Fumihiko Okumura

Phosphorylation-dependent degradation of c-Myc is mediated by the F-box protein Fbw7

The F‐box protein Skp2 mediates c‐Myc ubiquitylation by binding to the MB2 domain. However, the turnover of c‐Myc is largely dependent on phosphorylation of threonine‐58 and serine‐62 in MB1, residues that are often mutated in cancer. We now show that the F‐box protein Fbw7 interacts with and thereby destabilizes c‐Myc in a manner dependent on phosphorylation of MB1. Whereas wild‐type Fbw7 promoted c‐Myc turnover in cells, an Fbw7 mutant lacking the F‐box domain delayed it. Furthermore, depletion of Fbw7 by RNA interference increased both the abundance and transactivation activity of c‐Myc. Accumulation of c‐Myc was also apparent in mouse Fbw7−/− embryonic stem cells. These observations suggest that two F‐box proteins, Fbw7 and Skp2, differentially regulate c‐Myc stability by targeting MB1 and MB2, respectively.

Cytoplasmic ubiquitin ligase KPC regulates proteolysis of p27Kip1 at G1 phase

The cyclin-dependent kinase inhibitor p27Kip1 is degraded at the G0–G1 transition of the cell cycle by the ubiquitin–proteasome pathway. Although the nuclear ubiquitin ligase (E3) SCFSkp2 is implicated in p27Kip1 degradation, proteolysis of p27Kip1 at the G0–G1 transition proceeds normally in Skp2−/− cells. Moreover, p27Kip1 is exported from the nucleus to the cytoplasm at G0–G1 (refs 9–11). These data suggest the existence of a Skp2-independent pathway for the degradation of p27Kip1 at G1 phase. We now describe a previously unidentified E3 complex: KPC (Kip1 ubiquitination-promoting complex), consisting of KPC1 and KPC2. KPC1 contains a RING-finger domain, and KPC2 contains a ubiquitin-like domain and two ubiquitin-associated domains. KPC interacts with and ubiquitinates p27Kip1 and is localized to the cytoplasm. Overexpression of KPC promoted the degradation of p27Kip1, whereas a dominant-negative mutant of KPC1 delayed p27Kip1 degradation. The nuclear export of p27Kip1 by CRM1 seems to be necessary for KPC-mediated proteolysis. Depletion of KPC1 by RNA interference also inhibited p27Kip1 degradation. KPC thus probably controls degradation of p27Kip1 in G1 phase after export of the latter from the nucleus.

The E3 Ligase TTC3 Facilitates Ubiquitination and Degradation of Phosphorylated Akt

The serine threonine kinase Akt is a core survival factor that underlies a variety of human diseases. Although regulatory phosphorylation and dephosphorylation have been well documented, the other posttranslational mechanisms that modulate Akt activity remain unclear. We show here that tetratricopeptide repeat domain 3 (TTC3) is an E3 ligase that interacts with Akt. TTC3 contains a canonical RING finger motif, a pair of tetratricopeptide motifs, a putative Akt phosphorylation site, and nuclear localization signals, and is encoded by a gene within the Down syndrome (DS) critical region on chromosome 21. TTC3 is an Akt-specific E3 ligase that binds to phosphorylated Akt and facilitates its ubiquitination and degradation within the nucleus. Moreover, DS cells exhibit elevated TTC3 expression, reduced phosphorylated Akt, and accumulation in the G(2)M phase, which can be reversed by TTC3 siRNA or Myr-Akt. Thus, interaction between TTC3 and Akt may contribute to the clinical symptoms of DS.

Functional regulation of FEZ1 by the U-box-type ubiquitin ligase E4B contributes to neuritogenesis.

