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

Annat F Ikin^*¹ , Mirsada Causevic^*¹ , Steve Pedrini¹ , Lyndsey S Benson¹ , Joseph D Buxbaum² , Toshiharu Suzuki³ , Simon Lovestone⁴ , Shigeki Higashiyama⁵ , Tomas Mustelin⁶ , Robert D Burgoyne⁶ and Sam Gandy¹^,2

¹Farber Institute for Neurosciences of Thomas Jefferson University, 900 Walnut Street, Philadelphia, 19107, PA, USA

²Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, 10029, NY, USA

³Hokkaido University, Sapporo, Japan

⁴Ehime University School of Medicine, Ehime, Japan

⁵The Burnham Institute, La Jolla CA, USA

⁶Physiological Laboratory, University of Liverpool, Crown St, Liverpool, L69 3BX, UK

author email corresponding author email* Contributed equally

Molecular Neurodegeneration 2007, 2:23doi:10.1186/1750-1326-2-23


Published:	9 December 2007

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 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.

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 sAPPα.

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.