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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

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.
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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.

Mentions: We therefore tested the involvement of Eve-1 in phorbol-stimulated sAPPα generation by transiently expressing Eve-1-c and Eve-1-d (the two isoforms shown to bind ADAMs 9, 10 and 17) in HEK293-695 cells. We found no consistent effect of Eve-1c or Eve-1d on either basal or phorbol-stimulated sAPPα secretion (Figure 5), although, in some experiments, Eve-1c showed a nonsignificant trend toward potentiation of shedding. Further work on Eve-1 isoforms is ongoing. No 32PO4 incorporation into either Eve-1c or Eve-1d was detectable in response to phorbol ester treatment (not shown) despite the existence of consensus PKC phosphorylation sites. It is possible that separate adaptors exist that correspond to each protein that undergoes shedding (i.e., that Eve-1, the pro-EGF/ADAM adaptor, is not employed as an adaptor for APP/ADAM). To date, no adaptor has been identified that regulates α-secretase shedding of the APP ectodomain according to the phosphorylation state of the adaptor.


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)

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

License
Show All Figures
getmorefigures.php?uid=PMC2211485&req=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.
Mentions: We therefore tested the involvement of Eve-1 in phorbol-stimulated sAPPα generation by transiently expressing Eve-1-c and Eve-1-d (the two isoforms shown to bind ADAMs 9, 10 and 17) in HEK293-695 cells. We found no consistent effect of Eve-1c or Eve-1d on either basal or phorbol-stimulated sAPPα secretion (Figure 5), although, in some experiments, Eve-1c showed a nonsignificant trend toward potentiation of shedding. Further work on Eve-1 isoforms is ongoing. No 32PO4 incorporation into either Eve-1c or Eve-1d was detectable in response to phorbol ester treatment (not shown) despite the existence of consensus PKC phosphorylation sites. It is possible that separate adaptors exist that correspond to each protein that undergoes shedding (i.e., that Eve-1, the pro-EGF/ADAM adaptor, is not employed as an adaptor for APP/ADAM). To date, no adaptor has been identified that regulates α-secretase shedding of the APP ectodomain according to the phosphorylation state of the adaptor.

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