To investigate if such an effect is observed in native
To investigate if such an effect is observed in native GlyT1, we introduced S605D and S605A mutations into GlyT1 and transfected these DNA constructs into N2a cells. For an internal control of calpain cleavage, we also introduced the first 100 amino-acids of the rat GlyT2N terminus upstream of the mouse GlyT1b initiating methionine (see Section 2 for details). Both mutated transporters and chimerical transporters were normally transported into the plasma membrane and exhibited sodium dependent glycine uptake comparable to that of transporters with wild-type N-termini (Fig. 2). Removal of the N- and C-terminal parts of the rGlyT2N-mGlyT1b chimera through the GlyT1b N- and C-terminal calpain sites (Baliova and Jursky, 2005, Baliova and Jursky, 2010) resulted in a molecular weight shift sufficiently large to allow the extent of GlyT1 calpain cleavage to be followed. AntiGlyT1C603-626 src inhibitor were then used to study the effect of the introduced mutations on the T602/T603 calpain cleavage site. The fully shifted truncated mGlyT1 shown in Fig. 3A indicates that there was sufficient calpain activity to cleave the transporter quantitatively. Fig. 3 also shows that the S605D mutant largely preserved antiGlyT1C603-626 immnunoreactivity in the shifted band, but that the S605A mutant lost this immunoreactivity almost entirely. The wild-type protein preserved moderate immunoreactivity, suggesting that it was partially phosphorylated. These results were corroborated by others obtained using untagged GlyT1 variants (Fig. 3B). Normally, active endogenous phosphatases, kinases and an ATP pool can still be found in N2a cellular extracts after freezing and thawing. Supplementing these extracts with calcium and magnesium is expected to trigger a series of phosphorylation and dephosphorylation cycles on their various protein substrates, including GlyT1, until the remaining cellular ATP supply is exhausted. This should result in the majority of the substrates being present in the dephosphorylated form. If this is correct, then the process can be shifted to substantial increase of the phosphorylated substrates by adding external ATP and phosphatase inhibitors. Preincubation of the N2a extract supplemented with external ATP and phosphatase inhibitors did significantly inhibited loss of anti-GlyT1C603-626 immunoreactivity following calpain cleavage (Fig. 4). This suggests that phosphorylation might indeed inhibit calpain cleavage and that the phosphomimetic mutation S605D does correctly mimic phosphorylation. The second serine (S636) which is likely to be phosphorylated resides in a 12 amino-acid GlyT1 C-terminal peptide fragment, which is cleaved off by calpain and is a part of the GlyT1 C-terminal PDZ binding motif (Baliova and Jursky, 2010) (Fig. 1A). Introducing phosphomimetic mutations into this serine prevented the appearance of the double band normally produced by calpain cleavage at the very C-terminal calpain (G626/S627) cleavage site (Fig. 5A). This indicates that phosphorylation of serine 636 might block cleavage of GlyT1 at this site in vitro as well. To investigate the effect of a S636D mutation on the calpain cleavage of GlyT1 in cell lines, we introduced this mutation into wild-type GlyT1. To eliminate interference from the T602/T603 calpain cleavage site, we also created a double (S605D, S636D) mutant of GlyT1. Fig. 5B shows that both the wild-type and S636D mutants were intensively cleaved by calpain at the T602/T603 cleavage site and that T602/T603 cleavage, but not G626/S627 cleavage, was inhibited by the introduction of a S605D mutation. As shown in Fig. 5C, however, the replacement of serine 636 completely eliminated the anti-GlyT1C626-638S antibody signal, preventing us from investigating the effect of this mutation on the terminal GlyT1C calpain cleavage in vivo. Given that serine 636 has a lower probability of phosphorylation (Fig. 1) and that the GST fusion partner may alter the accessibility of this site, it is currently not clear whether in vivo phosphorylation of serine 636 will have similar effects on terminal calpain cleavage as these observed for the S636D phophomimetic mutation of recombinant GlyT1C fusion protein.