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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

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).
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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).

Mentions: The subcellular localization of APP was similar following phorbol ester treatment of cells transfected with either the wild type Munc13-1 or H567K Munc13-1 mutant (Figure 2, panels "b" and "d") whilst the characteristic difference in phorbol ester binding and intracellular localization between the wild type Munc13-1 and H567K Munc13-1 mutant was observed (Figure 2, panels "a" and "c"). As previously reported [11], in contrast to the wild type Munc13-1 (Figure 2, panel "a"), the H567K Munc13-1 mutant failed to translocate to the plasma membrane following phorbol ester application (Figure 2, panel "c").


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)

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

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

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).
Mentions: The subcellular localization of APP was similar following phorbol ester treatment of cells transfected with either the wild type Munc13-1 or H567K Munc13-1 mutant (Figure 2, panels "b" and "d") whilst the characteristic difference in phorbol ester binding and intracellular localization between the wild type Munc13-1 and H567K Munc13-1 mutant was observed (Figure 2, panels "a" and "c"). As previously reported [11], in contrast to the wild type Munc13-1 (Figure 2, panel "a"), the H567K Munc13-1 mutant failed to translocate to the plasma membrane following phorbol ester application (Figure 2, panel "c").

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