Kazunobu Saito

The novel lipid raft adaptor p18 controls endosome dynamics by anchoring the MEK–ERK pathway to late endosomes

The regulation of endosome dynamics is crucial for fundamental cellular functions, such as nutrient intake/digestion, membrane protein cycling, cell migration and intracellular signalling. Here, we show that a novel lipid raft adaptor protein, p18, is involved in controlling endosome dynamics by anchoring the MEK1–ERK pathway to late endosomes. p18 is anchored to lipid rafts of late endosomes through its N‐terminal unique region. p18−/− mice are embryonic lethal and have severe defects in endosome/lysosome organization and membrane protein transport in the visceral endoderm. p18−/− cells exhibit apparent defects in endosome dynamics through perinuclear compartment, such as aberrant distribution and/or processing of lysosomes and impaired cycling of Rab11‐positive recycling endosomes. p18 specifically binds to the p14–MP1 complex, a scaffold for MEK1. Loss of p18 function excludes the p14–MP1 complex from late endosomes, resulting in a downregulation of the MEK–ERK activity. These results indicate that the lipid raft adaptor p18 is essential for anchoring the MEK–ERK pathway to late endosomes, and shed new light on a role of endosomal MEK–ERK pathway in controlling endosome dynamics.

Recruitment of the autophagic machinery to endosomes during infection is mediated by ubiquitin

After bacterial invasion, ubiquitin is conjugated to host endosomal proteins and recognized by the autophagic machinery independent of LC3.

The Lipid Raft-Anchored Adaptor Protein Cbp Controls the Oncogenic Potential of c-Src

The tyrosine kinase c-Src is upregulated in various human cancers irrespective of its negative regulator Csk, but the regulatory mechanisms remain unclear. Here, we show that a lipid raft-anchored Csk adaptor, Cbp/PAG, is directly involved in controlling the oncogenicity of c-Src. Using Csk-deficient cells that can be transformed by c-Src overexpression, we found that Cbp expression is markedly downregulated by c-Src activation and re-expression of Cbp efficiently suppresses c-Src transformation as well as tumorigenesis. Cbp-deficient cells are more susceptible to v-Src transformation than their parental cells. Upon phosphorylation, Cbp specifically binds to activated c-Src and sequesters it in lipid rafts, resulting in an efficient suppression of c-Src function independent of Csk. In some human cancer cells and tumors, Cbp is downregulated and the introduction of Cbp significantly suppresses tumorigenesis. These findings indicate a potential role for Cbp as a suppressor of c-Src-mediated tumor progression.

Mechanism for Release of Alkaline Phosphatase Caused by Glycosylphosphatidylinositol Deficiency in Patients with Hyperphosphatasia Mental Retardation Syndrome

Background: Hyperphosphatasia was observed with glycosylphosphatidylinositol (GPI) deficiency due to mutation in PIGV, but not the PIGM gene. Results: Alkaline phosphatase was released from cells defective in late-stage GPI biosynthesis. Conclusion: In the presence of mannose-bearing GPI intermediates, GPI transamidase cleaves the C-terminal hydrophobic signal peptide and releases alkaline phosphatase. Significance: This study explains the mechanism for release of anchorless GPI-anchored protein. Hyperphosphatasia mental retardation syndrome (HPMR), an autosomal recessive disease characterized by mental retardation and elevated serum alkaline phosphatase (ALP) levels, is caused by mutations in the coding region of the phosphatidylinositol glycan anchor biosynthesis, class V (PIGV) gene, the product of which is a mannosyltransferase essential for glycosylphosphatidylinositol (GPI) biosynthesis. Mutations found in four families caused amino acid substitutions A341E, A341V, Q256K, and H385P, which drastically decreased expression of the PIGV protein. Hyperphosphatasia resulted from secretion of ALP, a GPI-anchored protein normally expressed on the cell surface, into serum due to PIGV deficiency. In contrast, a previously reported PIGM deficiency, in which there is a defect in the transfer of the first mannose, does not result in hyperphosphatasia. To provide insights into the mechanism of ALP secretion in HPMR patients, we took advantage of CHO cell mutants that are defective in various steps of GPI biosynthesis. Secretion of ALP requires GPI transamidase, which in normal cells, cleaves the C-terminal GPI attachment signal peptide and replaces it with GPI. The GPI-anchored protein was secreted substantially into medium from PIGV-, PIGB-, and PIGF-deficient CHO cells, in which incomplete GPI bearing mannose was accumulated. In contrast, ALP was degraded in PIGL-, DPM2-, or PIGX-deficient CHO cells, in which incomplete shorter GPIs that lacked mannose were accumulated. Our results suggest that GPI transamidase recognizes incomplete GPI bearing mannose and cleaves a hydrophobic signal peptide, resulting in secretion of soluble ALP. These results explain the molecular mechanism of hyperphosphatasia in HPMR.

