Marcks phosphorylation plays a regulatory role on PIP2 concentration in acrosomal exocytosis

RODRIGUEZ PEÑA M.; MAYORGA L.S.; MICHAUT M.A.

Villa Carlos Paz, Córdoba.

Congreso; XLIV REUNIÓN ANUAL / 2008, ANNUAL MEETING SOCIEDAD ARGENTINA DE INVESTIGACIÓN BIOQUÍMICA Y BIOLOGÍA MOLECULAR.; 2008

SOCIEDAD ARGENTINA DE INVESTIGACIÓN 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 and 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 (AE). We expressed MARCKS effector domain (ED) and assayed on the AE stimulated either by a PKC activator, PMA, or by calcium in a permeabilized sperm model. MARCKS ED inhibited specifically AE stimulated by PMA and calcium suggesting a role for MARCKS in AE. MARCKS ED has a high affinity for PIP2, and a model has emerged in which PKC-mediated regulation of MARCKS could control spatial availability of PIP2. We tested this model in our system by adding PIP2, which reverted MARCKS inhibitory effect in calcium-stimulated exocytosis. In addition, Western blot analysis using a specific phospho-MARCKS antibody shown that MARCKS phosphorylation increased after PMA treatment, and a N-terminal MARCKS antibody revealed that MARCKS was released from membrane following PMA treatment. Altogether, these results suggest that MARCKS phosphorylation might play a regulatory role on PIP2 concentration in acrosome exocytosis in human sperm.