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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

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).
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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).

Mentions: Figure 3A shows immunoblots of the lysates of cells expressing various combinations of the molecules of interest. The presence of X11 appeared to stabilize the APP holoprotein, as reported [32]. No Munc18 Ser306- or Ser313-phospho-state-specific modulation of APP holoprotein levels was evident in the absence (lanes 4–7) or presence (lanes 8–11) of X11. Figure 4B shows that all forms of Munc18 enhanced sAPPα release, but sensitivity of sAPPα secretion to stimulation by PMA was observed regardless of the integrity or phospho-mimetic mutation of either or both Munc18 PKC phosphorylation sites. Further, since serine-to-glutamate (S-to-E) phospho-site mutants are designed to mimic constitutively phosphorylated amino acids, one might predicted that S-to-E phospho-site mutants would yield effects resembling those caused by phospho-forms of Munc18. In this case, one might predict that single S-to-E mutant forms of Munc18 or double S-to-E mutant Munc18 might enhance basal sAPPα release to an extent that would be greater than that of wildtype Munc18, but such an effect was not observed (Figure 4B). In some experiments, S-to-E double mutant Munc18 was associated with lower fold-effects in response to phorbol esters as compared with single mutant forms. This altered pattern appeared not to be attributable to phorbol sensitivity per se, but to a relative instability of the double mutant or enhanced sensitivity to proteolysis, as revealed by immunoblotting for Munc18 (data not shown).


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)

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

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

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).
Mentions: Figure 3A shows immunoblots of the lysates of cells expressing various combinations of the molecules of interest. The presence of X11 appeared to stabilize the APP holoprotein, as reported [32]. No Munc18 Ser306- or Ser313-phospho-state-specific modulation of APP holoprotein levels was evident in the absence (lanes 4–7) or presence (lanes 8–11) of X11. Figure 4B shows that all forms of Munc18 enhanced sAPPα release, but sensitivity of sAPPα secretion to stimulation by PMA was observed regardless of the integrity or phospho-mimetic mutation of either or both Munc18 PKC phosphorylation sites. Further, since serine-to-glutamate (S-to-E) phospho-site mutants are designed to mimic constitutively phosphorylated amino acids, one might predicted that S-to-E phospho-site mutants would yield effects resembling those caused by phospho-forms of Munc18. In this case, one might predict that single S-to-E mutant forms of Munc18 or double S-to-E mutant Munc18 might enhance basal sAPPα release to an extent that would be greater than that of wildtype Munc18, but such an effect was not observed (Figure 4B). In some experiments, S-to-E double mutant Munc18 was associated with lower fold-effects in response to phorbol esters as compared with single mutant forms. This altered pattern appeared not to be attributable to phorbol sensitivity per se, but to a relative instability of the double mutant or enhanced sensitivity to proteolysis, as revealed by immunoblotting for Munc18 (data not shown).

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