MARCKS PHOSPHORYLATION: A NEW COMPONENT OF THE SIGNAL TRANSDUCTION PATHWAY IN ACROSOMAL EXOCYTOSIS

RODRIGUEZ PEÑA M.; CASTILLO BENNETT J.; MAYORGA L.S.; MICHAUT M.A.

Mar del Plata, Buenos Aires

Congreso; 2007, XLIII Reunión Anual/2007, Annual Meeting de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2007

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

MARCKS is a prominent substrate of PKC in many cell types; nevertheless the presence of MARCKS in sperm, as a possible PKC substrate has not been investigated. Using a specific antibody against MARCKS, Western blot analysis revealed the presence of MARCKS in human sperm. Furthermore, immunocytochemistry assays showed that MARCKS localized at the acrosomal region. This localization prompted us to investigate if MARCKS participate in acrosomal exocitosis (AE). To test this hypothesis, we expressed MARCKS effector domain (ED). Using the Streptolysin O-permeabilized sperm model, we investigated the effect of MARCKS ED on the AE stimulated by a PKC activator, phorbol 12-myristate 13-acetate (PMA). MARCKS ED inhibited specifically AE stimulated by PMA. To investigate if MARCKS phosphorylation might affect the AE stimulated by PMA, we generated two different mutants of MARCKS ED: the constitutively phosphorylated and the constitutively unphosphorylated MARCKS by replacement serines of the ED with aspartic acid and alanine, respectively. When these mutants were assayed in the AE, only the constitutively nonphosphorylated MARCKS inhibited the exocytosis stimulated by PMA suggesting that MARCKS phosphorylation is required during the progression of AE. These results show that MARCKS is expressed in human sperm and its phosphorylation is a new component of the signal transduction pathways in AE.