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

RODRIGUEZ PEÑA, MARCELO JAVIER; MAYORGA, LUIS SEGUNDO; MICHAUT, MARCELA ALEJANDRA

Mar del Plata, Bs. As, Argentina

Congreso; Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB); 2007

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 reveled the presence of MARCKS in human sperm. Furthermore, immunocytochemistry assays showed that MARCKS localized at the acrosomal region in human sperm. This localization prompted us to investigate if MARCKS might participate in acrosomal exocytosis. To test this hypothesis, we expressed MARCKS effector domain (ED) as a GST- fusion protein. Using the Streptolysin O-permeabilized sperm model, we investigated the effect of MARCKS ED on the acrosome exocytosis stimulated by a PKC activator, phorbol 12-myristate, 13-acetate (PMA). MARCKS ED inhibited specifically acrosomal exocytosis stimulated by PMA in a concentration-dependent manner. To investigate if MARCKS phosphorylation might affect the acrosomal exocytosis 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 acrosome exocytosis, only the constitutively nonphosphorylated MARCKS inhibited the exocytosis stimulated by PMA suggesting that MARCKS phosphorylation is required during the progression of this exocytosis. These results show that MARCKS is expressed in human sperm and MARCKS phosphorylation is a new component of the signal transduction pathways in acrosomal exocytosis.