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PTP-MEG2 regulates quantal size and fusion pore opening through two distinct structural bases and substrates

By Yun-Fei Xu, Xu Chen, Zhao Yang, Peng Xiao, Chun-Hua Liu, Kang-Shuai Li, Xiao-Zhen Yang, Yi-Jing Wang, Zhong-Liang Zhu, Zhi-Gang Xu, Sheng Zhang, Chuan Wang, You-Chen Song, Wei-Dong Zhao, Chang-He Wang, Zhi-Liang Ji, Zhong-Yin Zhang, Min Cui, Jin-Peng Sun, Xiao Yu

Posted 29 Oct 2019
bioRxiv DOI: 10.1101/822031

Tyrosine phosphorylation of secretion machinery proteins is a crucial regulatory mechanism for exocytosis. However, the participation of protein tyrosine phosphatases (PTPs) in different exocytosis stages has not been defined. Here we demonstrated that PTP-MEG2 controls multiple steps of catecholamine secretion. Biochemical and crystallographic analyses revealed key residues that the interactions between govern the PTP-MEG2 and NSF-pY83 site, specify PTP-MEG2 substrate selectivity and modulate the fusion of catecholamine-containing vesicles. Unexpectedly, delineation of PTP-MEG2 mutants along with the NSF binding interface revealed that PTP-MEG2 controls the fusion pore opening through non-NSF dependent mechanisms. Utilizing bioinformatics search and biochemical and electrochemical screening approaches, we discovered that PTP-MEG2 regulates the opening and extension of the fusion pore by dephosphorylating the DYNAMIN2-pY125 and MUNC18-1-pY145 site. Further structural and biochemical analysis confirmed the interaction of PTP-MEG2 with MUNC18-1-pY145 or DYNAMIN2-pY125 through a distinct structural basis compared with that of the NSF-pY83 site. Our studies extended mechanistic insights in complex exocytosis processes. HIGHLIGHTS PTP-MEG2 regulates multiple steps of exocytosis. A crystal structure of the PTP-MEG2/phosphor-NSF-pY83 segment was obtained. Functional delineation of the PTP-MEG2/NSF interface led to the discovery of new PTP-MEG2 substrates. PTP-MEG2 regulates fusion pore opening and extension through the DYNAMIN2-pY125 site and MUNC18-1 pY145 site. The distinct structural basis of the recognition of substrates by PTP-MEG2 allows selective inhibitor design. ### Competing Interest Statement The authors have declared no competing interest. * PTP : protein tyrosine phosphatase NSF : N-ethylmaleimide-sensitive fusion protein AngII : Angiotensin II EPI : epinephrine NE : norepinephrine CFE : carbon fiber electrode LDCVs : large-dense-core vesicles PSF : pre-spike foot SAF : stand-alone foot pNPP : p-nitrophenyl phosphate MUNC18 : Mammalian homolog of Unc-18 Cav1.2 : Voltage-gated calcium channel subunit alpha Cav1.2 VAMP7 : Vesicle-associated membrane protein 7 PPP3CA : Serine/threonine-protein phosphatase 2B catalytic subunit alpha isoform PM : plasma membrane LYP : Lymphoid phosphatase SKAP-HOM : Src kinase-associated phosphoprotein HER2 : Receptor tyrosine-protein kinase erbB-2 SHP1 : Protein-tyrosine phosphatase SHP-1 PTPH1 : Protein-tyrosine phosphatase H1.

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