Ordered Multisite Phosphorylation of Lethal Giant Larvae by Atypical Protein Kinase C

[Image: see text] In Par complex-mediated cell polarity, phosphorylation by atypical protein kinase C (aPKC) is coupled to substrate cortical displacement. Polarized substrates often contain multiple phosphorylation sites, but the role of multisite phosphorylation in Par-mediated polarity remains unclear. Here, we have dissected the role of the three aPKC phosphorylation sites within the tumor suppressor Lethal giant larvae. Using a cultured Drosophila S2 cell cortical displacement assay, we observed that phosphorylation at any one site causes only partial displacement. Complete displacement requires that all three sites be modified. We undertook a kinetic analysis to determine if aPKC phosphorylates each site equivalently. As the sites are closely spaced, we observed not only differences in the rate of phosphorylation but also interaction between the sites. A complete description of the rates reveals a preferential order of phosphorylation. Our results provide new insights into how multiple phosphorylations and phosphorylation rates could regulate localization behaviors of fate determinants at the cortex.