specifi- city tyrosine phosphorylation regulated kinase 1A (DYRK1A), which were selected both for the significance of the effect (based on p values for the ratio of 12E8/ total tau) and for the known biology of these candidates (see Discussion). The gene encoding DYRK1A is located within the Down Syndrome critical region (DSCR) on chromosome 21. Screening results showed 12E8 tau levels to be reduced to 77 ± 5% of control (p = 0.017), total tau to be 85 ± 10% of control (p = 0.127), which lead to a significant reduction in the ratio of 12E8 tau/ total tau to 86 ± 4% of control (p = 0.023; Table 1 and Figure 3A). We confirmed via western analyses that DYRK1A is required for efficient phosphorylation of 12E8 tau. We retransfected our cell line with siRNA tar- geting DYRK1A and quantitated effects on 12E8 tau and total tau levels via western (Figure 3B). Results con- firmed that DYRK1A is required for maintaining normal levels of 12E8 tau. The DYRK1A siRNA reduced DYRK1A protein expression by over 60%, causing a nearly 40% reduction of 12E8 tau compared to the non- silencing siRNA control. DYRK1A siRNA had no signifi- cant effect on total tau levels in this assay. Interestingly, the effects on tau phosphorylation are strikingly larger than observed in the fluorescence screening assay. Rea- sons for this are unclear, however it suggests that the screening assay may be underestimating the magnitude of effects on tau phosphorylation. Since DYRK1A is a proline- directed serine/threonine kinase and serine 262 is a non-proline directed site on tau, these effects of DYRK1A may result from indirect effects through addi- tional kinases (see Discussion). Tests of AKAP13 showed that this protein kinase A (PKA) associated protein is also involved in 12E8 tau phosphorylation. Screening results showed 12E8 tau levels to be reduced to 74 ± 7% (p = 0.023) of control non-silencing siRNA samples, total tau levels to be 93 ± 7% (p = 0.223) of control, leading to a reduction in the ratio of 12E8 tau/total tau to 79 ± 9% (p = 0.054) of con- trol (Table 1 and Figure 4A). Because this phosphoryla- tion profile resembled that of MARK2, a confirmed S262 tau kinase, we proceeded to confirm these effects via sec- ondary measures. Western results demonstrated a role for AKAP13 in maintaining 12E8 tau phosphorylation levels. The AKAP13 siRNA reduced AKAP13 protein expression to undetectable levels (Figure 4B), causing a nearly 50% reduction of 12E8 tau. AKAP13 siRNA had no significant effect on total tau levels in this assay. Some drosophila models have shown that increased tau expression can lead to neurodegeneration, albeit in the absence of NFT pathology[25]. Thus the potential for a tau therapeutic that affects both phosphorylated and total tau levels cannot at this point be diminished. We therefore performed a second analysis of the data to