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Doc2 and Munc13-4 Regulate Ca²⁺-Dependent Secretory Lysosome Exocytosis in Mast Cells¹

Hironori Higashio^*, Noriyuki Nishimura^*, Hiroyoshi Ishizaki, Jun Miyoshi, Satoshi Orita^*,, Ayuko Sakane^* and Takuya Sasaki^2,*

^* Department of Biochemistry, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan; Department of Molecular Biology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan; and Pharmaceutical Research Division, Shionogi and Company, Osaka, Japan

The Doc2 family comprises the brain-specific Doc2 and the ubiquitous Doc2β and Doc2. With the exception of Doc2, these proteins exhibit Ca²⁺-dependent phospholipid-binding activity in their Ca²⁺-binding C2A domain and are thought to be important for Ca²⁺-dependent regulated exocytosis. In excitatory neurons, Doc2 interacts with Munc13-1, a member of the Munc13 family, through its N-terminal Munc13-1-interacting domain and the Doc2-Munc13-1 system is implicated in Ca²⁺-dependent synaptic vesicle exocytosis. The Munc13 family comprises the brain-specific Munc13-1, Munc13-2, and Munc13-3, and the non-neuronal Munc13-4. We previously showed that Munc13-4 is involved in Ca²⁺-dependent secretory lysosome exocytosis in mast cells, but the involvement of Doc2 in this process is not determined. In the present study, we found that Doc2 but not Doc2β was endogenously expressed in the RBL-2H3 mast cell line. Doc2 colocalized with Munc13-4 on secretory lysosomes, and interacted with Munc13-4 through its two regions, the N terminus containing the Munc13-1-interacting domain and the C terminus containing the Ca²⁺-binding C2B domain. In RBL-2H3 cells, Ca²⁺-dependent secretory lysosome exocytosis was inhibited by expression of the Doc2 mutant lacking either of the Munc13-4-binding regions and the inhibition was suppressed by coexpression of Munc13-4. Knockdown of endogenous Doc2 also reduced Ca²⁺-dependent secretory lysosome exocytosis, which was rescued by re-expression of human Doc2 but not by its mutant that could not bind to Munc13-4. Moreover, Ca²⁺-dependent secretory lysosome exocytosis was severely reduced in bone marrow-derived mast cells from Doc2 knockout mice. These results suggest that the Doc2-Munc13-4 system regulates Ca²⁺-dependent secretory lysosome exocytosis in mast cells.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Grants-in-Aid for Scientific Research (18790204 to H.H., 18590271 to N.N., and 15079207, 18390089 to T.S.) from the Ministry of Education, Culture, Sport, Science, and Technology of Japan.

² Address correspondence and reprint requests to Dr. Takuya Sasaki, Department of Biochemistry, Institute of Health Biosciences, University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan. E-mail address: sasaki{at}basic.med.tokushima-u.ac.jp

³ Abbreviations used in this paper: SNARE, soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor; FHL, familial hemophagocytic lymphohistiocytosis; Mid, Munc13-1-interacting domain; HA, hemagglutinin; siRNA, small-interfering RNA; BMMC, bone marrow-derived mast cell; PVDF, polyvinylidene difluoride; TPA, 12-O-tetradecanoylphorbol-13-acetate; Syt, synaptotagmin.