Protein kinase C-mediated phosphorylation of the two polybasic regions of synaptotagmin VI regulates their function in acrosomal exocytosis

Carlos M. Roggero, Claudia N. Tomes, Gerardo A. De Blas, Jimena Castillo, Marcela A. Michaut, Mitsunori Fukuda, Luis S. Mayorga

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

We have previously reported that synaptotagmin VI is present in human sperm cells and that a recombinant protein containing the C2A and C2B domains abrogates acrosomal exocytosis in permeabilized spermatozoa, an effect that was regulated by phosphorylation. In this report, we show that each individual C2 domain blocks acrosomal exocytosis. The inhibitory effect was completely abrogated by phosphorylation of the domains with purified PKCβII. We found by site-directed mutagenesis that Thr418 and/or Thr419 in the polybasic region (KKKTTIK) of the C2B domain - a key region for the function of synaptotagmins - are the PKC target that regulates its inhibitory effect on acrosomal exocytosis. Similarly, we showed that Thr284 in the polybasic region of C2A (KCKLQTR) is the target for PKC-mediated phosphorylation in this domain. An antibody that specifically binds to the phosphorylated polybasic region of the C2B domain recognized endogenous phosphorylated synaptotagmin in the sperm acrosomal region. The antibody was inhibitory only at early stages of exocytosis in sperm acrosome reaction assays, and the immunolabeling decreased upon sperm stimulation, indicating that the protein is dephosphorylated during acrosomal exocytosis. Our results indicate that acrosomal exocytosis is regulated through the PKC-mediated phosphorylation of conserved threonines in the polybasic regions of synaptotagmin VI.

Original languageEnglish
Pages (from-to)422-435
Number of pages14
JournalDevelopmental Biology
Volume285
Issue number2
DOIs
Publication statusPublished - 2005 Sep 15
Externally publishedYes

Keywords

  • Acrosome reaction
  • Human sperm
  • PKC
  • Phosphorylation
  • Spermatozoon
  • Synaptotagmin

ASJC Scopus subject areas

  • Molecular Biology
  • Developmental Biology
  • Cell Biology

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