Zarelli Valeria E P, Ruete Maria C, Roggero Carlos M, Mayorga Luis S, Tomes Claudia N
Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM-CONICET), Facultad de Ciencias Médicas, CC 56, Universidad Nacional de Cuyo, 5500 Mendoza, Argentina.
J Biol Chem. 2009 Apr 17;284(16):10491-503. doi: 10.1074/jbc.M807614200. Epub 2009 Feb 10.
The reversible phosphorylation of tyrosyl residues in proteins is a cornerstone of the signaling pathways that regulate numerous cellular responses. Protein tyrosine phosphorylation is controlled through the concerted actions of protein-tyrosine kinases and phosphatases. The goal of the present study was to unveil the mechanisms by which protein tyrosine dephosphorylation modulates secretion. The acrosome reaction, a specialized type of regulated exocytosis undergone by sperm, is initiated by calcium and carried out by a number of players, including tyrosine kinases and phosphatases, and fusion-related proteins such as Rab3A, alpha-SNAP, N-ethylmaleimide-sensitive factor (NSF), SNAREs, complexin, and synaptotagmin VI. We report here that inducers were unable to elicit the acrosome reaction when permeabilized human sperm were loaded with anti-PTP1B antibodies or with the dominant-negative mutant PTP1B D181A; subsequent introduction of wild type PTP1B or NSF rescued exocytosis. Wild type PTP1B, but not PTP1B D181A, caused cis SNARE complex dissociation during the acrosome reaction through a mechanism involving NSF. Unlike its non-phosphorylated counterpart, recombinant phospho-NSF failed to dissociate SNARE complexes from rat brain membranes. These results strengthen our previous observation that NSF activity is regulated rather than constitutive during sperm exocytosis and indicate that NSF must be dephosphorylated by PTP1B to disassemble SNARE complexes. Interestingly, phospho-NSF served as a substrate for PTP1B in an in vitro assay. Our findings demonstrate that phosphorylation of NSF on tyrosine residues prevents its SNARE complex dissociation activity and establish for the first time a role for PTP1B in the modulation of the membrane fusion machinery.
蛋白质中酪氨酸残基的可逆磷酸化是调节众多细胞反应的信号通路的基石。蛋白质酪氨酸磷酸化通过蛋白酪氨酸激酶和磷酸酶的协同作用来控制。本研究的目的是揭示蛋白质酪氨酸去磷酸化调节分泌的机制。顶体反应是精子经历的一种特殊类型的受调控胞吐作用，由钙引发，并由多种参与者执行，包括酪氨酸激酶和磷酸酶，以及与融合相关的蛋白质，如Rab3A、α-SNAP、N-乙基马来酰亚胺敏感因子（NSF）、SNAREs、结合蛋白和突触结合蛋白VI。我们在此报告，当用抗PTP1B抗体或显性负性突变体PTP1B D181A加载通透的人类精子时，诱导剂无法引发顶体反应；随后引入野生型PTP1B或NSF可挽救胞吐作用。野生型PTP1B而非PTP1B D181A在顶体反应期间通过涉及NSF的机制导致顺式SNARE复合体解离。与其非磷酸化对应物不同，重组磷酸化NSF未能从大鼠脑膜中解离SNARE复合体。这些结果加强了我们之前的观察，即NSF活性在精子胞吐过程中是受调节的而非组成性的，并表明NSF必须被PTP1B去磷酸化才能拆解SNARE复合体。有趣的是，在体外试验中，磷酸化NSF作为PTP1B的底物。我们的研究结果表明，NSF酪氨酸残基的磷酸化会阻止其SNARE复合体解离活性，并首次确立了PTP1B在调节膜融合机制中的作用。
