FIGURE 1.

Serine 14 is phosphorylated by CKII and is a key regulator of interaction with Munc18-1 at the plasma membrane. a, sequence alignment of syntaxin 1 orthologs and paralogs. The casein kinase II consensus site is highly conserved throughout evolution but is less conserved between mammalian paralogs. This may be indicative of a divergence of function between syntaxin isoforms. b, structural alignment of Munc18-1 (dark gray; Protein Data Bank code 3C98) (16) and Munc18-3 (light gray; Protein Data Bank code 2PJX) (15) of the region involved in mode 2/3 interaction with syntaxin. This region exhibits a very high degree of structural conservation indicative of an important functional role. Shown in red is the N-terminal peptide of syntaxin 4 bound to Munc18-3 with the serine residue highlighted in a space fill representation. c, to confirm that Ser¹⁴ is a casein kinase II phosphorylation site in syntaxin 1, multiple syntaxins were expressed and purified from bacteria (GST-Syx^1–261). These were subjected to in vitro phosphorylation with detection using a fluorescent in-gel phosphostain. Total protein was observed by Coomassie staining. The cytoplasmic domain of syntaxin was readily phosphorylated by CKII in vitro, and this was blocked by mutation of Ser¹⁴ or the acidic consensus site at Asp¹⁷. d, to test these mutations in a cellular context, phosphorylation of heterologously expressed proteins was detected by immunoblotting (IB) using a Ser¹⁴ phospho-specific antibody. This demonstrated that heterologous syntaxin was phosphorylated on this site in vivo and was perturbed by modification of Ser¹⁴ or the CKII consensus site.