Time-resolved imaging of protein kinase C activation during sea urchin egg fertilization

To study protein kinase C (PKC) activation during sea urchin egg fertilization we used three different fluorescent probes specific for PKC, namely, fim-1, which recognizes the catalytic site of the enzyme, and BODIPY- and NBD-phorbol esters interacting with the PKC regulatory domain. We were able to follow PKC activation during the early steps of fertilization, the three different probes giving the same fluorescent pattern. Within 120 s following insemination, the fluorescent signal increased and clustered in the cortical zone of the cell. The process was Ca2+ dependent and was inhibited in the presence of staurosporine, a PKC inhibitor. According to our in vitro probe characterization, this signal increase is due to PKC activation. These findings were further confirmed by Western blot analysis. This initial phase was followed by a rapid decrease which might be attributed to PKC hydrolysis by Ca2(+)-dependent proteases. The kinetics and the site distribution of PKC activation appear in complete agreement with the putative functions previously suggested for PKC during fertilization.