Abstract
The majority of familial Alzheimer's disease cases have been attributed to mutations in the presenilin 1 (PS1) gene. PS1 is synthesized as an inactive holoprotein that undergoes endoproteolytic processing to generate a functional N- and C-terminal heterodimer (NTF and CTF, respectively). We identified a single residue in PS1, Ser(397), which regulates the CTF levels in a population of dimer that has a rapid turnover. This residue is part of a highly conserved glycogen synthase kinase-3beta (GSK-3beta) consensus phosphorylation site within the loop domain of PS1. Site-directed mutagenesis at the Ser(397) position increased levels of PS1 CTF but not NTF or holoprotein. Similar increases in only CTF levels were seen when cells expressing wild type PS1 were treated with lithium chloride, an inhibitor of GSK-3beta. Both wild type and PS1 S397A CTF displayed a biphasic turnover, reflecting rapidly degraded and stable populations. Rapid turnover was delayed for mutant PS1 S397A, causing increased CTF. These data demonstrate that PS1 NTF.CTF endoproteolytic fragments are generated in excess, that phosphorylation at Ser(397) by GSK-3beta regulates the discard of excess CTF, and that the disposal of surplus NTF is mediated by an independent mechanism. Overall, the results indicate that production of active NTF.CTF dimer is more complex than limited endoproteolysis of PS1 holoprotein and instead involves additional regulatory events.
Highlights
Alignment of the hydrophilic loop domain of presenilin 1 (PS1) from several species revealed that this domain contains three highly conserved and potential GSK-3 consensus phosphorylation sites: TERES324, STPES357, and SATAS401, two of which are completely conserved in all species examined (Ref. 23 and Fig. 1)
The sequence (S/T)XXXS is known to be a consensus sequence for GSK-3 phosphorylation. Since this motif is highly conserved in PS1, it seems likely that these identified GSK-3 consensus phosphorylation sites mediate specific PS1 function(s)
Endoproteolysis of the 46-kDa PS1 holoprotein to a heterodimer composed of 20kDa CTF and 30-kDa NTF has been linked to the stabilization of PS1 [6, 7, 13], to its incorporation into a high molecular weight complex [9, 10], and to its maturation as a functional entity [11]
Summary
Cell Culture—Human embryonic kidney 293T (293HEK) cells were grown in Dulbecco’s modified Eagle’s medium-21 containing 10% fetal bovine serum, 1% L-glutamine, and antibiotics (50 units/ml penicillin and 50 g/ml streptomycin). Transient transfection was carried out with 293HEK cells using the calcium phosphate precipitation method with the indicated DNA constructs as previously described [23]. Western Blot and Co-immunoprecipitation Analyses—293HEK cells were transiently transfected with the indicated constructs and were harvested 24 – 48 h post-transfection. The PS1 monoclonal antibody directed to the C-terminal “loop” domain (3.6.1) was obtained by immunizing mice with a synthetic peptide spanning PS1 residues 309 –331. The monoclonal antibody directed to the N terminus (614.1) was raised a glutathione S-transferase fusion protein expressed in bacteria containing the N-terminal 77 residues of PS1. Twenty-four hours post-transfection, cells were washed twice with phosphate-buffered saline, and growth medium was replaced with methionine-, cysteine-, and serum-free medium and incubated at 37 °C for 60 min. Western blot analysis was used to ensure equivalent levels of exogenous PS1 expression
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