Fig. 9.

Direct interaction of DYRK1A and SYN1, phosphorylation of SYN1 by DYRK1A. (A) DYRK1A and SYN1 were immunoblotted following immunoprecipitation from wt mice brain extracts. DYRK1A or SYN1 present in the starting material (Input) were recovered in the IPs. SYN1 (74 kDa) was present in the DYRK1A IP and DYRK1A (85 kDa) was detected in the SYN1 IP, suggesting that these two proteins interact directly. Positive control of the SYN1 IP was performed using an anti-CAMKII antibody. As expected, CAMKII (50 kDa) was present in the SYN1 IP. DYRK1A IP also brought down CAMKII, suggesting complexes between SYN1, CAMKII and DYRK1A. (B) Sequence of SYN1 in the vicinity of Ser551 matches with the consensus DYRK1A phosphorylation site. Based on this sequence, three peptides were synthesized and used as potential substrates: SYN1, SYN1-S551A and SYN1-S553A. (C) Kinase activity of recombinant DYRK1A towards the three different SYN1peptides. SYN1 and SYN1-S553A peptides were phosphorylated at the same level as Woodtide by recombinant DYRK1A (71.7%±5.2%, 70.1% and 78.4%±11.4%, respectively). No significant catalytic activity was measured with the SYN1-S551A peptide (7.9%±1.2%).