E4B (also known as UFD2a) is a mammalian homolog of Saccharomyces cerevisiae Ufd2, which was originally described as a ubiquitin chain assembly factor (E4). E4B is a U-box-type ubiquitin-protein isopeptide ligase (E3) and likely functions as either an E3 or an E4. With a yeast two-hybrid screen, we have now identified FEZ1 (fasciculation and elongation protein zeta 1) as a protein that interacts with E4B. FEZ1 is implicated in neuritogenesis when phosphorylated by protein kinase Cζ (PKCζ). Interaction between E4B and FEZ1 in mammalian cells was enhanced by coexpression of constitutively active PKCζ. E4B mediated the polyubiquitylation of FEZ1 but did not affect its intracellular stability, suggesting that such modification of FEZ1 is not a signal for its proteolysis. Polyubiquitylation of FEZ1 by E4B required Lys27 of ubiquitin. Expression of a dominant-negative mutant of E4B in rat pheochromocytoma PC12 cells resulted in inhibition of neurite extension induced either by nerve growth factor or by coexpression of FEZ1 and constitutively active PKCζ. These findings indicate that E4B serves as a ubiquitin ligase for FEZ1 and thereby regulates its function but not its degradation.

Citrin/Mitochondrial Glycerol-3-phosphate Dehydrogenase Double Knock-out Mice Recapitulate Features of Human Citrin Deficiency

Citrin is the liver-type mitochondrial aspartate-glutamate carrier that participates in urea, protein, and nucleotide biosynthetic pathways by supplying aspartate from mitochondria to the cytosol.Citrin also plays a role in transporting cytosolic NADH reducing equivalents into mitochondria as a component of the malate-aspartate shuttle. In humans, loss-of-function mutations in the SLC25A13 gene encoding citrin cause both adult-onset type II citrullinemia and neonatal intrahepatic cholestasis, collectively referred to as human citrin deficiency. Citrin knock-out mice fail to display features of human citrin deficiency. Based on the hypothesis that an enhanced glycerol phosphate shuttle activity may be compensating for the loss of citrin function in the mouse, we have generated mice with a combined disruption of the genes for citrin and mitochondrial glycerol 3-phosphate dehydrogenase. The resulting double knock-out mice demonstrated citrullinemia, hyperammonemia that was further elevated by oral sucrose administration, hypoglycemia, and a fatty liver, all features of human citrin deficiency. An increased hepatic lactate/pyruvate ratio in the double knock-out mice compared with controls was also further elevated by the oral sucrose administration, suggesting that an altered cytosolic NADH/NAD+ ratio is closely associated with the hyperammonemia observed. Microarray analyses identified over 100 genes that were differentially expressed in the double knock-out mice compared with wild-type controls, revealing genes potentially involved in compensatory or downstream effects of the combined mutations. Together, our data indicate that the more severe phenotype present in the citrin/mitochondrial glycerol-3-phosphate dehydrogenase double knock-out mice represents a more accurate model of human citrin deficiency than citrin knock-out mice.

TRIM40 promotes neddylation of IKKγ and is downregulated in gastrointestinal cancers

Gastrointestinal neoplasia seems to be a common consequence of chronic inflammation in the gastrointestinal epithelium. Nuclear factor-kappaB (NF-κB) is an important transcription factor for carcinogenesis in chronic inflammatory diseases and plays a key role in promoting inflammation-associated carcinoma in the gastrointestinal tract. Activation of NF-κB is regulated by several posttranslational modifications including phosphorylation, ubiquitination and neddylation. In this study, we showed that tripartite motif (TRIM) 40 is highly expressed in the gastrointestinal tract and that TRIM40 physically binds to Nedd8, which is conjugated to target proteins by neddylation. We also found that TRIM40 promotes the neddylation of inhibitor of nuclear factor kappaB kinase subunit gamma, which is a crucial regulator for NF-κB activation, and consequently causes inhibition of NF-κB activity, whereas a dominant-negative mutant of TRIM40 lacking the RING domain does not inhibit NF-κB activity. Knockdown of TRIM40 in the small intestinal epithelial cell line IEC-6 caused NF-κB activation followed by increased cell growth. In addition, we found that TRIM40 is highly expressed in normal gastrointestinal epithelia but that TRIM40 is downregulated in gastrointestinal carcinomas and chronic inflammatory lesions of the gastrointestinal tract. These findings suggest that TRIM40 inhibits NF-κB activity via neddylation of inhibitor of nuclear factor kappaB kinase subunit gamma and that TRIM40 prevents inflammation-associated carcinogenesis in the gastrointestinal tract.

