Azorsa et al. BMC Genomics 2010, 11:25 http://www.biomedcentral.com/1471-2164/11/25 [11]. There are numerous tau phosphorylation sites associated with tau dysfunction and neurodegeneration [12]. Phosphorylation of tau protein on serine 262 has been demonstrated to significantly reduce the affinity of tau protein for microtubules[13] and this serine is hyperphosphorylated early in progression of disease pathology, before mature NFTs form[11]. Thus, increased ser262 phosphorylation is an important initial step in the pathological progression to cytoskeletal dys- function and NFT formation in Alzheimer’s disease, although full neurodegenerative effects likely require hyperphosphorylation of multiple sites[14]. Identifying the kinases involved in ser262 hyperphosphorylation will increase our understanding of the mechanisms causing tau and cytoskeletal dysfunction in AD, and could pro- vide new targets for the discovery of tau-modifying AD treatments. In vitro, numerous Ser/Thr kinases phosphorylate tau protein[15]. Kinases reported to phosphorylate tau on ser262, either directly or indirectly, include calcium/cal- modulin dependent protein kinase 2[16,17], protein kinase A[18], microtubule affinity regulating kinase 2 [19], phosphorylase kinase[20], and glycogen synthase kinase 3b [21,22]. However, the in vivo role of these kinases in the etiology of neurofibrillary tangle forma- tion remains unclear. We report the use of a high-content siRNA based screening strategy, surveying 572 kinases throughout the human genome, to identify the kinases involved in ser262 tau phosphorylation. After screening validated siRNAs, we identified candidate kinases that either increased or decreased pS262 tau levels. We provide evi- dence that the eukaryotic translation initiation factor 2 a kinase 2 (EIF2AK2), dual-specificity tyrosine-(Y)-phos- phorylation regulated kinase 1A (DYRK1A), and a- kinase anchor protein 13 (AKAP13) proteins contribute to phosphorylation of tau at the 12E8 epitope (pS262/ pS356). Results High-content siRNA screen of the kinome To identify the kinases that are important in AD rele- vant pathologic phosphorylation of tau protein, we developed a cell-based, high-throughput immunofluores- cence assay for the rapid detection and quantitation of both total tau and 12E8 tau (pS262 and pS356) protein expression. The assay uses an H4 neuroglioma cell line engineered to overexpress four repeat tau (4R0N). We confirmed that the assay was sensitive to changes in both total tau protein levels and phosphorylated tau protein levels using siRNA directed at the tau (MAPT) mRNA (Figure 1A). Importantly, we are able to achieve >95% siRNA transfection efficiency with the H4-tau cell line as assessed by transfection with a lethal siRNA Page 2 of 10 (data not shown). The accuracy of the immunofluores- cence quantitation was confirmed by measuring the effects of the MAPT siRNA on total and phosphorylated tau protein levels using western analysis (Figure 1B). We next tested if reduced 12E8 tau levels could be detected using siRNA directed against microtubule affinity regu- lating kinase 2 (MARK2), which has previously been shown to phosphorylate tau protein on serine 262[19]. The MARK2 siRNA, which reduced MARK2 expression >95% at the mRNA level, led to a 26% reduction in 12E8 phosphorylated tau (p = 0.029 for three replicates) and to an insignificant change in total tau protein expression (Figure 2). This result demonstrates that this immunofluorescence assay can detect changes to phos- phorylated tau protein independently of significant changes in total tau protein levels. siRNA screening results Following assay validation, we screened the validated kinome siRNA library (Qiagen) for kinases that affect tau phosphorylation status. Cataloging of kinases from the human genome has identified at least 518 kinases [23,24]. This library contains siRNAs to 572 known and predicted kinases. Two siRNAs per target kinase were screened in triplicate for a total of 3,432 target siRNAs screened. Standard paired, two-tailed T-tests were used to determine significant effects relative to non-silencing siRNA controls, which were present in triplicate on each plate. All siRNA screening data are presented as Additional File 1. To identify potential therapeutic targets that might modify the course of tau phosphorylation and dysfunc- tion in AD, we first identified those kinases that signifi- cantly affected the ratio of 12E8 tau to total tau. The ratio of 12E8 tau/total tau could be altered in several ways, either by changes to phosphorylated tau, by changes to total tau, or by a combination of changes to both phosphorylated tau and total tau. For this reason we initially identified siRNAs that significantly reduced 12E8 tau levels with no significant effects on total tau levels relative to non-silencing siRNA controls. Of these kinases, we identified those that significantly affected the ratio of 12E8 tau/total tau relative to the non-silencing siRNA control. These kinases are listed in Table 1 and are candidate kinases acting in specific tau phosphoryla- tion pathways. Notably, the microtubule affinity regulating kinase 2 (MARK2) protein, which has been shown to phosphory- late serine 262 of tau protein[19], showed the largest reductions in the ratio of 12E8 tau/total tau when knocked down (69% of control; p = 0.012). This is com- parable to the effects seen in our validation assays with MARK2 siRNA, suggesting that the screening assay is performing as expected. Because this kinase is already well known to be a serine 262 tau kinase, we did not