VopV, an F-Actin-Binding Type III Secretion Effector, Is Required for Vibrio parahaemolyticus-Induced Enterotoxicity

Vibrio parahaemolyticus, a Gram-negative halophilic bacterium that causes acute gastroenteritis in humans, is characterized by two type III secretion systems (T3SS), namely T3SS1 and T3SS2. T3SS2 is indispensable for enterotoxicity but the effector(s) involved are unknown. Here, we identify VopV as a critical effector that is required to mediate V. parahaemolyticus T3SS2-dependent enterotoxicity. VopV was found to possess multiple F-actin-binding domains and the enterotoxicity caused by VopV correlated with its F-actin-binding activity. Furthermore, a T3SS2-related secretion system and a vopV homologous gene were also involved in the enterotoxicity of a non-O1/non-O139 V. cholerae strain. These results indicate that the F-actin-targeting effector VopV is involved in enterotoxic activity of T3SS2-possessing bacterial pathogens.

Identification of telomere-associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP)

Biochemical analysis of molecular interactions in specific genomic regions requires their isolation while retaining molecular interactions in vivo. Here, we report isolation of telomeres by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using a transcription activator-like (TAL) protein recognizing telomere repeats. Telomeres recognized by the tagged TAL protein were immunoprecipitated with an antibody against the tag and subjected to identification of telomere-binding molecules. enChIP-mass spectrometry (enChIP-MS) targeting telomeres identified known and novel telomere-binding proteins. The data have been deposited to the ProteomeXchange with identifier PXD000461. In addition, we showed that RNA associated with telomeres could be isolated by enChIP. Identified telomere-binding molecules may play important roles in telomere biology. enChIP using TAL proteins would be a useful tool for biochemical analysis of specific genomic regions of interest.

Transforming Potential of Src Family Kinases Is Limited by the Cholesterol-Enriched Membrane Microdomain

ABSTRACT The upregulation of Src family kinases (SFKs) has been implicated in cancer progression, but the molecular mechanisms regulating their transforming potentials remain unclear. Here we show that the transforming ability of all SFK members is suppressed by being distributed to the cholesterol-enriched membrane microdomain. All SFKs could induce cell transformation when overexpressed in C-terminal Src kinase (Csk)-deficient fibroblasts. However, their transforming abilities varied depending on their affinity for the microdomain. c-Src and Blk, with a weak affinity for the microdomain due to a single myristate modification at the N terminus, could efficiently induce cell transformation, whereas SFKs with both myristate and palmitate modifications were preferentially distributed to the microdomain and required higher doses of protein expression to induce transformation. In contrast, disruption of the microdomain by depleting cholesterol could induce a robust transformation in Csk-deficient fibroblasts in which only a limited amount of activated SFKs was expressed. Conversely, the addition of cholesterol or recruitment of activated SFKs to the microdomain via a transmembrane adaptor, Cbp/PAG1, efficiently suppressed SFK-induced cell transformation. These findings suggest that the membrane microdomain spatially limits the transforming potential of SFKs by sequestering them away from the transforming pathways.

Identification of functional domains and novel binding partners of STIM proteins

With a signal trap method, we previously identified stromal interaction molecule (STIM: originally named as SIM) as a protein, which has a signal peptide in 1996. However, recent works have accumulated evidences that STIM1 and STIM2 reside in endoplasmic reticulum (ER) and that both mainly sense ER Ca2+ depletion, which plays an essential role in store operated calcium entry. In the present study, we extensively analyzed the domain functions and associated molecules of STIMs. A STIM1 mutant lacking the coiled‐coil domains was massively expressed on the cell surface while mutants with the coiled‐coil domains localized in ER. In addition, STIM1 mutants with the coiled‐coil domains showed a longer half‐life of proteins than those without them. These results are likely to indicate that the coiled‐coil domains of STIM1 are essential for its ER‐retention and its stability. Furthermore, we tried to comprehensively identify STIM1‐associated molecules with mass spectrometry analysis of co‐immunoprecipitated proteins for STIM1. This screening clarified that both STIM1 and STIM2 have a capacity to bind to a chaperone, calnexin as well as two protein‐transporters, exportin1 and transportin1. Of importance, our result that glycosylation on STIM1 was not required for the association between STIM1 and calnexin seems to indicate that calnexin might function on STIM1 beyond a chaperone protein. Further information concerning regulatory mechanisms for STIM proteins including the data shown here will provide a model of Ca2+ control as well as a useful strategy to develop therapeutic drugs for intracellular Ca2+‐related diseases including inflammation and allergy. J. Cell. Biochem. 112: 147–156, 2011.

