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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.

Ikin AF, Causevic M, Pedrini S, Benson LS, Buxbaum JD, Suzuki T, Lovestone S, Higashiyama S, Mustelin T, Burgoyne RD, Gandy S - Mol Neurodegener (2007)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Farber Institute for Neurosciences of Thomas Jefferson University, 900 Walnut Street, Philadelphia, 19107, PA, USA. samgandy@earthlink.net.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: Introduction of either Munc13-1 wild type or Munc13-1 H567K mutant resulted in a significant, 3–5 fold increase in basal sAPPα release (Figure 1, lanes 1, 3, 5). Munc13-1 wild type and the Munc13-1 H567K mutant molecules were identical in their effects on basal (constitutive) sAPPα secretion (Figure 1, lanes 1, 3, 5). Since Munc13-1 is primarily a receptor for phorbol esters, which mimic the effects of endogenous diacylglycerol, we applied the phorbol ester PMA in the presence of either the wild type or mutant Munc13. Our results showed a typical increase in sAPPα release [29] in both Munc13-1 wild type and Munc13-1 H567K mutant transfected cells (Figure 1, compare lane 1 vs 2, lane 3 vs 4, and lane 5 vs 6; Table 1) indicating that phorbol ester interaction with histidine-567 of the C1 domain of Munc13-1 is not a key step in regulated shedding.


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.

Ikin AF, Causevic M, Pedrini S, Benson LS, Buxbaum JD, Suzuki T, Lovestone S, Higashiyama S, Mustelin T, Burgoyne RD, Gandy S - Mol Neurodegener (2007)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211485&req=5

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.
Mentions: Introduction of either Munc13-1 wild type or Munc13-1 H567K mutant resulted in a significant, 3–5 fold increase in basal sAPPα release (Figure 1, lanes 1, 3, 5). Munc13-1 wild type and the Munc13-1 H567K mutant molecules were identical in their effects on basal (constitutive) sAPPα secretion (Figure 1, lanes 1, 3, 5). Since Munc13-1 is primarily a receptor for phorbol esters, which mimic the effects of endogenous diacylglycerol, we applied the phorbol ester PMA in the presence of either the wild type or mutant Munc13. Our results showed a typical increase in sAPPα release [29] in both Munc13-1 wild type and Munc13-1 H567K mutant transfected cells (Figure 1, compare lane 1 vs 2, lane 3 vs 4, and lane 5 vs 6; Table 1) indicating that phorbol ester interaction with histidine-567 of the C1 domain of Munc13-1 is not a key step in regulated shedding.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Farber Institute for Neurosciences of Thomas Jefferson University, 900 Walnut Street, Philadelphia, 19107, PA, USA. samgandy@earthlink.net.

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.

No MeSH data available.


Related in: MedlinePlus