Proximity interactions of the ubiquitin ligase Mind bomb 1 reveal a role in regulation of epithelial polarity complex proteins.

MIB1 belongs to the RING domain containing family of E3 ubiquitin ligases. In vertebrates, MIB1 plays an essential role in activation of Notch signaling during development, through the ubiquitination and endocytosis of Notch ligands. More recently, Notch independent functions for MIB1 have been described in centriole homeostasis, dendritic spine outgrowth and directional cell migration. Here we use proximity-dependent biotin identification (BioID) to define the MIB1 interactome that included 163 high confidence interactions with polypeptides linked to centrosomes and cilia, endosomal trafficking, RNA and DNA processing, the ubiquitin system, and cell adhesion. Biochemical analysis identified several proteins within these groups including CCDC14 and EPS15 that were ubiquitinated but not degraded when co-expressed with MIB1. The MIB1 interactome included the epithelial cell polarity protein, EPB41L5. MIB1 binds to and ubiquitinates EPB41L5 resulting in its degradation. Furthermore, MIB1 ubiquitinates the EPB41L5-associated polarity protein CRB1, an important determinant of the apical membrane. In polarized cells, MIB1 localized to the lateral membrane with EPB41L5 and to the tight junction with CRB1, CRB3 and ZO1. Furthermore, over expression of MIB1 resulted in altered epithelial cell morphology and apical membrane expansion. These results support a role for MIB1 in regulation of polarized epithelial cell morphology.

SLAP Is a Negative Regulator of FcεRI Receptor-Mediated Signaling and Allergic Response.

Binding of antigen to IgE-high affinity FcεRI complexes on mast cells and basophils results in the release of preformed mediators such as histamine and de novo synthesis of cytokines causing allergic reactions. Src-like adapter protein (SLAP) functions co-operatively with c-Cbl to negatively regulate signaling downstream of the T cell receptor, B cell receptor, and receptor tyrosine kinases (RTK). Here, we investigated the role of SLAP in FcεRI-mediated mast cell signaling, using bone marrow derived mast cells (BMMCs) from SLAP knock out (SLAP KO) mice. Mature SLAP-KO BMMCs displayed significantly enhanced antigen induced degranulation and synthesis of IL-6, TNFα, and MCP-1 compared to wild type (WT) BMMCs. In addition, SLAP KO mice displayed an enhanced passive cutaneous anaphylaxis response. In agreement with a negative regulatory role, SLAP KO BMMCs showed enhanced FcεRI-mediated signaling to downstream effector kinases, Syk, Erk, and Akt. Recombinant GST-SLAP protein binds to the FcεRIß chain and to the Cbl-b in mast cell lysates, suggesting a role in FcεRI down regulation. In addition, the ubiquitination of FcεRIγ chain and antigen mediated down regulation of FcεRI is impaired in SLAP KO BMMCs compared to the wild type. In line with these findings, stimulation of peripheral blood human basophils with FcεRIα antibody, or a clinically relevant allergen, resulted in increased SLAP expression. Together, these results indicate that SLAP is a dynamic regulator of IgE-FcεRI signaling, limiting allergic responses.

The E3 ubiquitin ligases RNF126 and Rabring7 regulate endosomal sorting of the epidermal growth factor receptor.

Activation of the epidermal growth factor receptor (EGFR) results in internalization and ubiquitin-dependent endosomal sorting, leading to lysosomal degradation. Here we describe the role of the RING-finger-domain-containing protein RNF126 and the related protein, Rabring7 in EGFR endosomal sorting. We demonstrate that RNF126 specifies K48-linked chains with UbcH5b and also functions with Ubc13/Uev1a to form K63-linked chains in vitro. RNF126 and Rabring7 associate with the EGFR through a ubiquitin-binding zinc finger domain and both E3 ubiquitin ligases promote ubiquitylation of EGFR. In the absence of c-Cbl or in cells expressing Cbl-70Z, the binding of RNF126 and Rabring7 to the EGFR is reduced, suggesting that RNF126 and Rabring7 function downstream of c-Cbl. In HeLa cells depleted of either RNF126 or Rabring7 the EGFR is retained in a late endocytic compartment and is inefficiently degraded. In addition, depletion of RNF126 or Rabring7 destabilizes ESCRT-II and reduces the number of multivesicular bodies formed after EGF stimulation. We also show that the depletion of Rabring7 attenuates the degradation of MET and that both RNF126 and Rabring7 regulate the sorting of CXCR4 from an early endocytic compartment. Together these data suggest that RNF126 and Rabring7 play a role in the ubiquitin-dependent sorting and downregulation of membrane receptors.

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

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.

Essential role for caspase 8 in T-cell homeostasis and T-cell-mediated immunity.

Defects in death receptor-mediated apoptosis have been linked to cancer and autoimmune disease in humans. The in vivo role of caspase 8, a component of this pathway, has eluded analysis in postnatal tissues because of the lack of an appropriate animal model. Targeted disruption of caspase 8 is lethal in utero. We generated mice with a targeted caspase 8 mutation that is restricted to the T-cell lineage. Despite normal thymocyte development in the absence of caspase 8, we observed a marked decrease in the number of peripheral T-cells and impaired T-cell response ex vivo to activation stimuli. caspase 8 ablation protected thymocytes and activated T-cells from CD95 ligand but not anti-CD3-induced apoptosis, or apoptosis activated by agents that are known to act through the mitochondria. caspase 8 mutant mice were unable to mount an immune response to viral infection, indicating that caspase 8 deletion in T-cells leads to immunodeficiency. These findings identify an essential, cell-stage-specific role for caspase 8 in T-cell homeostasis and T-cell-mediated immunity. This is consistent with the recent identification of caspase 8 mutations in human immunodeficiency.

