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Control of vesicle fusion by a tyrosine phosphatase

Abstract

The tyrosine phosphatase PTP-MEG2 is targeted by its amino-terminal Sec14p homology domain to the membrane of secretory vesicles. There it regulates vesicle size by promoting homotypic vesicle fusion by a mechanism that requires its catalytic activity. Here, we identify N-ethylmaleimide-sensitive factor (NSF), a key regulator of vesicle fusion, as a substrate for PTP-MEG2. PTP-MEG2 reduced the phosphotyrosine content of NSF and co-localized with NSF and syntaxin 6 in intact cells. Furthermore, endogenous PTP-MEG2 co-immunoprecipitated with endogenous NSF. Phosphorylation of NSF at Tyr 83, as well as an acidic substitution at the same site, increased its ATPase activity and prevented αSNAP binding. Conversely, expression of a Y83F mutant of NSF caused spontaneous fusion events. Our results suggest that the molecular mechanism by which PTP-MEG2 promotes secretory vesicle fusion involves the local release of NSF from a tyrosine-phosphorylated, inactive state. This represents a novel mechanism for localized regulation of NSF and the first demonstrated role for a protein tyrosine phosphatase in the regulated secretory pathway.

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Figure 1: Identification of NSF as a substrate for PTP-MEG2.
Figure 2: PTP-MEG2 co-localizes with syntaxin 6 and NSF.
Figure 3: The Fes and Fer PTKs phosphorylate NSF on Tyr 83.
Figure 4: Effects of tyrosine phosphorylation of NSF at Tyr 83.
Figure 5: Tyrosine phosphorylation of NSF at Tyr 83 prevents αSNAP binding.
Figure 6: In vivo effects of tyrosine phosphorylation of NSF at Tyr 83.

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Acknowledgements

We are grateful to G. Reed and S. Whiteheart for anti-NSF antibodies and cDNA, to S. Tooze for anti-syntaxin-6 antibodies and to T. Tsuji for advice on ATPase assays. This work was supported by fellowships from the American Italian Cancer Foundation, the Litta Foundation and by grants AI55741, AI35603, AI48032, AI53585, CA96949 (to T.M.) and AG00252 (to H.H.) from the National Institutes of Health.

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Correspondence to Tomas Mustelin.

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Huynh, H., Bottini, N., Williams, S. et al. Control of vesicle fusion by a tyrosine phosphatase. Nat Cell Biol 6, 831–839 (2004). https://doi.org/10.1038/ncb1164

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