This work defines the regions of mammalian Numb required for its localization to the cell cortex and demonstrates that extrinsic signals regulate this process. We show that both the PTB domain and the α-adaptin–binding motifs are required for localization of Numb to cortical membrane patches and vesicles. This cortical membrane pool of Numb is dynamically regulated by GPCR signaling through activated Gαq and Gα13 and by direct activation of PKC. Our results indicate that PKC-dependent phosphorylation events regulate the movement of Numb proteins between the cell cortex and the cytosol in mammalian cells.
Previous studies have shown that the p66 and p72 isoforms of mammalian Numb are predominantly localized to the cortical membrane and intracellular vesicles. These two isoforms contain an alternatively spliced exon within the PTB domain (PTBi) possessing three lysine residues that, in addition to the five lysine residues located on either side, form a basic patch that could mediate electrostatic interactions with acidic membrane phospholipids. Indeed, we have previously shown that the PTBi domain preferentially binds to liposomes containing PI(4)P and PI(4,5)P2
(Dho et al., 1999
). Lipid-binding regions are a common feature among other endocytic adaptors, including Dab1 and -2, AP180, epsin, and ARH (Mishra et al., 2002
; Aguilar et al., 2003
; Yun et al., 2003
; Balla, 2005
), and our analysis of a series of Numb localization mutants indicates that membrane localization of Numb p66 and p72 requires the PTBi domain.
Whereas the PTBi domain alone is sufficient for association with the PM, further compartmentalization of Numb into membrane patches and vesicles requires the adaptin-binding DPF motifs. This is mediated primarily by the DPF2 motif, because loss of the more amino-terminal DPF1 motif alone had no effect on membrane localization to AP2-positive puncta. Indeed, previous studies suggest that DPF1 does not bind to α-adaptin (Santolini et al., 2000
). However, our studies indicate that DPF1-mediated interactions likely facilitate and/or stabilize Numb localization in cortical patches, because deletion of both DPF1 and DPF2 (either alone or in the context of a C-terminal truncation) has a more dramatic effect on the localization of Numb compared with loss of DPF2 alone. We have also shown that these motifs, which mediate the association of Numb with the AP2 complex, on their own are not sufficient for optimal localization of Numb to the cell cortex. Therefore, we propose that the PTBi domain promotes Numb association with membrane phospholipids and that additional protein–protein interactions through α-adaptin–binding motifs stabilize and promote Numb compartmentalization into membrane patches and vesicles that also contain AP2. Complete loss of Numb localization to membrane puncta occurred only when all sequences downstream of the PTBi domain were removed, suggesting that additional weak protein–protein interactions may further stabilize Numb localization. In support of this idea, several reports have suggested that Numb binds to Src homology (SH) 3 domain-containing proteins through a proline-rich region that contains putative binding sites for SH3 as well as WW domains (Verdi et al., 1996
; Tang et al., 2005
). A similar cooperative model of PM recruitment has recently been described, in which regulated protein–lipid and protein–protein interactions target and stabilize AP2 complexes in PM patches that mediate clathrin coat assembly (Honing et al., 2005
The interaction between Numb and α-adaptin is conserved in Drosophila
, and epistasis experiments have shown that α-adaptin acts downstream of dNumb in asymmetric cell divisions of the peripheral nervous system. Although Numb is not required for the cortical localization of α-adaptin in sensory organ precursor (SOP) cells undergoing asymmetric cell division, its polarized distribution is Numb dependent (Berdnik et al., 2002
). In our analysis, Numb did not seem to play a role in localization of AP2 to the cortex, because expression of Numb localization mutants had no effect on the PM staining of α-adaptin. In addition, analysis of HeLa cells made Numb-deficient by RNA interference (RNAi), showed no change in α-adaptin localization (our unpublished data), as would be expected if Numb was important in the subcellular distribution of AP2. In contrast, in HeLa cells made AP2 deficient by α-adaptin RNAi, we observed a marked decrease in the association of Numb at the cortical membrane. Therefore, in nonpolarized mammalian cells, Numb is not required for localization of AP2 to the PM or cortical patches or vesicles. Our studies, however, cannot preclude the possibility that, in polarized or asymmetrically dividing mammalian cells, Numb may influence the distribution of AP2.
Many studies have shown that in Drosophila
, Numb localization is dynamically regulated. In developing neuroblasts, dNumb is evenly distributed along the cell membrane until late prophase, when it forms a basal crescent overlying one of the centrosomes. dNumb remains asymmetric through mitosis when it is segregated into one of the daughter cells, after which it becomes homogeneously distributed again (Knoblich et al., 1995
). Recent studies by Mayer et al. (2005)
have shown that Drosophila
Numb is rapidly exchanged between a cytoplasmic pool and the cell cortex, and during asymmetric cell division it is preferentially recruited to one side of the cell cortex. The formation of the Numb crescent is dependent on the activity of the PAR3/PAR6/aPKC polarity complex, which localizes to the opposite pole (Roegiers et al., 2001
). In the neuroblast, this seems to be mediated in part through the aPKC-dependent phosphorylation of the cytoskeletal protein Lgl, which prevents localization of determinants such as Numb to the apical cell cortex (Betschinger et al., 2003
). However, in SOP cells, Numb localization is independent of Lgl, suggesting a distinct mechanism of regulation by the PAR3/PAR6/aPKC polarity complex (Justice et al., 2003
; Langevin et al., 2005
Similarly, mammalian Numb proteins have been reported to localize asymmetrically in mitotic neuroepithelial cells in the chicken (Wakamatsu et al., 1999
) and dividing mouse neural progenitor cells (Zhong et al., 1996
). Polarized distribution of Numb not restricted to mitotic cells, because asymmetric localization of Numb is also observed in postmitotic retinal cells (Dooley et al., 2003
), and as shown in our study, polarized MDCK cells. Although the factors that regulate asymmetric distribution of Numb in mammals have not been defined, Klezovitch et al. (2004)
observed that in cortical progenitors, loss of the vertebrate homologue of Lgl results in a loss of asymmetric distribution of Numb. This finding suggests that the machinery responsible for directing asymmetric distribution of Numb may be conserved.
