For inhibition of Cdk5, brain protein extracts were pre-treated with 100 of roscovitine (EMD Millipore, Darmstadt, Germany) for 1 h at room temperature. Mass spectrometry analysis Phosphorylation assay and protein purification were performed as described above. Cdk5-mediated phosphorylation of EFhd2 affected its calcium binding activity. Finally, a phospho-specific antibody was generated against EFhd2 phosphorylated at S74 and was used to detect this phosphorylation event in postmortem brain tissue from Alzheimer’s disease and normal-aging control cases. Results demonstrated that EFhd2 is phosphorylated at S74. These results imply that EFhd2’s physiological and/or pathological function could be CAL-130 regulated by its phosphorylation state. studies indicated that GSK3 prime tau proteins for subsequent phosphorylation by Cdk5/p35 or Cdk5/p25.22 Bioinformatics analysis of EFhd2’s protein sequence indicates that it has several regions with the consensus P-(S/T) sequence that is phosphorylated by proline-directed kinases, such as Cdk5/p35 and GSK3. Therefore, based on the association of EFhd2 to tau-mediated neurodegeneration, it is plausible to hypothesize that EFhd2 could be a substrate of either or both of these kinases. In this study, this hypothesis was directly tested using brain extract from a transgenic mouse that overexpresses p25 (CK-p25) and phosphorylation assays. Additionally, it was determined the effect that EFhd2 phosphorylation exerts on its calcium binding. Finally, a phospho-specific antibody was generated and evaluated to determine the phosphorylation of EFhd2 in AD and normal aging control. Results CK-p25 brain extract phosphorylates EFhd2 protein CAL-130 CK-p25 is a transgenic mouse that inducibly overexpresses human p25 protein under the control of the CamKII alpha promoter, restraining the expression to the forebrain.30 Neurodegeneration was detected after only 2 weeks of p25 DDPAC overexpression in the forebrain of transgenic mice.30 Concurrently with the overexpression of p25, phosphorylation of known Cdk5 substrates, such as tau, neurofilament H, and Amyloid precursor protein, was detected.30 Thus, CAL-130 subcortical and cortical brain regions from non-transgenic and CK-p25 mice after 2 (2W) or 4 (4W) weeks of induction were homogenized. No change in the level of Cdk5 was detected [Fig. 1(A)]. As expected, however, overexpression of p25 was detected in the cortical region of CK-p25 mice after 2W and 4W of induction [Fig. 1(A)]. After 4W of induction, p25 was also detected in the subcortical brain region [Fig. 1(A)]. Open in a separate window Figure 1 CK-p25 brain extract phosphorylates EFhd2. (A) Western blot analysis of brain extract derived from CK-p25 mice using anti-Cdk5 and anti-p25 confirmed the induction of p25 at 2 weeks (2W) and 4 weeks (4W), preferentially in the cortical region. The level of the Cdk5 protein did not change. (B) HIS-EFhd2 full length (FL) was exposed to cortical (cort) and sub-cortical (sub) brain extract after 2W or 4W of induction in absence (?) or presence (+) of 100 roscovitine. The level of phosphorylated HIS-EFhd2 was detected using Pro-Q diamond phospho-protein staining and coomassie blue (CB) staining was used to detect total protein used. Beads alone (beads) were used as negative control for non-specific binding and to detect background levels of the Pro-Q diamond staining. The brain extracts were incubated with purified recombinant HIS-EFhd2 for kinase assay [Fig. 1(B)]. After incubation with brain extract, the purified recombinant proteins were resolved in SDS-PAGE and visualized using coomassie blue [Fig. 1(B)]. ProQ-diamond phospho-specific dye was used to detect phosphorylated proteins. The results indicated that HIS-EFhd2 is phosphorylated after incubation with brain extract, especially by the protein extract derived from cortical region where p25 is predominantly overexpressed [Fig. 1(B)]. Cdk5 or a kinase activated by it could mediate the detected phosphorylation of EFhd2. To corroborate that Cdk5 mediates EFhd2 phosphorylation, roscovitine, a potent Cdk5 inhibitor,31,32 was added to the reaction. Roscovitine blocked the phosphorylation of the recombinant HIS-EFhd2 when the 2W induced brain extract was used [Fig. 1(B)]. However, roscovitine CAL-130 only reduced the phosphorylation of HIS-EFhd2 detected upon incubation with the 4W induced brain extract [Fig. 1(B)]. It is possible that after 4 weeks of p25 induction, the hyperactivity of Cdk5 could not be completely inhibited by roscovitine. Alternatively, the hyperactivity Cdk5 may activate other kinases that also mediate the phosphorylation of EFhd2 proteins. Nevertheless, the results demonstrate that EFhd2 can be phosphorylated kinase assays were performed and the level of EFhd2 phosphorylation.