Protein Kinase M, the Cytosolic Counterpart of Protein Kinase C, Remodels the Internal Cytoskeleton of the Mammalian Egg during Activation

AbstractWe have investigated mechanisms by which intracellular signals act to restructure the spatial organization of the cytoskeleton as the mammalian egg is converted into the zygote. Four distinct approaches (one cytological, two biochemical, and one pharmacological) demonstrate protein kinase C (PKC) and its cytosolic active counterpart, PKM, act in succession at the time of egg activation. PKM serves to remodel the internal cytoskeleton. The cytological approach mapped the distribution of kinase over time using the PKC reporter dye, Rim-1, which demonstrated a temporal shift in kinase distribution from its initial site of activation at the plasma membrane to its subsequent association with a cross-linked network of intermediate filaments referred to as sheets. The first of two biochemical analyses, Western blot analysis, demonstrated that eggs activated with calcium ionophore contained PKC in the detergent-soluble fraction and PKM in the sheet enriched fraction. Prior to egg activation, PKM is not detected in the sheet-enriched fraction; only PKC is detected in the detergent-soluble fraction of these eggs. The second biochemical analysis demonstrated, via [32P]ATP labeling of a known PKC substrate, that the PKM generated from activation of PKC in response to activation of eggs with calcium ionophore is an active kinase and is located in the sheet-enriched fraction. In addition, purified forms of PKM, but not PKC, could be shown to act on the internal cytoskeleton when perfused into a permeabilized egg system. Pharmacological treatments demonstrate that elevation of [Ca2+]i does not act directly to alter the internal cytoskeleton of the egg. Our results suggest that this kinase is employed at the time of fertilization to provide an internal chronometer acting first at the cell periphery as PKC and subsequently in the cell interior as PKM