A high-affinity Arg-X-X-Lys SH3 binding motif confers specificity for the interaction between Gads and SLP-76 in T cell signaling.

A critical event in T cell receptor (TCR)-mediated signaling is the recruitment of hematopoietic-specific adaptor proteins that collect and transmit signals downstream of the TCR. Gads, a member of the Grb2 family of SH2 and SH3 domain-containing adaptors, mediates the formation of a complex between LAT and SLP-76 that is essential for signal propagation from the TCR. Here we examine the binding specificity of the Gads and Grb2 SH3 domains using peptide arrays and find that a nonproline-based R-X-X-K motif found in SLP-76 binds to the Gads carboxy-terminal SH3 domain with high affinity (K(D) = 240 +/- 45 nM). The Grb2 C-terminal SH3 domain also binds this motif, but with a 40-fold lower affinity than Gads. Single point mutations in either the relevant R (237) or K (240) completely abrogated SLP-76 association with Gads in vivo and impaired SLP-76 function. A chimeric Grb2 protein, possessing the C-terminal SH3 domain of Gads, was able to partially substitute for Gads in signaling downstream of the T cell receptor. These results provide a molecular explanation for the specific role of Gads in T cell receptor signaling, and identify a discrete subclass of SH3 domains whose binding is dependent on a core R-X-X-K motif.

Functional cooperation between c-Cbl and Src-like adaptor protein 2 in the negative regulation of T-cell receptor signaling.

Adaptor proteins assemble multiprotein signaling complexes, enabling the transduction of intracellular signals. While many adaptor proteins positively regulate signaling in this manner, a subgroup of adaptors function as negative regulators. Here we report the identification of a hematopoiesis-specific adaptor protein that we have designated Src-like adaptor protein 2 (SLAP-2). SLAP-2 is most closely related to SLAP and contains a Src homology 3 (SH3) domain and an SH2 domain, as well as an amino-terminal myristoylation site that mediates SLAP-2 association with membranes. Following stimulation of primary thymocytes with anti-CD3 and anti-CD28, SLAP-2 coimmunoprecipitates with tyrosine-phosphorylated c-Cbl and an unidentified protein of approximately 72 kDa. In activated Jurkat T cells, SLAP-2 also binds an additional 70-kDa phosphoprotein, identified as ZAP-70. Binding of SLAP-2 to both p72 and ZAP-70 is dependent on its SH2 domain, while c-Cbl interacts with the carboxy-terminal region. Overexpression of wild-type SLAP-2 alone or in combination with c-Cbl in Jurkat T cells leads to inhibition of T-cell antigen receptor-induced activation of nuclear factor of activated T cells. The inhibitory effect of SLAP-2 requires the carboxy-terminal c-Cbl binding region. Expression of SLAP-2 with SYK or ZAP-70 in COS cells or Jurkat T cells causes the degradation of these kinases, and SLAP-2 overexpression in Jurkat T cells reduces the surface expression of CD3. These results suggest that the mechanism of action of SLAP-2 and the related protein SLAP is to promote c-Cbl-dependent degradation of the tyrosine kinases SYK and ZAP-70 and down-regulation of CD3 at the cell surface.

1alpha,25-dihydroxyvitamin D3 stimulates phosphorylation of IkappaBalpha and synergizes with TPA to induce nuclear translocation of NFkappaB during monocytic differentiation of NB4 leukemia cells.

Treatment of NB4 acute promyelocytic leukemia cells with 1,25-dihydroxyvitamin D3 (1,25D3) or analogs 20-epi-22-oxa-24a,26a,27a-trihomo-1alpha,25-dihydroxyvitamin D3, 1,24-dihydroxy-22-ene-24-cyclopropylvitamin D3, 1alpha,25-dihydroxylumisterol3, or 1alpha,25(OH)2-d5-previtamin D3 in combination with TPA induces monocytic differentiation. The role of 1,25D3 in the induction of maturation has been shown to be a priming effect. Differentiation in response to these agents requires VDR-independent signaling of 1,25D3, PKC signaling, intracellular calcium, and calpain activity. In this study we identify the NFkappaB/IkappaB signaling pathway as a target of 1,25D3 and TPA action. One of the priming effects of 1,25D3 appears to be the rapid phosphorylation of serine residues on IkappaBalpha. On their own, 1,25D3, its analogs, and TPA do not alter IkappaBalpha expression; however, combinations of analogs with TPA result in a synergistic decrease in IkappaBalpha expression. Decreased expression of IkappaBalpha likely results from enhanced degradation, which allows the observed subsequent nuclear translocation of NFkappaB subunit p65. Since nuclear-localized NFkappaB was observed only in combination-treated cells, it is proposed that nuclear targets of NFkappaB are required for monocytic differentiation. Intracellular calcium and proteolytic activity are both necessary for the induction of IkappaB regulation and translocation of NFkappaB and are critical components of the nongenomic signaling cascades of the 1,25D3-induced differentiation pathway.