In this study, we have identified a novel mitochondrial ubiquitin ligase, designated MITOL, which is localized in the mitochondrial outer membrane. MITOL possesses a Plant Homeo-Domain (PHD) motif responsible for E3 ubiquitin ligase activity and predicted four-transmembrane domains. MITOL displayed a rapid degradation by autoubiquitination activity in a PHD-dependent manner. HeLa cells stably expressing a MITOL mutant lacking ubiquitin ligase activity or MITOL-deficient cells by small interfering RNA showed an aberrant mitochondrial morphology such as fragmentation, suggesting the enhancement of mitochondrial fission by MITOL dysfunction. Indeed, a dominant-negative expression of Drp1 mutant blocked mitochondrial fragmentation induced by MITOL depletion. We found that MITOL associated with and ubiquitinated mitochondrial fission protein hFis1 and Drp1. Pulse-chase experiment showed that MITOL overexpression increased turnover of these fission proteins. In addition, overexpression phenotype of hFis1 could be reverted by MITOL cooverexpression. Our finding indicates that MITOL plays a critical role in mitochondrial dynamics through the control of mitochondrial fission proteins.

IntroductionAggregation of the high-affinity IgE receptor (Fc⑀RI) on mast cells mediates the allergic response by the release of inflammatory mediators by degranulation and production of cytokines and arachidonic acid metabolites. 1 Fc⑀RI signals by activating nonreceptor-family proteintyrosine kinases (PTKs) Lyn, Syk, and Btk. On aggregation of Fc⑀RI␣ subunits, the Src family PTK Lyn phosphorylates the associated Fc⑀RI␤ and ␥ subunits on tyrosine residues in the immunoreceptor tyrosinebased activating motif. This leads to the recruitment of Syk to the plasma membrane, which is essential for Fc⑀RI-mediated degranulation by phosphorylating downstream molecules. 2 Similarly, Btk is membranetargeted by phosphatidylinositol-3,4,5-triphosphate to its pleckstrinhomology (PH) domain. Two types of kinase Syk and Btk contribute tyrosine phosphorylation and activation of phospholipase C-␥ (PLC-␥) and calcium mobilization. 1,3 Several adaptors and scaffolds lacking enzymatic and transcriptional domains have been identified in the immune receptormediated signaling pathway. Proximal adaptor proteins, which are the substrates of PTKs, have important roles in early Fc⑀RI signaling. They have multiple motifs and domains that allow their binding to the other molecules. Activated PTKs phosphorylate the substrate adaptor proteins to assemble the cytoplasmic signaling molecules in the juxtamembrane region for effective interaction, presumably in glycolipid enriched microdomains (GEMs). Mice deficient in the adaptor protein linker for activation of T cells (LAT) SLP-76 (SH2-containing leukocyte phosphoprotein of 76 kDa) or Gab2 (Grb2-associated binder-2) were resistant to IgE-mediated passive systemic anaphylaxis. 4-6 Analysis of bone marrow-derived mast cells (BMMCs) from mice revealed their essential roles in Fc⑀RI-mediated degranulation and/or cytokine production.3BP2 was originally isolated as an Abl SH3-binding protein of unknown function. 7 In addition to the proline-rich region that mediates SH3 binding, it has PH and Src homology 2 (SH2) domains. 3BP2 was also identified as one of the Syk kinaseinteracting proteins by a yeast 2-hybrid screen. 8 C-terminal SH2 domain of 3BP2 is required to interact with Syk in yeast. Expression of 3BP2 has been reported in T, B, natural killer (NK), and monocytic cell lines. Transient overexpression of 3BP2 in T cells induces transcriptional activation of the interleukin 2 (IL-2) gene. In NK cells, overexpression of 3BP2 by vaccinia virus enhances cytotoxicity. 9 Prior to these findings, a specific motif recognized by 3BP2-SH2 domain was examined by in vitro degenerated peptide library screening. 10 The optimal sequence for the 3BP2-SH2 domain is Tyr-Glu-Asn (YEN) motif.The present experiments demonstrated the expression of 3BP2 in mast cells and the functional importance of 3BP2-SH2 domain in Fc⑀RI-mediated signal transduction. Overexpression of 3BP2-SH2 domains in the rat basophilic leukemia RBL-2H3 cells resulted in a dramatic suppression of the Fc⑀RI-mediated tyrosine phosphorylation of P...

