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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

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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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.

Mentions: Because of the biological and potential clinical importance of regulated shedding, we embarked upon two strategies to elucidate its molecular basis. First, we identified the molecular machinery responsible for α-secretase cleavage in Saccharomyces cerevisiae [43]. In that case, endogenous α-secretase was identified as yapsin [44], a PI-linked intravesicular/cell surface protease rather than the integral metalloproteinases that catalyze the reaction in mammalian cells. Thus, this molecular diversity thwarted our strategy of discovering the basis for regulated shedding via a yeast genetics approach. In the current study, we took a "candidate molecule approach", examining four molecules that might plausibly underlie phorbol- or PKC-regulated APP ectodomain shedding. Potential mechanisms for how each of the four PMES candidates tested herein might act to regulate APP shedding are depicted in cartoon form in Figure 6; however, none of these four proteins appears to fulfill the essential criteria for a PMES.


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)

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

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

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.
Mentions: Because of the biological and potential clinical importance of regulated shedding, we embarked upon two strategies to elucidate its molecular basis. First, we identified the molecular machinery responsible for α-secretase cleavage in Saccharomyces cerevisiae [43]. In that case, endogenous α-secretase was identified as yapsin [44], a PI-linked intravesicular/cell surface protease rather than the integral metalloproteinases that catalyze the reaction in mammalian cells. Thus, this molecular diversity thwarted our strategy of discovering the basis for regulated shedding via a yeast genetics approach. In the current study, we took a "candidate molecule approach", examining four molecules that might plausibly underlie phorbol- or PKC-regulated APP ectodomain shedding. Potential mechanisms for how each of the four PMES candidates tested herein might act to regulate APP shedding are depicted in cartoon form in Figure 6; however, none of these four proteins appears to fulfill the essential criteria for a PMES.

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