The cortical membrane pool of Numb is redistributed to the cytosol in response to activation of Gαq
-coupled GPCRs, and this cytosolic pool is subsequently recruited back to the cortical membrane. This effect is mediated by two distinct signaling events, which independently contribute to the loss of Numb association with the PM and have distinct effects on the rapidity with which it is subsequently recruited from the cytosol. First, the PTBi domain mediates a response with a rapid, transient time course, similar to that observed with the isolated PH domain of PLCδ, which is mobilized from the PM in response to hydrolysis of PI(4,5)P2
(Stauffer et al., 1998
). Because the Numb PTBi domain binds PIP2
, the PTB-dependent dissociation of Numb from the membrane is most likely a consequence of PLC-dependent PIP2
hydrolysis. Replenishment of PIP2
levels through the action of phosphatidylinositol phosphate kinases would allow redistribution of Numb back to the PM.
The region between amino acids 218 and 366 mediates the response of Numb to both GPCR stimulation and direct activation of PKC. This region encompasses at least 12 serine residues that are putative PKC phosphorylation sites, and it is required for the TPA-stimulated redistribution of Numb. Deletion of this serine-rich region not only blunts the response of Numb to TPA treatment but also enhances membrane association under basal conditions. This observation suggests that this region is not required for membrane binding but rather it plays a role in the relocalization of Numb in response to cellular signaling. In addition, the presence of this region slows the kinetics of redistribution of Numb from the cytosol to the membrane. Therefore, this region likely contains phosphorylation sites that could promote the binding of Numb to a cytosolic protein that facilitates its loss from the membrane or regulates membrane reassociation. In support of this model, we have used mass spectral analyses to identify nine sites of Numb phosphorylation, four of which lie within this region (Smith, Lau, Rahmani, Dho, Brothers, She, Berry, Bonneil, Thibault, Schweisguth, Le Borgne, and McGlade, unpublished data). In addition, a Ca2+
and calmodulin-dependent protein kinase 1 phosphorylation site was recently identified at amino acid 276, within the amino acid 218–366 region, and reported to mediate binding to 14-3-3 proteins in vitro (Tokumitsu et al., 2005
), although the consequence of phosphorylation of this site on Numb localization was not evaluated. Together, these data suggest that, in mammalian cells, phosphorylation of Numb plays a role in regulating its distribution between a cytosolic pool and the membrane cortex. Phosphorylation may be a mechanism, analogous to that described for Lgl, by which Numb is asymmetrically restricted to specific regions of a cell, for example, the basalateral membrane of MDCK cells.
In addition to phosphorylation, a change in the solubility of Numb in Triton X-100 was also associated with TPA stimulation. A number of proteins bound to clathrin-coated pits, including Dab2, CALM, Epsin, and α-adaptin, are resistant to extraction with nonionic detergents such as Triton X-100 and saponin (Woodward and Roth, 1978
; Tebar et al., 1999
; Morris and Cooper, 2001
). The observation that stimulation increases the solubility of Numb in detergent supports the view that Numb dissociates from a multimolecular complex associated with clathrin-coated pits.
Mammalian Numb functions as an endocytic adaptor protein and has been demonstrated to regulate the endocytosis and trafficking of surface molecules such as EGF receptor, Tac, L1, and Notch (Santolini et al., 2000
; Nishimura et al., 2003
; Smith et al., 2004
; McGill and McGlade, unpublished data). Although we have shown that activation of GPCRs leads to changes in the subcellular distribution of Numb, we did not find any evidence that Numb was cotrafficked with these receptors, nor did we observe any effect of Numb expression or knockdown on the internalization or recycling of the substance P receptor (Dho, unpublished data). Therefore, we propose that the effects of GPCR activation on Numb localization are not secondary to its endocytic function but rather reflect a direct role for signaling events in controlling the distribution of Numb proteins. Heterotrimeric G proteins have emerged as important mediators of polarity and asymmetric cell divisions in Drosophila
(Hampoelz and Knoblich, 2004
; McCudden et al., 2005
). In particular, overexpression of the activated form of the trimeric G protein Go (GoGTP
) in Drosophila
mitotic SOPs results in severe defects in Numb localization, including near-ubiquitous cortical Numb staining or loss from the PM (Katanaev and Tomlinson, 2006
). Our results raise the possibility that activation of heterotrimeric G proteins is a conserved mechanism regulating the subcellular distribution of Numb. As such, the localization of Numb and, by extension its function, may be under the control of both extrinsic and intrinsic signaling cues, which regulate cell polarity.