Epithelial sheets are barriers that subdivide the body into physiologically distinct compartments. To achieve this, epithelial cells acquire apical-basal polarity. Polarity regulators associated with the apical membrane include atypical protein kinase C (aPKC), Cdc42, and the transmembrane protein Crumbs. Moreover, the vesicle trafficking machinery contributes to epithelial polarization by facilitating the transport of proteins and lipids to apical and basolateral domains. The mechanisms by which polarity proteins interact with the trafficking machinery remain unclear. Our previous work showed that the regulation of Crumbs endocytosis or recycling by Cdc42 and its downstream effector aPKC is important for epithelial integrity This led us to hypothesize that aPKC has phosphorylation targets in the vesicle trafficking machinery.
Bioinformatic analysis suggests that aPKC may have several phosphorylation targets in the trafficking machinery, including the retromer component Vps26. The retromer retrieves proteins from endosomes and transports them back to the Golgi or to the plasma membrane. Our data suggest that aPKC, when activated by Cdc42, phosphorylates Vps26, and as a result increases the activity of the retromer complex. We generated Vps26 mutations and found that Vps26 maternal/zygotic mutants show loss of epithelial integrity and defects that resemble those observed in crumbs or other polarity mutants. Normal, phosphomimic and non-phosphorylatable isoforms of Vps26 form a complex with aPKC in vivo, and, expression of phosphomimic Vps26 suppresses the Cdc42 loss-of-function phenotype. Furthermore, Cdc42 or aPKC compromised embryos show loss of Crumbs from the plasma membrane, and its accumulation in Vps26 positive endosomes. These findings suggest that the retromer is recruited to endosomes in order to recycle Crumbs but is unable to do so in the absence of aPKC phosphorylation of Vps26. We propose therefore that aPKC phosphorylation of the retromer is essential mechanism for maintaining epithelial polarity.