title: Effect of phosphomimetic mutations on the C-terminal sensitivity of glycine transporter GlyT1 to calpain.
abstract: Previously, we found that the C-terminus of the glycine transporter GlyT1 loses the most of its epitopes during pathological calcium increase in rodent synaptosomes but that the more internal epitopes are spared. We also found that epitope immunoreactivity likely decreases via both phosphorylation and calpain-mediated proteolysis. Here we show that the predicted phosphomimetic mutation S605D fully blocks the adjacent (T602/T603) internal calpain cleavage in the mouse GlyT1b C-terminal fusion protein, but that the neutral S605A mutation does not. Consistent with this, the phophomimetic mutation, but not the neutral mutation, significantly protected the internal GlyT1 C-terminal antiGlyT1C603-626 epitopes against calpain when introduced into tissue culture expressed GlyT1b. Because similar effects can be obtained using phosphatase inhibitors, it may be that phosphorylation of S605 protects the GlyT1 C-terminal sequences from calpain cleavage in vivo.
authors.affiliation: Laboratory of Neurobiology, Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 845 51 Bratislava, Slovakia.
authors.forename: Andrea
authors.initials: A
authors.lastname: Mihalikova
authors.affiliation: Laboratory of Neurobiology, Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 845 51 Bratislava, Slovakia.
authors.forename: Martina
authors.initials: M
authors.lastname: Baliova
authors.affiliation: Laboratory of Neurobiology, Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 845 51 Bratislava, Slovakia. Electronic address: Frantisek.Jursky@savba.sk.
authors.forename: Frantisek
authors.initials: F
authors.lastname: Jursky
mesh_terms: D000818:Animals; D002154:Calpain / Q000378:metabolism*; D045744:Cell Line, Tumor; D050487:Glycine Plasma Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D010766:Phosphorylation
chemical_list: D050487:Glycine Plasma Membrane Transport Proteins; C493267:Slc6a9 protein, mouse; D002154:Calpain
keywords: Calpain; GlyT1; Glycine transporter