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

ROGGERO, CARLOS M.; TOMES, CLAUDIA N.; DE BLAS, GERARDO A.; CASTILLO, JIMENA; MICHAUT, MARCELA A.; FUKUDA, MITSUNORI; MAYORGA, LUIS S.

DEVELOPMENTAL BIOLOGY

ACADEMIC PRESS INC ELSEVIER SCIENCE

Año: 2005 vol. 285 p. 422 - 435

0012-1606

We have previously reported that synaptotagmin VI is present in human sperm cells and that a recombinant protein containing the C2Aand C2B domains abrogates acrosomal exocytosis in permeabilized spermatozoa, an effect that was regulated by phosphorylation. In thisreport, we show that each individual C2 domain blocks acrosomal exocytosis. The inhibitory effect was completely abrogated byphosphorylation of the domains with purified PKChII. We found by site-directed mutagenesis that Thr418 and/or Thr419 in the polybasicregion (KKKTTIK) of the C2B domain ? a key region for the function of synaptotagmins ? are the PKC target that regulates its inhibitoryeffect on acrosomal exocytosis. Similarly, we showed that Thr284 in the polybasic region of C2A (KCKLQTR) is the target for PKCmediatedphosphorylation in this domain. An antibody that specifically binds to the phosphorylated polybasic region of the C2B domainrecognized endogenous phosphorylated synaptotagmin in the sperm acrosomal region. The antibody was inhibitory only at early stages ofexocytosis in sperm acrosome reaction assays, and the immunolabeling decreased upon sperm stimulation, indicating that the protein isdephosphorylated during acrosomal exocytosis. Our results indicate that acrosomal exocytosis is regulated through the PKC-mediatedphosphorylation of conserved threonines in the polybasic regions of synaptotagmin VI.