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. 2007 Dec 9:2:23.
doi: 10.1186/1750-1326-2-23.

Evidence against roles for phorbol binding protein Munc13-1, ADAM adaptor Eve-1, or vesicle trafficking phosphoproteins Munc18 or NSF as phospho-state-sensitive modulators of phorbol/PKC-activated Alzheimer APP ectodomain shedding

Annat F Ikin  1 , Mirsada Causevic, Steve Pedrini, Lyndsey S Benson, Joseph D Buxbaum, Toshiharu Suzuki, Simon Lovestone, Shigeki Higashiyama, Tomas Mustelin, Robert D Burgoyne, Sam Gandy
Affiliations

Evidence against roles for phorbol binding protein Munc13-1, ADAM adaptor Eve-1, or vesicle trafficking phosphoproteins Munc18 or NSF as phospho-state-sensitive modulators of phorbol/PKC-activated Alzheimer APP ectodomain shedding

Annat F Ikin et al. Mol Neurodegener. .

Abstract

Background: Shedding of the Alzheimer amyloid precursor protein (APP) ectodomain can be accelerated by phorbol esters, compounds that act via protein kinase C (PKC) or through unconventional phorbol-binding proteins such as Munc13-1. We have previously demonstrated that application of phorbol esters or purified PKC potentiates budding of APP-bearing secretory vesicles at the trans-Golgi network (TGN) and toward the plasma membrane where APP becomes a substrate for enzymes responsible for shedding, known collectively as alpha-secretase(s). However, molecular identification of the presumptive "phospho-state-sensitive modulators of ectodomain shedding" (PMES) responsible for regulated shedding has been challenging. Here, we examined the effects on APP ectodomain shedding of four phorbol-sensitive proteins involved in regulation of vesicular membrane trafficking of APP: Munc13-1, Munc18, NSF, and Eve-1.

Results: Overexpression of either phorbol-sensitive wildtype Munc13-1 or phorbol-insensitive Munc13-1 H567K resulted in increased basal APP ectodomain shedding. However, in contrast to the report of Rossner et al (2004), phorbol ester-dependent APP ectodomain shedding from cells overexpressing APP and Munc13-1 wildtype was indistinguishable from that observed following application of phorbol to cells overexpressing APP and Munc13-1 H567K mutant. This pattern of similar effects on basal and stimulated APP shedding was also observed for Munc18 and NSF. Eve-1, an ADAM adaptor protein reported to be essential for PKC-regulated shedding of pro-EGF, was found to play no obvious role in regulated shedding of sAPPalpha.

Conclusion: Our results indicate that, in the HEK293 system, Munc13-1, Munc18, NSF, and EVE-1 fail to meet essential criteria for identity as PMES for APP.

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Figures

Figure 1
Figure 1
Munc13-1 increases constitutive and phorbol-stimulated sAPPα secretion in a fashion that is independent of the integrity of its phorbol-sensing C1 domain. Levels of soluble APPα (sAPPα) ectodomain were measured by Western blotting of cell supernatants with anti-APP antibody, 6E10, following the treatment of cells with DMSO (-) or 100 nM PMA (+) for 2 hours in a 37°C, 5% CO2 cell culture incubator. Levels of holoAPP were measured from cell lysates with anti-APP antibody 369. Levels of Munc13-1 wild type and Munc13-1 H567K mutant proteins were measured by anti-GFP antibody. Equal protein loading was verified by measuring the levels of actin protein in all cell lysates.
Figure 2
Figure 2
Localization of APP following PMA treatment. HEK293 cells were co-transfected with the following cDNAs: (a, b) APPSWE-pPrk5 and Munc13-1WT-pEGFP-N1 and (c, d) APPSWE-pPrk5 and Munc13-1H567K-pEGFP-N1. Treatment with 100 nM PMA was carried out for 2 hours. GFP immunofluorescence allowed visualization of (a) Munc13-1WT and (c) Munc13-1H567K mutant molecules (green). (b, d) APP was immunolabeled with rabbit polyclonal anti-APP-specific antibody 369 followed by rhodamine red conjugated secondary antibody (red).
Figure 3
Figure 3
APP metabolism and sAPPα release are not modulated by the phospho-state of Munc18 at serine 306, serine 313, or by dual phosphorylation at both residues. (A) HoloAPP levels (top panel) were determined in the absence (lane 2) or presence of wildtype or phospho-site mutant forms of Munc18 (lanes 4–7) and in the presence of X11 alone (lane 3) or the combination of X11 plus each form of Munc18 (lanes 8–11). (B) Basal and PMA-stimulated sAPPα release were determined in the absence (lanes 1 and 6) or presence of wildtype (lanes 2 and 7) or phospho-site mutant forms of Munc18 (lanes 3–5 and 8–10).
Figure 4
Figure 4
Phorbol-stimulated sAPPα secretion is not sensitive to the phospho-state of NSF at tyrosine 83. HEK293 cells stably expressing APP695 (HEK293-695) were transiently transfected with either wild type NSF (WT), the tyrosine 83 phospho state mimetic NSF mutant (Y83E), or the the tyrosine 83 dephospho state mimetic NSF mutant (Y83F). Empty vector was used as control (Cont). Cells were treated with PDBu for one hour after which media were collected and immunoprecipitated with antibody 6E10. Western blotting for sAPPα was performed using 6E10. (A) Representative Western blot for sAPPα in the absence (-) or presence (+) of PDBu. (B) Quantification of 3 such experiments.
Figure 5
Figure 5
Phorbol-stimulated sAPPα secretion is not modulated through Eve-1. HEK293-695 were transiently transfected with either Eve1-b or Eve1-c. Empty vector was used as control (Cont). Cells were treated with PDBu and processed as above. (A) Representative Western blot for sAPPα in the absence (-) or presence (+) of PDBu. (B) Quantification of 3 such experiments.
Figure 6
Figure 6
Possible mechanisms for how the PMES candidates tested in this study might modulate shedding of the APP ectodomain. a, This cartoon depicts how phorbol esters (PMA) might promote translocation of wildtype but not H567K Munc13-1 to the PM. b, This cartoon depicts how phosphorylation of Munc18 by PKC might facilitate fusion of APP transport vesicles with α-secretase transport vesicles, thereby facilitating shedding. c, This cartoon depicts how dephosphorylation of phospho-NSF might disinhibit NSF-mediated vesicle fusion; as in b, the notion is that APP and α-secretase might be traveling in separate vesicles prior to phosphorylation-state mediated facilitation of vesicle fusion. d, This cartoon depicts how the phosphorylation state of Eve-1 (or some theoretical accessory Eve-1 binding protein) might modulate vesicle interactions with the PM or they might modulate the physical docking between APP and α-secretase.
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