aPKC-mediated phosphorylation regulates asymmetric membrane localization of the cell fate determinant Numb

Abstract

In Drosophila, the partition defective (Par) complex containing Par3, Par6 and atypical protein kinase C (aPKC) directs the polarized distribution and unequal segregation of the cell fate determinant Numb during asymmetric cell divisions. Unequal segregation of mammalian Numb has also been observed, but the factors involved are unknown. Here, we identify in vivo phosphorylation sites of mammalian Numb and show that both mammalian and Drosophila Numb interact with, and are substrates for aPKC in vitro. A form of mammalian Numb lacking two protein kinase C (PKC) phosphorylation sites (Numb2A) accumulates at the cell membrane and is refractory to PKC activation. In epithelial cells, mammalian Numb localizes to the basolateral membrane and is excluded from the apical domain, which accumulates aPKC. In contrast, Numb2A is distributed uniformly around the cell cortex. Mutational analysis of conserved aPKC phosphorylation sites in Drosophila Numb suggests that phosphorylation contributes to asymmetric localization of Numb, opposite to aPKC in dividing sensory organ precursor cells. These results suggest a model in which phosphorylation of Numb by aPKC regulates its polarized distribution in epithelial cells as well as during asymmetric cell divisions.

PKCζ regulates Notch receptor routing and activity in a Notch signaling-dependent manner

“…Numb is a negative regulator of Notch signaling [47] and regulates Notch endocytosis [48]. Numb has recently been shown to be phosphorylated by aPKCs [49], which prompted us to test whether the effect of PKCζ on Notch was influenced by Numb. Overexpression of Numb reduced Notch activity (Supplementary information, Figure S2), in keeping with previous reports [48].…”

Partitioning-defective Protein 6 (Par-6) Activates Atypical Protein Kinase C (aPKC) by Pseudosubstrate Displacement

“…Miranda associates with the cortex via its cortical localization domain, but once this domain is phosphorylated by aPKC it is released into the cytoplasm leading to their mutually exclusive localization. Cortical association of the protein Numb is also modulated by aPKC phosphorylation, both in Drosophila and polarized mammalian cells (9), suggesting that coupling of aPKC-mediated phosphorylation to cortical displacement may be a general mechanism for Parmediated polarity.…”

“…Through mechanisms that are still being elucidated, the Par complex becomes polarized to one cortical domain and keeps other, cell type-specific factors, localized to an opposite cortical domain (10 -12). The activity of aPKC is a key output of the Par complex because the association of substrates with the cortex can be modulated by phosphorylation (7)(8)(9). For example, in Drosophila neuroblasts the protein Miranda localizes to a cortical domain opposite the Par complex, and its polarization requires aPKC activity (8).…”

“…The Par complex functions to create distinct, polarized cortical domains by coupling phosphorylation to cortical release (7)(8)(9). Through mechanisms that are still being elucidated, the Par complex becomes polarized to one cortical domain and keeps other, cell type-specific factors, localized to an opposite cortical domain (10 -12).…”

PARtitioning Numb

“…In the 24 January issue of The EMBO Journal, Jane McGlade and colleagues reported that Numb is a substrate for aPKC, and that this phosphorylation is necessary to maintain its asymmetric localization in both mammalian epithelial cells and mitotic Drosophila SOP cells (Smith et al, 2007).…”