TLR4-induced NF-κB and MAPK signaling regulate the IL-6 mRNA stabilizing protein Arid5a

Abstract The AT-rich interactive domain-containing protein 5a (Arid5a) plays a critical role in autoimmunity by regulating the half-life of Interleukin-6 (IL-6) mRNA. However, the signaling pathways underlying Arid5a-mediated regulation of IL-6 mRNA stability are largely uncharacterized. Here, we found that during the early phase of lipopolysaccharide (LPS) stimulation, NF-κB and an NF-κB-triggered IL-6-positive feedback loop activate Arid5a gene expression, increasing IL-6 expression via stabilization of the IL-6 mRNA. Subsequently, mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) promotes translocation of AU-rich element RNA-binding protein 1 (AUF-1) from the nucleus to the cytoplasm, where it destabilizes Arid5a mRNA by binding to AU-rich elements in the 3΄ UTR. This results in downregulation of IL-6 mRNA expression. During the late phase of LPS stimulation, p38 MAPK phosphorylates Arid5a and recruits the WW domain containing E3 ubiquitin protein ligase 1 (WWP1) to its complex, which in turn ubiquitinates Arid5a in a K48-linked manner, leading to its degradation. Inhibition of Arid5a phosphorylation and degradation increases production of IL-6 mRNA. Thus, our data demonstrate that LPS-induced NF-κB and MAPK signaling are required to control the regulation of the IL-6 mRNA stabilizing molecule Arid5a. This study therefore substantially increases our understanding of the mechanisms by which IL-6 is regulated.

Hepatocyte Factor JMJD5 Regulates Hepatitis B Virus Replication through Interaction with HBx

ABSTRACT Hepatitis B virus (HBV) is a causative agent for chronic liver diseases such as hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). HBx protein encoded by the HBV genome plays crucial roles not only in pathogenesis but also in replication of HBV. Although HBx has been shown to bind to a number of host proteins, the molecular mechanisms by which HBx regulates HBV replication are largely unknown. In this study, we identified jumonji C-domain-containing 5 (JMJD5) as a novel binding partner of HBx interacting in the cytoplasm. DNA microarray analysis revealed that JMJD5-knockout (JMJD5KO) Huh7 cells exhibited a significant reduction in the expression of transcriptional factors involved in hepatocyte differentiation, such as HNF4A, CEBPA, and FOXA3. We found that hydroxylase activity of JMJD5 participates in the regulation of these transcriptional factors. Moreover, JMJD5KO Huh7 cells exhibited a severe reduction in HBV replication, and complementation of HBx expression failed to rescue replication of a mutant HBV deficient in HBx, suggesting that JMJD5 participates in HBV replication through an interaction with HBx. We also found that replacing Gly135 with Glu in JMJD5 abrogates binding with HBx and replication of HBV. Moreover, the hydroxylase activity of JMJD5 was crucial for HBV replication. Collectively, these results suggest that direct interaction of JMJD5 with HBx facilitates HBV replication through the hydroxylase activity of JMJD5. IMPORTANCE HBx protein encoded by hepatitis B virus (HBV) plays important roles in pathogenesis and replication of HBV. We identified jumonji C-domain-containing 5 (JMJD5) as a novel binding partner to HBx. JMJD5 was shown to regulate several transcriptional factors to maintain hepatocyte function. Although HBx had been shown to support HBV replication, deficiency of JMJD5 abolished contribution of HBx in HBV replication, suggesting that HBx-mediated HBV replication is largely dependent on JMJD5. We showed that hydroxylase activity of JMJD5 in the C terminus region is crucial for expression of HNF4A and replication of HBV. Furthermore, a mutant JMJD5 with Gly135 replaced by Glu failed to interact with HBx and to rescue the replication of HBV in JMJD5-knockout cells. Taken together, our data suggest that interaction of JMJD5 with HBx facilitates HBV replication through the hydroxylase activity of JMJD5.