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

RODRIGUEZ PEÑA MJ; MAYORGA LS; MICHAUT MA

Carlos Paz

Congreso; SAIB XLIV; 2008

MARCKS is a prominent substrate of PKC in many cell types;nevertheless the presence of MARCKS in sperm, as a possible PKCsubstrate has not been investigated. Using a specific antibodyagainst MARCKS, Western blot analysis revealed the presence ofMARCKS in human sperm and immunocytochemistry assaysshowed that MARCKS localized at the acrosomal region in humansperm. This localization prompted us to investigate if MARCKSmight participate in acrosomal exocytosis (AE). We expressedMARCKS effector domain (ED) and assayed on the AE stimulatedeither by a PKC activator, PMA, or by calcium in a permeabilizedsperm model. MARCKS ED inhibited specifically AE stimulatedby PMA and calcium suggesting a role for MARCKS in AE.MARCKS ED has a high affinity for PIP2, and a model hasemerged in which PKC-mediated regulation of MARCKS couldcontrol spatial availability of PIP2. We tested this model in oursystem by adding PIP2, which reverted MARCKS inhibitory effectin calcium-stimulated exocytosis. In addition, Western blotanalysis using a specific phospho-MARCKS antibody shown thatMARCKS phosphorylation increased after PMA treatment, and aN-terminal MARCKS antibody revealed that MARCKS wasreleased from membrane following PMA treatment. Altogether,these results suggest that MARCKS phosphorylation might play aregulatory role on PIP2 concentration in acrosome exocytosis inhuman sperm.