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.

Non-receptor type of protein-tyrosine kinase Syk contains 2 Src homology 2 (SH2) domains in tandem and multiple autophosphorylation sites. Syk is activated upon binding of tandem SH2 domains to immunoreceptor tyrosine-based activating motif (ITAM) and plays an essential role in lymphocyte development and activation of immune cells. Syk is critical for tyrosine phosphorylation of multiple proteins which regulate important pathways leading from the receptor, such as Ca(2+) mobilization and mitogen-activated protein kinase (MAPK) cascades. Recent findings reveal that expression of Syk appears to be involved in a wide variety of cellular functions and pathogenesis of malignant cancer. These observations have demonstrated that Syk is a key molecule that controls multiple physiological functions in cells.

SummaryTo explore the mechanism(s) by which the Syk protein tyrosine kinase participates in B cell antigen receptor (BCR) signaling, we have studied the function of various Syk mutants in B cells made Syk deficient by homologous recombination knockout . Both Syk SH2 domains were required for BCR-mediated Syk and phospholipase C (PLC)-y2 phosphorylation, inositol 1,4,5-triphosphate release, and Ca" mobilization. A possible explanation for this requirement was provided by findings that recruitment of Syk to tyrosine-phosphorylated immunoglobulin (Ig) o and Igo requires both Syk SH2 domains . A Syk mutant in which the putative autophosphorylation site (Y518/Y519) of Syk was changed to phenylalanine was also defective in signal transduction ; however, this mutation did not affect recruitment to the phosphorylated immunoreceptor family tyrosine-based activation motifs (ITAMs) . These findings not only confirm that both SH2 domains are necessary for Syk binding to tyrosine-phosphorylated Iga and Ig[3 but indicate that this binding is necessary for Syk (Y518/519) phosphorylation after BCR ligation. This sequence of events is apparently required for coupling the BCR to most cellular protein tyrosine phosphorylation, to the phosphorylation and activation of PLC-y2, and to Ca 21 mobilization .

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