TRIM24 mediates ligand-dependent activation of androgen receptor and is repressed by a bromodomain-containing protein, BRD7, in prostate cancer cells

The androgen receptor (AR) is a ligand-dependent transcription factor that belongs to the family of nuclear receptors, and its activity is regulated by numerous AR coregulators. AR plays an important role in prostate development and cancer. In this study, we found that TRIM24/transcriptional intermediary factor 1alpha (TIF1alpha), which is known as a ligand-dependent nuclear receptor co-regulator, interacts with AR and enhances transcriptional activity of AR by dihydrotestosterone in prostate cancer cells. We showed that TRIM24 functionally interacts with TIP60, which acts as a coactivator of AR and synergizes with TIP60 in the transactivation of AR. We also showed that TRIM24 binds to bromodomain containing 7 (BRD7), which can negatively regulate cell proliferation and growth. A luciferase assay indicated that BRD7 represses the AR transactivation activity upregulated by TRIM24. These findings indicate that TRIM24 regulates AR-mediated transcription in collaboration with TIP60 and BRD7.

TRIM8 modulates STAT3 activity through negative regulation of PIAS3

TRIM8 is a member of the protein family defined by the presence of a common domain structure composed of a tripartite motif: a RING-finger, one or two B-box domains and a coiled-coil motif. Here, we show that TRIM8 interacts with protein inhibitor of activated STAT3 (PIAS3), which inhibits IL-6-dependent activation of STAT3. Ectopic expression of TRIM8 cancels the negative effect of PIAS3 on STAT3, either by degradation of PIAS3 through the ubiquitin-proteasome pathway or exclusion of PIAS3 from the nucleus. Furthermore, expression of TRIM8 in NIH3T3 cells enhances Src-dependent tumorigenesis. These findings indicate that TRIM8 enhances the STAT3-dependent signal pathway by inhibiting the function of PIAS3.

t(8;21)(q22;q22) fusion proteins preferentially bind to duplicated AML1/RUNX1 DNA-binding sequences to differentially regulate gene expression

Chromosome abnormalities are frequently associated with cancer development. The 8;21(q22;q22) chromosomal translocation is one of the most common chromosome abnormalities identified in leukemia. It generates fusion proteins between AML1 and ETO. Since AML1 is a well-defined DNA-binding protein, AML1-ETO fusion proteins have been recognized as DNA-binding proteins interacting with the same consensus DNA-binding site as AML1. The alteration of AML1 target gene expression due to the presence of AML1-ETO is related to the development of leukemia. Here, using a 25-bp random double-stranded oligonucleotide library and a polymerase chain reaction (PCR)-based DNA-binding site screen, we show that compared with native AML1, AML1-ETO fusion proteins preferentially bind to DNA sequences with duplicated AML1 consensus sites. This finding is further confirmed by both in vitro and in vivo DNA-protein interaction assays. These results suggest that AML1-ETO fusion proteins have a selective preference for certain AML1 target genes that contain multimerized AML1 consensus sites in their regulatory elements. Such selected regulation provides an important molecular mechanism for the dysregulation of gene expression during cancer development.

The Role of Elongin BC-Containing Ubiquitin Ligases

The Elongin complex was originally identified as a positive regulator of RNA polymerase II and is composed of a transcriptionally active subunit (A) and two regulatory subunits (B and C). The Elongin BC complex enhances the transcriptional activity of Elongin A. “Classical” SOCS box-containing proteins interact with the Elongin BC complex and have ubiquitin ligase activity. They also interact with the scaffold protein Cullin (Cul) and the RING domain protein Rbx and thereby are members of the Cullin RING ligase (CRL) superfamily. The Elongin BC complex acts as an adaptor connecting Cul and SOCS box proteins. Recently, it was demonstrated that classical SOCS box proteins can be further divided into two groups, Cul2- and Cul5-type proteins. The classical SOCS box-containing protein pVHL is now classified as a Cul2-type protein. The Elongin BC complex containing CRL family is now considered two distinct protein assemblies, which play an important role in regulating a variety of cellular processes such as tumorigenesis, signal transduction, cell motility, and differentiation.