Tohru Ichimura

Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages

Beclin 1, a protein essential for autophagy, binds to hVps34/Class III phosphatidylinositol-3-kinase and UVRAG. Here, we have identified two Beclin 1 associated proteins, Atg14L and Rubicon. Atg14L and UVRAG bind to Beclin 1 in a mutually exclusive manner, whereas Rubicon binds only to a subpopulation of UVRAG complexes; thus, three different Beclin 1 complexes exist. GFP–Atg14L localized to the isolation membrane and autophagosome, as well as to the ER and unknown puncta. Knockout of Atg14L in mouse ES cells caused a defect in autophagosome formation. GFP–Rubicon was localized at the endosome/lysosome. Knockdown of Rubicon caused enhancement of autophagy, especially at the maturation step, as well as enhancement of endocytic trafficking. These data suggest that the Beclin 1–hVps34 complex functions in two different steps of autophagy by altering the subunit composition.

Brain 14-3-3 protein is an activator protein that activates tryptophan 5-monooxygenase and tyrosine 3-monooxygenase in the presence of Ca2+,calmodulin-dependent protein kinase II.

We have found that the 14‐3‐3 protein, an acidic neuronal protein, is substantially identical to the ‘activator’ protein [(1981) J. Biol. Chem. 256, 5404–5409] that activates tryptophan 5‐monooxygenase and tyrosine 3‐monooxygenase in the presence of Ca2+,calmodulin dependent protein kinase II. This finding is based on the remarkable similarity of both these proteins in physicochemical, biochemical and immunochemical properties, as well as on detection for the 14‐3‐3 protein of an activator activity towards tryptophan 5‐monooxygenase. The result suggests that the 14‐3‐3 protein plays a role in the regulation of serotonin and noradrenaline biosynthesis in brain.

Hepatitis C virus RNA replication is regulated by FKBP8 and Hsp90

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a component of viral replicase and is well known to modulate the functions of several host proteins. Here, we show that NS5A specifically interacts with FKBP8, a member of the FK506‐binding protein family, but not with other homologous immunophilins. Three sets of tetratricopeptide repeats in FKBP8 are responsible for interactions with NS5A. The siRNA‐mediated knockdown of FKBP8 in a human hepatoma cell line harboring an HCV RNA replicon suppressed HCV RNA replication, and this reduction was reversed by the expression of an siRNA‐resistant FKBP8 mutant. Furthermore, immunoprecipitation analyses revealed that FKBP8 forms a complex with Hsp90 and NS5A. Treatment of HCV replicon cells with geldanamycin, an inhibitor of Hsp90, suppressed RNA replication in a dose‐dependent manner. These results suggest that the complex consisting of NS5A, FKBP8, and Hsp90 plays an important role in HCV RNA replication.

14-3-3 Proteins Modulate the Expression of Epithelial Na+ Channels by Phosphorylation-dependent Interaction with Nedd4-2 Ubiquitin Ligase

The ubiquitin E3 protein ligase Nedd4-2 is a physiological regulator of the epithelial sodium channel ENaC, which is essential for transepithelial Na+ transport and is linked to Liddles syndrome, an autosomal dominant disorder of human salt-sensitive hypertension. Nedd4-2 function is negatively regulated by phosphorylation via a serum- and glucocorticoid-inducible protein kinase (Sgk1), which serves as a mechanism to inhibit the ubiquitination-dependent degradation of ENaC. We report here that 14-3-3 proteins participate in this regulatory process through a direct interaction with a phosphorylated form of human Nedd4-2 (a human gene product of KIAA0439, termed hNedd4-2). The interaction is dependent on Sgk1-catalyzed phosphorylation of hNedd4-2 at Ser-468. We found that this interaction preserved the activity of the Sgk1-stimulated ENaC-dependent Na+ current while disrupting the interaction decreased ENaC density on the Xenopus laevis oocytes surface possibly by enhancing Nedd4-2-mediated ubiquitination that leads to ENaC degradation. Our findings suggest that 14-3-3 proteins modulate the cell surface density of ENaC cooperatively with Sgk1 kinase by maintaining hNedd4-2 in an inactive phosphorylated state.

