Malmersjö Seth, Di Palma Serena, Diao Jiajie, Lai Ying, Pfuetzner Richard A, Wang Austin L, McMahon Moira A, Hayer Arnold, Porteus Matthew, Bodenmiller Bernd, Brunger Axel T, Meyer Tobias
Department of Chemical and Systems Biology, Stanford University, Stanford, CA, USA
Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland Functional Genomics Center Zurich, ETH Zurich/University of Zurich, Zurich, Switzerland.
EMBO J. 2016 Aug 15;35(16):1810-21. doi: 10.15252/embj.201694071. Epub 2016 Jul 11.
Membrane fusion is essential for eukaryotic life, requiring SNARE proteins to zipper up in an α-helical bundle to pull two membranes together. Here, we show that vesicle fusion can be suppressed by phosphorylation of core conserved residues inside the SNARE domain. We took a proteomics approach using a PKCB knockout mast cell model and found that the key mast cell secretory protein VAMP8 becomes phosphorylated by PKC at multiple residues in the SNARE domain. Our data suggest that VAMP8 phosphorylation reduces vesicle fusion in vitro and suppresses secretion in living cells, allowing vesicles to dock but preventing fusion with the plasma membrane. Markedly, we show that the phosphorylation motif is absent in all eukaryotic neuronal VAMPs, but present in all other VAMPs. Thus, phosphorylation of SNARE domains is a general mechanism to restrict how much cells secrete, opening the door for new therapeutic strategies for suppression of secretion.
膜融合对于真核生物的生命活动至关重要，它需要SNARE蛋白以α-螺旋束的形式“拉链式”结合在一起，从而将两个膜拉近。在此，我们表明囊泡融合可通过SNARE结构域内核心保守残基的磷酸化来抑制。我们采用蛋白质组学方法，利用PKCB基因敲除的肥大细胞模型，发现关键的肥大细胞分泌蛋白VAMP8在SNARE结构域的多个残基处被蛋白激酶C（PKC）磷酸化。我们的数据表明，VAMP8的磷酸化在体外降低了囊泡融合，并在活细胞中抑制了分泌，使得囊泡能够停靠，但阻止了与质膜的融合。值得注意的是，我们发现所有真核生物神经元VAMP中均不存在磷酸化基序，但在所有其他VAMP中都存在。因此，SNARE结构域的磷酸化是一种限制细胞分泌量的普遍机制，为抑制分泌的新治疗策略打开了大门。
