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The Journal of Neuroscience, January 2, 2008, 28(1):21-30; doi:10.1523/JNEUROSCI.2352-07.2008

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Cellular/Molecular
Phosphorylation of SNAP-25 at Ser187 Mediates Enhancement of Exocytosis by a Phorbol Ester in INS-1 Cells

Yilong Shu,^1,2 Xin Liu,² Yan Yang,² Masami Takahashi,⁴ and Kevin D. Gillis^1,2,3

¹Interdisciplinary Neuroscience Program, ²Dalton Cardiovascular Research Center, and ³Department of Biological Engineering, University of Missouri-Columbia, Columbia, Missouri 65211, and ⁴Department of Biochemistry, Kitasato University School of Medicine, Kanagawa 228-8555, Japan

Correspondence should be addressed to Kevin D. Gillis, Dalton Cardiovascular Research Center, University of Missouri-Columbia, 134 Research Park Drive, Columbia, MO 65211. Email: gillisk{at}missouri.edu

Activation of diacylglycerol (DAG) signaling pathways with phorbol esters dramatically enhances Ca²⁺-triggered exocytosis from both endocrine cells and neurons, however the relevant targets of DAG are controversial. A possible effector mechanism for this signaling pathway is phosphorylation of SNAP-25 (25 kDa synaptosome-associated protein) at Ser187 by PKC. Here, we investigated the role of Ser187 in the enhancement of exocytosis by the phorbol ester PMA (phorbol 12-myristate 13-acetate). We used patch-clamp measurements of membrane capacitance together with photorelease of caged-Ca²⁺ and membrane depolarization to study exocytosis. Expression of the nonphosphorylatable S187C SNAP-25 mutant did not attenuate the enhancement of exocytosis by PMA in either bovine chromaffin cells or the INS-1 insulin-secreting cell line. To test the effects of Ser187 mutations under conditions in which the endogenous SNAP-25 is disabled, we expressed botulinum toxin serotype E to cleave SNAP-25 in INS-1 cells. Coexpression of a toxin-resistant mutant (TR), but not wild-type SNAP-25, was able to rescue PMA-modulated exocytosis. Coexpression of the toxin with the TR-S187C SNAP-25 mutant was able to completely block the enhancement of exocytosis by PMA in response to photoelevation of [Ca²⁺]_i to low µM levels or to a depolarizing train. The phospho-mimetic S187E mutation enhanced the small, fast burst of exocytosis evoked by photelevation of Ca²⁺, but, like PMA, had smaller effects on exocytosis evoked by a depolarizing train. This work supports the hypothesis that phosphorylation of Ser187 of SNAP-25 by PKC is a key step in the enhancement of exocytosis by DAG.

Key words: phorbol esters; PKC; SNAP-25; exocytosis; BoNT/E; phosphomimetic mutations

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Received May 23, 2007; revised Oct. 11, 2007; accepted Nov. 6, 2007.

Correspondence should be addressed to Kevin D. Gillis, Dalton Cardiovascular Research Center, University of Missouri-Columbia, 134 Research Park Drive, Columbia, MO 65211. Email: gillisk{at}missouri.edu

This article has been cited by other articles:

				X. Lou, N. Korogod, N. Brose, and R. Schneggenburger Phorbol Esters Modulate Spontaneous and Ca2+-Evoked Transmitter Release via Acting on Both Munc13 and Protein Kinase C J. Neurosci., August 13, 2008; 28(33): 8257 - 8267. [Abstract] [Full Text] [PDF]

				L. Eliasson, F. Abdulkader, M. Braun, J. Galvanovskis, M. B. Hoppa, and P. Rorsman Novel aspects of the molecular mechanisms controlling insulin secretion J. Physiol., July 15, 2008; 586(14): 3313 - 3324. [Abstract] [Full Text] [PDF]

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