14-3-3 Protein Binds to Insulin Receptor Substrate-1, One of the Binding Sites of Which Is in the Phosphotyrosine Binding Domain

Insulin binding to its receptor induces the phosphorylation of cytosolic substrates, insulin receptor substrate (IRS)-1 and IRS-2, which associate with several Src homology-2 domain-containing proteins. To identify unique IRS-1-binding proteins, we screened a human heart cDNA library with32P-labeled recombinant IRS-1 and obtained two isoforms (ε and ζ) of the 14-3-3 protein family. 14-3-3 protein has been shown to associate with IRS-1 in L6 myotubes, HepG2 hepatoma cells, Chinese hamster ovary cells, and bovine brain tissue. IRS-2, a protein structurally similar to IRS-1, was also shown to form a complex with 14-3-3 protein using a baculovirus expression system. The amount of 14-3-3 protein associated with IRS-1 was not affected by insulin stimulation but was increased significantly by treatment with okadaic acid, a potent serine/threonine phosphatase inhibitor. Peptide inhibition experiments using phosphoserine-containing peptides of IRS-1 revealed that IRS-1 contains three putative binding sites for 14-3-3 protein (Ser-270, Ser-374, and Ser-641). Among these three, the motif around Ser-270 is located in the phosphotyrosine binding domain of IRS-1, which is responsible for the interaction with the insulin receptor. Indeed, a truncated mutant of IRS-1 consisting of only the phosphotyrosine binding domain retained the capacity to bind to 14-3-3 protein in vivo. Finally, the effect of 14-3-3 protein binding on the insulin-induced phosphorylation of IRS-1 was investigated. Phosphoamino acid analysis revealed that IRS-1 coimmunoprecipitated with anti-14-3-3 antibody to be weakly phosphorylated after insulin stimulation, on tyrosine as well as serine residues, compared with IRS-1 immunoprecipitated with anti-IRS-1 antibody. Thus, the association with 14-3-3 protein may play a role in the regulation of insulin sensitivity by interrupting the association between the insulin receptor and IRS-1.

Distinct forms of the protein kinase-dependent activator of tyrosine and tryptophan hydroxylases.

Tyrosine and tryptophan hydroxylases are the key enzymes in the regulation of catecholamine and serotonin levels in neurons and other endocrine cells. Among the mechanisms proposed for the modulation of activity, phosphorylation of the enzyme is believed to be of functional significance with respect to the stimulus-response coupling, but the precise mechanism is unknown. Here, we show the existence of multiple, distinct forms of the 14-3-3 activator protein, a neuronal protein essential for activation of tyrosine and tryptophan hydroxylases by Ca2+/calmodulin-dependent protein kinase type II. Bovine brain 14-3-3 protein was resolved by reversed-phase chromatography into seven polypeptides (alpha to eta), all of which were active towards tryptophan hydroxylase when the renatured preparations were assayed in the presence of Ca2+, calmodulin and the protein kinase. Determination of the amino acid sequences of the beta and gamma chains and comparison of the sequences with the previously determined sequence of the eta chain revealed that these molecules are highly homologous, and share a common structural feature in containing an extremely acidic C-terminal region predicted as a domain for interaction with the phosphorylated hydroxylases. Northern blot analysis indicated that the beta, gamma and eta chain are expressed abundantly in the brain; however, these polypeptides appear to be expressed with different tissue specificities because gamma mRNA is found only in the brain, while lower levels of beta and eta mRNAs are detected in several other tissues. These findings suggest the involvement of a diverse family of the activator protein in the stimulus-coupled, Ca2(+)-dependent regulation of monoamine biosynthesis.

E6AP Ubiquitin Ligase Mediates Ubiquitylation and Degradation of Hepatitis C Virus Core Protein

ABSTRACT Hepatitis C virus (HCV) core protein is a major component of viral nucleocapsid and a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis. We previously showed that the HCV core protein is degraded through the ubiquitin-proteasome pathway. However, the molecular machinery for core ubiquitylation is unknown. Using tandem affinity purification, we identified the ubiquitin ligase E6AP as an HCV core-binding protein. E6AP was found to bind to the core protein in vitro and in vivo and promote its degradation in hepatic and nonhepatic cells. Knockdown of endogenous E6AP by RNA interference increased the HCV core protein level. In vitro and in vivo ubiquitylation assays showed that E6AP promotes ubiquitylation of the core protein. Exogenous expression of E6AP decreased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected Huh-7 cells. Furthermore, knockdown of endogenous E6AP by RNA interference increased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected cells. Taken together, our results provide evidence that E6AP mediates ubiquitylation and degradation of HCV core protein. We propose that the E6AP-mediated ubiquitin-proteasome pathway may affect the production of HCV particles through controlling the amounts of viral nucleocapsid protein.

