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Comparative Study
. 1997 May 13;94(10):5349-54.
doi: 10.1073/pnas.94.10.5349.

Proteolytic processing of the Alzheimer disease-associated presenilin-1 generates an in vivo substrate for protein kinase C

J Walter  1 , J Grünberg, A Capell, B Pesold, A Schindzielorz, M Citron, K Mendla, P S George-Hyslop, G Multhaup, D J Selkoe, C Haass
Affiliations
Comparative Study

Proteolytic processing of the Alzheimer disease-associated presenilin-1 generates an in vivo substrate for protein kinase C

J Walter et al. Proc Natl Acad Sci U S A. .

Abstract

The majority of familial Alzheimer disease mutations are linked to the recently cloned presenilin (PS) genes, which encode two highly homologous proteins (PS-1 and PS-2). It was shown that the full-length PS-2 protein is phosphorylated constitutively within its N-terminal domain by casein kinases, whereas the PS-1 protein is not. Full-length PS proteins undergo endoproteolytic cleavage within their hydrophilic loop domain resulting in the formation of approximately 20-kDa C-terminal fragments (CTF) and approximately 30-kDa N-terminal fragments [Thinakaran, G., et al. (1996) Neuron 17, 181-190]. Here we describe the surprising finding that the CTF of PS-1 is phosphorylated by protein kinase C (PKC). Stimulation of PKC causes a 4- to 5-fold increase of the phosphorylation of the approximately 20-kDa CTF of PS-1 resulting in reduced mobility in SDS gels. PKC-stimulated phosphorylation occurs predominantly on serine residues and can be induced either by direct stimulation of PKC with phorbol-12,13-dibutyrate or by activation of the m1 acetylcholine receptor-signaling pathway with the muscarinic agonist carbachol. However, phosphorylation of full-length PS-1 and PS-2 is not altered upon PKC stimulation. In addition, a mutant form of PS-1 lacking exon 10, which does not undergo endoproteolytic cleavage [Thinakaran, G., et al. (1996) Neuron 17, 181-190] is not phosphorylated by PKC, although it still contains all PKC phosphorylation sites conserved between different species. These results show that PKC phosphorylates the PS-1 CTF. Therefore, endoproteolytic cleavage of full-length PS-1 results in the generation of an in vivo substrate for PKC. The selective phosphorylation of the PS-1 CTF indicates that the physiological and/or pathological properties of the CTF are regulated by PKC activity.

