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. 2001 Mar 19;152(6):1159–1168. doi: 10.1083/jcb.152.6.1159

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© 2001 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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PACS-1 binding to the VMAT2 COOH terminus. (A) GST fusion proteins containing the COOH terminus of wild-type VMAT2 or furin were bound to glutathione-sepharose beads and phosphorylated using casein kinase 2. Unphosphorylated and phosphorylated fusion proteins bound to glutathione-sepharose were then incubated with a poly-His–tagged version of the PACS-1 furin-binding domain and the bound protein detected using an antipolyhistidine antibody (PACS-1). Autoradiography was used to assess protein phosphorylation (³²P). Western analysis for GST confirmed the presence of equal amounts of unphosphorylated and phosphorylated GST fusion proteins. Similar to phosphorylated furin, phosphorylated VMAT2 binds PACS-1 more strongly than the unphosphorylated protein. (B) GST fusion proteins containing the COOH termini of wild-type, AA, DD, or 507* VMAT2 were incubated with the furin-binding domain of PACS-1 as described in A. Western analysis was then used as described above to detect bound PACS-1 and confirm the presence of equal amounts of GST fusions. PACS-1 binds strongly to the DD mutant, but poorly to wild-type VMAT2 and the AA mutant.