Azorsa et al. BMC Genomics 2010, 11:25 http://www.biomedcentral.com/1471-2164/11/25 levels of phosphorylated tau protein sufficient enough for detection of modulation of phosphorylation status. Because of this overexpression there is the possibility of creating a significant cytoplasmic pool of tau protein that is not bound to microtubules. This raises the possi- bility that some phospho-epitopes that are normally pro- tected (via microtubule binding or interactions with other proteins at physiologic concentrations) may be exposed and phosphorylation pathways that do not nor- mally regulate tau would have an effect in this assay. However, the identification of MARK2 as a significant pS262 tau phosphorylator (Table 1) suggests that tau regulatory pathways are functioning on the 12E8 epitope in this cell line. Nevertheless, the true applicability of the kinases that we identify here will emerge only after subsequent follow up studies in neuronal cell lines and in vivo in animal models of AD and of tauopathies. Both the eukaryotic translation initiation factor 2 a kinase 2 gene (EIF2AK2) and the dual specificity tyro- sine phosphorylation regulated kinase 1A gene (DYRK1A) have been implicated previously in neurode- generation in AD. Polymorphisms within the eukaryotic translation initiation factor 2 a kinase 2 (EIF2AK2) gene have been genetically associated with AD[26]. In addi- tion, EIF2AK2 has been shown to be activated in AD brain[27,28] and has been implicated in neuronal apop- tosis resulting from toxic b-amyloid peptides[28]. EIF2AK2 has also been implicated in the extrastriatal neurodegeneration of Parkinson’s disease and Hunting- ton’s disease[29]. In our study, two siRNAs that reduced EIF2AK2 protein expression by 95% caused a significant reduction of pS262 tau levels and a lesser but significant reduction in total tau levels (Table 2). These findings suggest an important role for EIF2AK2 in the expression of total and 12E8 phosphorylated tau protein. Moreover, the comparable effects seen for total tau and 12E8 tau suggest the possibility that EIF2AK2 may be required for the maintenance of multiple forms of phosphory- lated tau, in addition to 12E8 tau. Deciphering the mechanisms through which this regulation occurs will require additional experimentation. Polymorphisms within the DYRK1A locus also have been associated with AD in some AD patient popula- tions[30]. Moreover, DYRK1A has been implicated in phosphorylation of tau protein on threonine 212, serine 202, and serine 404[31,32]. In this work we demonstrate a role for DYRK1A in the phosphorylation of tau pro- tein on the 12E8 epitope, independent of significant effects on total tau protein expression. Interestingly, DYRK1A is a proline- directed serine/threonine kinase. Serine 262 is not a proline-directed phosphorylation site on tau. One possible scenario is that DYRK1A effects on S262 could occur through activities of additional kinases downstream of DYRK1A function. Presumably, Page 7 of 10 silencing of those kinases would also affect 12E8 tau levels in our assay and strong candidates for down- stream kinases through which DYRK1A may be signal- ing are those additional kinases in Table 1. Ongoing work is directed at identifying the additional signaling components involved in mediating the effects of DYRK1A on 12E8 tau phosphorylation and at determin- ing if DYRK1A is capable of directly phosphorylating tau protein on serine 262/serine 356. A possible role for AKAP13 in the regulation of tau phosphorylation Although it is clear that AKAP13 has significant effects on 12E8 tau levels in our cell line, the mechanisms through which this protein may affect tau are unclear. The AKAP13 protein is an ~320 kD protein that func- tions as an anchor protein for the regulatory subunit of protein kinase A, effectively localizing the PKA holoen- zyme to discrete locations within the cell. Protein kinase A has been reported to increase tau phosphorylation in vitro on the 12E8 epitope under some conditions, such as in response to the binding of a-synuclein to tau pro- tein[33]. Additionally, AKAP13, and other members of the AKAP family, have a Dbl homology (DH) domain that functions as a guanine nucleotide exchange activa- tion domain for the Rho/Rac family of GTP binding proteins. Thus, AKAP13 coordinates a Rho signaling pathway that ultimately leads to cytoskeletal reorganiza- tion. In addition, other AKAP family members have been implicated in regulating synaptic plasticity and long-term memory formation[34,35]. These combined observations suggest interesting possibilities wherein AKAP13 could anchor a signaling complex at the cell membrane that ultimately regulates tau phosphorylation and microtubule dynamics in response to extracellular stimuli. However, direct connections to tau phosphory- lation based on the currently known functions of AKAP13 are unclear but certainly worthy of further study. Conclusions One additional implication of our findings is that phos- phorylation of the 12E8 epitope is likely controlled by multiple redundant signaling pathways. This conclusion is supported by the fact that silencing of no single kinase fully blocked 12E8 tau phosphorylation. In addi- tion, the complexity of these signaling pathways is also evident in the observation that knockdown of multiple kinases leads to significant increases in 12E8 tau levels (Table 1). When coupled with recent findings showing that phosphorylation of tau at multiple sites appears to be required for neurotoxicity of tau[14], it is becoming clear that the regulatory pathways controlling cellular tau function and dysfunction in disease are complex. Our results were generated in a cell line that