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Figures

Figure 1
Figure 1
(A) Schematic drawing showing the potential eight-trans-membrane domain structure (32) and the proteolytic processing of the PS proteins (30). The proteolytic cleavage (indicated by an arrow) occurs within the large hydrophilic loop between TM6 and TM7 generating an ≈30-kDa N-terminal fragment and an ≈20-kDa CTF (30). The epitopes of the antibodies used are indicated. (B) PDBu stimulates phosphorylation of the CTF of PS-1. Untransfected COS-7 cells were radiolabeled in the presence (+) or absence (−) of PDBu with [35S]methionine (35S-Met) as well as [32P]orthophosphate (32P). Cell lysates were immunoprecipitated with antibody 3027. After [35S]methionine labeling a major polypeptide of ≈20 kDa (CTF) was observed under control conditions. Upon PKC stimulation with PDBu the ≈20-kDa band shifted to ≈23 kDa. Labeling with [32P]orthophosphate revealed two weakly phosphorylated polypeptides of ≈22 kDa and ≈23 kDa under control conditions. PDBu treatment resulted in the detection of a highly phosphorylated polypeptide of ≈23 kDa and a weaker band of ≈24 kDa [CTF(P)]. (C) Results very similar to B were obtained with two independent antibodies. Untransfected COS-7 cells were radiolabeled in the presence (+) or absence (−) of PDBu with [35S]methionine (35S-Met) as well as [32P]orthophosphate (32P). Cell lysates were immunoprecipitated with antibodies BOS 4627 (26) and PS-1loop (30).
Figure 5
Figure 5
Conservation of potential PKC phosphorylation sites within the loop domain of PS-1 from different species and of human PS-2. The potential phosphorylation sites, with minimal consensus sequence for PKC [R/K-X-S∗/T∗ or S∗/T∗-X-R/K (46); phosphate acceptor sites marked by asterisks] are in bold. Amino acid numbering is for the human sequence of PS-1 (9). The sequence encoded by exon 10 (290–319) is marked by a solid bar. Two potential phosphorylation sites (Ser310 and Ser313) are missing in the exon 10-deleted splicing variant. Both sites are not well conserved, in contrast to those downstream of exon 10. Note that the loop domain of human PS-2 contains no potential PKC phosphorylation sites, whereas that of human PS-1 contains seven.
Figure 3
Figure 3
PKC selectively phosphorylates the PS-1 CTF but not the full-length protein. Human kidney 293 cells stably expressing PS-1 wild-type (A) or PS-1 Δexon 10 (17, 38) (B) were radiolabeled with [35S]methionine (35S-Met) as well as [32P]orthophosphate (32P) in the presence (+) or absence (−) of PDBu, and PS proteins were isolated by immunoprecipitation using antibody BOS4627. Full-length PS proteins are indicated by an arrow marked fl-PS. Arrowheads indicate dimeric forms of PS proteins (26). Note that no increased phosphorylation of both overexpressed full-length proteins, wild-type and Δexon 10, is observed after PDBu treatment, whereas phosphorylation of the PS-1 CTF is strongly increased in cells expressing wild-type PS-1. Note that no phosphorylated endogenous PS-1 CTF is detected in cells expressing the PS-1 Δexon 10 protein (B). The weak band (∗) observed after 32P-labeling in cells transfected with the PS-1 is a nonspecific protein that does not comigrate with PS-1 (26).
Figure 2
Figure 2
(A) Dephosphorylation reverses the molecular mass shift of the PS-1 CTF. Immunoprecipitates from PDBu-treated COS-7 cells labeled with [35S]methionine were incubated in the presence or absence of alkaline phosphatase. Phosphatase treatment induces a shift of the phosphorylated 23-kDa CTF observed after PDBu treatment back to 20 kDa, which comigrates with the unphosphorylated CTF from unstimulated cells. (B) Inhibition of PKC suppresses phosphorylation of the 20-kDa PS-CTF. Untransfected COS-7 cells were radiolabeled in the presence (+) or absence (−) of PDBu with or without (±) the PKC-specific inhibitor GF109203X with [32P]orthophosphate. Cell lysates were immunoprecipitated with antibody 3027. The phosphorylated ≈23-kDa and ≈24-kDa polypeptides are marked by arrowheads [CTF(P)]; the position of the unphosphorylated CTF, which is not visible by 32P labeling is marked by an arrow. This band was detected by parallel labeling with [35S]methionine (data not shown). (C) Quantification of PKC-mediated phosphorylation of the PS-1 CTF. Untransfected COS-7 cells were radiolabeled in the presence (+) or absence (−) of PDBu with [32P]orthophosphate (32P). Cell lysates were immunoprecipitated with antibody 3027 and 32P incorporation was quantified by phosphorimaging (34). (Bars = ± SE; n = 3.) (D) Phosphoamino acid analysis of PKC-phosphorylated CTF. Untransfected COS-7 cells were radiolabeled in the presence of PDBu with [32P]orthophosphate. The CTF was isolated by immunoprecipitation using antibody 3027. The position of phosphoserine (P-S), phosphothreonine (P-T), and phosphotyrosine (P-Y) is indicated.
Figure 4
Figure 4
Stimulation of m1 muscarinic acetylcholine receptors results in the phosphorylation of the CTF of PS-1 by PKC. U373 cells stably transfected with the m1 receptor cDNA were labeled with [35S]methionine (35S-Met; Upper) for 14 h and then incubated for 2 h in the absence or presence of carbachol alone or in combination with either atropine or a PKC inhibitor. To activate PKC directly, cells were treated with PDBu for 2 h. The CTFs of PS-1 were immunoprecipitated with antibody 3027. The positions of unphosphorylated CTF (CTF) and phosphorylated CTF [CTF(P)] are marked by arrowheads. To analyze phosphate incorporation, cells were incubated in the presence of [32P]orthophosphate during drug treatments. The CTFs of PS-1 were precipitated from cell lysates with antibody 3027. The 32P-labeled ≈23-kDa and ≈24-kDa polypeptides are marked by arrowheads [CTF(P)], and the position of unphosphorylated CTF (not detectable by 32P labeling) is marked by an arrow. Note that stimulation of m1 receptors with carbachol results in a marked increase in phosphate incorporation as well as a molecular mass shift, similar to that observed after PDBu-mediated PKC stimulation.
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