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

<p>Abstract</p> <p>Background</p> <p>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 <it>trans</it>-Golgi network (TGN) and toward the plasma membrane where APP becomes a substrate for enzymes responsible for shedding, known collectively as α-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.</p> <p>Results</p> <p>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 <it>et al </it>(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 sAPPα.</p> <p>Conclusion</p> <p>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.</p

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-5

<p><b>Copyright information:</b></p><p>Taken from "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"</p><p>http://www.molecularneurodegeneration.com/content/2/1/23</p><p>Molecular Neurodegeneration 2007;2():23-23.</p><p>Published online 9 Dec 2007</p><p>PMCID:PMC2211485.</p><p></p>ture 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

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-4

<p><b>Copyright information:</b></p><p>Taken from "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"</p><p>http://www.molecularneurodegeneration.com/content/2/1/23</p><p>Molecular Neurodegeneration 2007;2():23-23.</p><p>Published online 9 Dec 2007</p><p>PMCID:PMC2211485.</p><p></p>esence (+) of PDBu. (B) Quantification of 3 such experiments

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-1

<p><b>Copyright information:</b></p><p>Taken from "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"</p><p>http://www.molecularneurodegeneration.com/content/2/1/23</p><p>Molecular Neurodegeneration 2007;2():23-23.</p><p>Published online 9 Dec 2007</p><p>PMCID:PMC2211485.</p><p></p>ualization of (a) Munc13-1and (c) Munc13-1mutant molecules (green). (b, d) APP was immunolabeled with rabbit polyclonal anti-APP-specific antibody 369 followed by rhodamine red conjugated secondary antibody (red)

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-6

<p><b>Copyright information:</b></p><p>Taken from "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"</p><p>http://www.molecularneurodegeneration.com/content/2/1/23</p><p>Molecular Neurodegeneration 2007;2():23-23.</p><p>Published online 9 Dec 2007</p><p>PMCID:PMC2211485.</p><p></p>ualization of (a) Munc13-1and (c) Munc13-1mutant molecules (green). (b, d) APP was immunolabeled with rabbit polyclonal anti-APP-specific antibody 369 followed by rhodamine red conjugated secondary antibody (red)

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-2

<p><b>Copyright information:</b></p><p>Taken from "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"</p><p>http://www.molecularneurodegeneration.com/content/2/1/23</p><p>Molecular Neurodegeneration 2007;2():23-23.</p><p>Published online 9 Dec 2007</p><p>PMCID:PMC2211485.</p><p></p>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)