Resumen de Analysis of CK2-dependent regulation of Myo5-induced actin polymerization during the endocytic uptake in S. Cerevisiae / Análisis de la regulación mediada por CK2 de la polimerización de actina inducida por Myo5 durante la internalización endocítica en “S. Cerevisiae”
Isabel Fernández Golbano
S. cerevisiae (budding yeast) is particularly powerful to study the relation between the molecular mechanisms of actin regulation and its biological significance due to its powerful molecular and genetic methods, well-established biochemical techniques, and development of live cell imaging and immuno-electron microscopy. Further, budding yeast has a simple cytoskeleton but the molecular mechanisms controlling its remodeling are conserved from yeast to mammals. Interestingly, a dynamic actin cytoskeleton is mandatory for endocytosis in S. cerevisiae. In this organism the formation of the primary endocytic profiles in yeast occurs concomitant with the assembly of a complex actin structure (the endocytic actin module), which includes the actin nucleator Arp2/3 complex. Consistent with observations indicating that the myosin-I Myo5 in complex with the WIP homolog Vrp1 is a major Arp2/3-dependent actin nucleating promoting factor (NPF) driving productive membrane invagination, our laboratory has demonstrated that the C-terminal domain of Myo5 immobilized on the surface of Sepharose beads induces assembly of the endocytic actin module in the presence of yeast extracts. Analysis of the in vitro actin assembly assay demonstrated that Myo5 is phosphorylated by the casein kinase CK2 at the serine 1205 during the assay. In this study, we have characterized the CK2 activity that phosphorylates Myo5 at the C-terminus and investigated the functional significance of this phosphorylation in vivo and in vitro. Our results indicate that a non-tetrameric and particulate-associated CK2 activity that includes the catalytic subunit Cka2 but not Cka1 or the regulatory subunits Ckb1 and Ckb2 phosphorylates Myo5 S1205. Our results also indicate that this phosphorylation event down-regulates the assembly of complex actin structures in vitro and slows down the internalization process and the dissociation of the myosin from the plasma membrane in vivo. The Cka2-mediated phosphorylation at Myo5 S1205 does not seem to regulate the affinity of the NPF for the Arp2/3 complex but rather down-regulates the interaction of Myo5 with its co-activator Vrp1. This phosphorylation also increases directly or indirectly the binding of the myosin to the endocytic coat components Sla1 and Pan1 and to the syndapin Bzz1, suggesting that the phosphorylation event occurs late during the maturation of the endocytic invagination. Finally, our data suggest that Cka2 have endocytic targets other than Myo5 and that its activity might also regulate early steps during the assembly of the endocytic coat.