Adaptor protein 3BP2 was originally isolated as a proteintyrosine kinase c-Abl-Src homology 3 (SH3) 1 domain-binding protein of unknown function (1). 3BP2 was also identified as a Syk kinase-interacting protein by the yeast two-hybrid screening (2). Transient overexpression of 3BP2 resulted in a transcriptional activation of nuclear factor of activated T cell and activator protein 1, which is induced by T-cell receptor aggregation. Ser 225 and Ser 277 of 3BP2 were identified as essential sites for interacting with 14-3-3 to negatively regulate the function of 3BP2 in lymphocytes (3). Moreover, infection of 3BP2 wild type into NK cells by vaccinia virus enhanced cell cytotoxicity. An in vitro binding study suggested that phosphorylation of Tyr 183 of 3BP2 could be associated with Vav and phospholipase C-␥ (4). In mast cells, overexpression of the 3BP2-SH2 domain suppressed high affinity IgE receptor (Fc⑀RI)-mediated tyrosine phosphorylation of phospholipase C-␥, Ca 2ϩ mobilization, and degranulation (5). These findings have demonstrated that 3BP2 plays a critical role in hematopoietic cells.To propagate the immunoreceptor signal, adaptor proteins contribute to protein-protein and protein-lipid interactions through multiple domains and/or specific phosphotyrosine-containing sequences. Tyrosine phosphorylation of 3BP2 was observed in NK cells and mast cells by cross-linking Fc␥R and Fc⑀RI, respectively (4, 5). To elucidate the function of 3BP2, it is necessary to determine the protein-tyrosine kinase (PTK) that phosphorylates 3BP2 and its binding partner to assemble a signaling complex through specific phosphotyrosine-containing motifs in 3BP2.The Src family PTK Lyn is associated with Fc⑀RI␤. Upon aggregation of Fc⑀RI, Lyn is critical for phosphorylating Fc⑀RI␤ and -␥ subunits on Tyr residues within the immunoreceptor tyrosine-based activating motif (ITAM) (6 -8). By analogy with studies on Hck, Lyn is thought to be activated by the disassembly of the closed intramolecular interaction by (i) CD45-mediated dephosphorylation of C-terminal regulatory Tyr residue, (ii) binding to SH3 and SH2 ligands, and (iii) autophosphorylation of Tyr in the activation loop (9). What is the binding ligand of the SH3 and SH2 domains of Lyn in Fc⑀RI signaling pathway? Pull-down experiments using glutathione S-transferase (GST)-Lyn-SH2 fusion protein indicated that there were multiple phosphoproteins interacting with the Lyn-SH2 after the antigen stimulation of RBL-2H3 cells (10). In addition, although the displacement of intramolecular SH3 interaction is not well understood, it seems likely that some aggregation-induced change in an associated molecule provides a higher affinity SH3 ligand that binds to the Lyn-SH3 domain (11). The SH3 domain is directed toward the proline-rich region, but such a ligand has not been identified yet in the Fc⑀RI signaling.We isolated nonreceptor type PTK Syk from porcine spleen (12). Syk is expressed in hematopoietic, epithelial, and endothelial cells (13)(14)(15)(16). When the ITAM of Fc⑀RI␥ subunits is...

IntroductionMast cells play a central role in inflammatory and immediate allergic reactions. The aggregation of Fc⑀RI triggers the activation of Lyn protein-tyrosine kinase (PTK), resulting in the rapid tyrosine phosphorylation of its ␤ and ␥ subunits. 1 We isolated nonreceptor type PTK Syk. 2,3 Tyrosine phosphorylated ␥ subunit of Fc⑀RI then recruits and activates Syk, the essential PTK for antigen-induced Ca ϩϩ influx, degranulation, and cytokine production in mast cells. [3][4][5][6][7][8][9] Syk phosphorylates an adaptor protein linker for activation of T cells (LAT) which is located in the lipid raft (also referred to as the glycolipid-enriched microdomains [GEMs]) to accumulate the signaling molecules and is essential for Fc⑀RI-mediated mast cell activation. 6,7,10 In contrast to this Lyn-Syk-LAT pathway, recent findings by using the genetic approach have revealed the existence of another complementary signaling pathway. Expression of the adaptor/scaffold protein Gab2 is necessary for Fc⑀RI-mediated activation of phosphatidylinositol 3-kinase (PI3-kinase) and downstream mast cell activation. 11 Src family PTK Fyn is required for tyrosine phosphorylation of Gab2, independent of Lyn and LAT. 12 Both of these 2 pathways are important for the Fc⑀RI-induced degranulation and cytokine production.Cbl-b, a second member of the Cbl-family of E3 ubiquitinprotein ligase, was originally isolated from the human breast cancer cells and is expressed in a variety of healthy tissues and cancer cells. 13 It consists of an amino-terminal variant Src homology domain 2 (SH2) domain, a RING finger domain, a proline-rich region, a carboxyl-terminal leucine-zipper domain, and potential tyrosine phosphorylation sites. Genetic studies revealed that c-Cbl and Cbl-b have the distinct and overlapped functions in T-cell regulations. 14 Although c-Cbl regulates thymocyte development and cell surface T-cell receptor (TCR) expression, Cbl-b Ϫ/Ϫ mice display a normal development of thymocytes and impaired peripheral T-cell activation mediated by CD28. 15,16 Cbl-b targets PI3-kinase by ubiquitination in T cells. 17,18 In addition, lack of Cbl-b results in the enhanced tyrosine phosphorylation and guanosine diphosphate/guanosine triphosphate (GDP/GTP) exchanging activity of Vav1. 15,16 Therefore, Cbl-b might indirectly regulate the activation of Vav1 through phosphatidylinositol 3-phosphate, the product of PI3-kinase. 19 Analysis of B cells from Cbl-b-deficient mice showed that lack of Cbl-b results in the sustained phosphorylation of Ig␣, Syk, phospholipase C-␥2 (PLC-␥2), and prolonged Ca ϩϩ mobilization. 20 However, Cbl-b-deficient avian pro-B cells display reduced PLC-␥2 activation and Ca ϩϩ mobilization, suggesting that Cbl-b may play a different role in immature and mature B cells. 21 In mast cells, c-Cbl catalyzes Fc⑀RI-mediated ubiquitination of Fc⑀RI and Syk, resulting in an inhibition of the downstream inflammatory mediator release. [22][23][24] However, the function of Cbl-b in mast cells has not been demonstrated yet.The lipid raf...

Background : Recent studies have demonstrated that c-Cbl functions as a ubiquitin-protein ligase toward immune receptors and non-receptor protein-tyrosine kinase Syk by facilitating their ubiquitination and subsequent targeting to proteasomes. However, it was not clear whether Src family kinase Lyn is regulated by the Cbl family of ubiquitin-protein ligases.