Activation of protein kinase C by the 14-3-3 proteins homologous with exol protein that stimulates calcium-dependent exocytosis

The 14‐3‐3 proteins are a family of acidic proteins found mainly in the brain and are suggested to have a role in monoamine synthesis based on their ability to activate tyrosine and tryptophan hydroxylases in the presence of type II Ca2+/calmodulin‐dependent protein kinase. Recently, however, it has been demonstrated that a member of the 14‐3‐3 family, termed Exol, stimulates Ca2+‐dependent exocytosis in permeabilized adrenal chromaffin cells, suggesting that this protein family may influence the protein kinase C‐mediated control of Ca2+‐dependent exocytosis. Here we show that the 14‐3‐3 proteins activate protein kinase C at about 2‐fold more than the known level of the activated protein kinase, i.e. the activity of protein kinase C in the presence of Ca2+ and phospholipids. This raises the possibility that the cellular activity of protein kinase C is regulated by diverse members of the 14‐3‐3 family and that the reported ability of Exol to reactivate Ca2+‐dependent exocytosis is based on its stimulatory effect on protein kinase C activity. The 14‐3‐3 family, therefore, appears to be a multifunctional regulator of cell signalling processes mediated by two types of Ca2+‐dependent protein kinase, protein kinase C and type II calmodulin‐dependent protein kinase.

Identification of the Site of Interaction of the 14-3-3 Protein with Phosphorylated Tryptophan Hydroxylase

The 14-3-3 protein family plays a role in a wide variety of cell signaling processes including monoamine synthesis, exocytosis, and cell cycle regulation, but the structural requirements for the activity of this protein family are not known. We have previously shown that the 14-3-3 protein binds with and activates phosphorylated tryptophan hydroxylase (TPH, the rate-limiting enzyme in the biosynthesis of neurotransmitter serotonin) and proposed that this activity might be mediated through the COOH-terminal acidic region of the 14-3-3 molecules. In this report we demonstrate, using a series of truncation mutants of the 14-3-3 isoform expressed in Escherichia coli, that the COOH-terminal region, especially restricted in amino acids 171-213, binds indeed with the phosphorylated TPH. This restricted region, which we termed 14-3-3 box I, is one of the structural regions whose sequence is highly conserved beyond species, allowing that the plant 14-3-3 isoform (GF14) could also activate rat brain TPH. The 14-3-3 box I is the first functional region whose activity has directly been defined in the 14-3-3 sequence and may represent a common structural element whereby 14-3-3 interacts with other target proteins such as Raf-1 kinase. The result is consistent with the recently published crystal structure of this protein family, which suggests the importance of the negatively charged groove-like structure in the ligand binding.

Molecular cloning of rat cDNAs for β and γ subtypes of 14-3-3 protein and developmental changes in expression of their mRNAs in the nervous system

We isolated cDNAs to beta and gamma subtypes of 14-3-3 protein, a putative regulatory protein for protein kinase C, from the brain and clarified a high homology in sequences of nucleotides and deduced amino acids between the two rat subtypes and the bovine counterparts and even reciprocally between the two rat subtypes. In Northern blot analysis, the gene expression of the two subtypes was detected weakly at E13, increased progressively after birth and reached a maximum at P7-P14. Thereafter it decreased slightly. In situ hybridization analysis allowed detection of the beta but not the gamma subtype in the matrix cells of the ventricular germinal zone of the neural wall. In post-mitotic neurons in the mantle zone and maturing brain loci, genes of the two subtypes were expressed in patterns similar to each other, and three neuron types were identified: type I neurons with high levels of expression throughout development; type II neurons showing high expression during the early developmental stages with a subsequent decrease in the expression at maturing and adult stages; and type III neurons showing consistently low levels of expression throughout development. The wider and more highly-patterned expression of the 14-3-3 protein family than expected suggests that this protein may be involved in the elaborate regulation of some fundamental cellular activities and differentiation of neurons.