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Septin4 was previously identified as a substrate of DYRK1A (Sitz et al., 2008). Nonetheless, the respective phosphorylation site and functional consequences of this phosphorylation have remained unknown. Therefore, the Septin4 amino acid sequence (based on Septin4_v1, see chapter 7.3, table 19) was analyzed for putative DYRK1A consensus phosphorylation motifs, revealing three potential matching proline directed sites as illustrated in figure 32A. Although Ser68 reflects an optimal DYRK1A consensus motif, this residue is not conserved in human Septin4. By site directed mutagenesis, single or combined phosphorylation resistant alanine point mutants of Flag-tagged mouse Septin4 (hereafter named Septin4) at Ser68, Ser100 and Ser107 were constructed. Co- transfection of GFP-DYRK1A with Septin4-wt or the serine to alanine point mutants into HeLa cells revealed that the point mutations increased the mobility of Septin4 in SDS-gel electrophoresis compared to Sept4-wt (figure 32B).

Figure 32: Ser107 is the major DYRK1A phosphorylation site in Septin4.

(A) Potential DYRK1A phosphorylation sites in murine Septin4. The Septin4_v1 amino acid sequence from position 60-110 is shown. Potential DYRK1A consensus motifs and serine positions are printed in bold. The general DYRK1A consensus phosphorylation sequence is illustrated below (Himpel et al., 2000). (B) HeLa cells were cotransfected with GFP-DYRK1A (GFP-D1A) and Flag- Septin4-wt or different phosphorylation resistant serine (S) to alanine (A) point mutants as indicated. Untransfected cells were used as negative control (-). Total protein was extracted, subjected to SDS-PAGE, and analyzed by western blotting. GFP-DYRK1A overexpression was detected with an anti-GFP, and Septin-4 with an anti-Flag antibody. A customized antibody detecting phosphorylated Ser107 in Septin4 was tested. Migration of mass standards is indicated in kDa (left).

Of all three mutated putative phosphorylation sites, Ser107 mutation to alanine showed the largest effect on Septin4 electrophoretic mobility, which was not enhanced by

additional mutations of Ser68 and Ser100. This result strongly suggested Ser107 as the major DYRK1A phosphorylation site in murine Septin4. Furthermore, to analyze Septin4 Ser107 phosphorylation, a specific antibody was raised against phosphorylated Ser107. The custom-made antibody detected Septin4-wt and the S68A mutant but not Septin4- S107A (figure 32B), indicating functionality and specificity.

To evaluate DYRK1A dependent Septin4 Ser107 phosphorylation, Septin4-wt was cotransfected with GFP, GFP-DYRK1A or GFP-DYRK1A-K188R into HeLa cells. Additionally, endogenous or overexpressed DYRK1A activity was inhibited with harmine for 24 h (figure 33A).

Figure 33: Analysis of DYRK1A dependent Septin4 Ser107 phosphorylation in HeLa cells. (A) HeLa cells were cotransfected with Flag-Septin4-wt (Flag-S4-wt) and GFP, GFP-DYRK1A (GFP-D1A) or GFP-DYRK1A-K188R (GFP-KR) and treated with harmine for 24 h, if indicated, starting at the day after transfection. (B) Septin4-wt was cotransfected with GFP or GFP-D1A into HeLa cells and treated with 1 µM of the DYRK1A inhibitors AnnH31 (AH31), harmine (Har) or iodotubercidin (Iodo) for 5 h starting the day after transfection. Untreated cells served as control (-). Densitometric evaluation of relative Ser107 phosphorylation is indicated below. Tubulin served as loading control. Migration of mass standards is indicated in kDa (left). Symbols (*, #, ▪) indicate band patterns and are defined in the text.

Analysis with the phosphospecific antibody revealed that GFP-DYRK1A overexpression increases phosphorylation of Ser107. In contrast, inhibition of endogenous as well as overexpressed DYRK1A with harmine resulted in a slight decrease of Ser107 phosphorylation. Notably, when Septin4 was cotransfected with GFP, detection of pSer107 immunoreactivity already revealed a high basal phosphorylation. In contrast to DYRK1A-wt, the kinase deficient mutant DYRK1A-K188R did not increase Septin4 Ser107 phosphorylation. Immunodetection with a Septin4 antibody showed a double band at approximately 63 kDa when Septin4 was cotransfected with GFP or GFP- DYRK1A- K188R, whereof the lower band (marked with #) showed less immunoreactivity if Septin4

correlated in shape and migration pattern with the signal gained by the anti-pSer107 antibody. These results indicate that overexpressed Septin4 occurs at least in two different phosphorylated states in HeLa cells, as reflected by the band-shift after GFP- DYRK1A cotransfection in comparison to GFP transfection control. Ser107 phosphorylation seemed to contribute to the phosphorylation dependent shift, as the phosphospecific antibody did not detect the lower band. Furthermore, potential fragmentation of overexpressed Septin4 is observed, supported by the observation that both antibodies independently showed similar immunoreactivity at a molecular weight of approximately 48 kDa (marked with ▪) which correlated in its signal intensity with total Septin4 levels.

As basal Ser107 phosphorylation was already high when Septin4 was overexpressed in HeLa cells, the effect of pharmacological inhibition of endogenous and overexpressed DYRK1A on Ser107 phosphorylation was verified. Therefore, Septin4 was coexpressed with GFP or GFP-DYRK1A in HeLa cells and kinase activity of endogenous as well as of overexpressed DYRK1A was reduced with three different inhibitors for 5 h (figure 33B). Densitometric evaluation revealed a reduced Ser107 phosphorylation after inhibition of endogenous DYRK1A, although a residual phosphorylation of 30% remained if compared to control. Consistent with the former results, coexpression of GFP-DYRK1A increased Ser107 phosphorylation and generated the characteristic band shift described above (indicated by * and #). Inhibition of exogenous DYRK1A activity decreased Ser107 phosphorylation and increased the intensity of the faster migrating band (#) if detected with the total Septin4 antibody. Nonetheless, Ser107 phosphorylation was not reduced like in the GFP cotransfection control, probably because of residual DYRK1A activity due to the strong overexpression. Anyway, the results indicate that DYRK1A contributes to Septin4 Ser107 phosphorylation in a cellular environment. However, the observation that inhibition of endogenous DYRK1A could not totally abolish Ser107 phosphorylation raised the question if other kinases GMGC group might contribute to Septin4 phosphorylation at this residue.