Shin-ichi Hoshino

Heterodimeric Phosphoinositide 3-Kinase Consisting of p85 and p110β Is Synergistically Activated by the βγ Subunits of G Proteins and Phosphotyrosyl Peptide

Phosphoinositide 3-kinase (PI 3-kinase) is a key signaling enzyme implicated in variety of receptor-stimulated cell responses. Receptors with intrinsic or associated tyrosine kinase activity recruit heterodimeric PI 3-kinases consisting of a 110-kDa catalytic subunit (p110) and an 85-kDa regulatory subunit (p85). We separated a PI 3-kinase that could be stimulated by the βγ subunits of G protein (Gβγ) from rat liver. The Gβγ-sensitive PI 3-kinase appeared to be a heterodimer consisting of p110β and p85 (or their related subunits). The stimulation by Gβγ was inhibited by the GDP-bound α subunit of the inhibitory GTP-binding protein. Moreover, the stimulatory action of Gβγ was markedly enhanced by the simultaneous addition of a phosphotyrosyl peptide synthesized according to the amino acid sequence of the insulin receptor substrate-1. Such enzymic properties could be observed with a recombinant p110β/p85α expressed in COS-7 cells with their cDNAs. In contrast, another heterodimeric PI 3-kinase consisting of p110α and p85 in the same rat liver, together with a recombinant p110α/p85α, was not activated by Gβγ, although their activities were stimulated by the phosphotyrosyl peptide. These results indicate that p110β/p85 PI 3-kinase may be regulated in a cooperative manner by two different types of membrane receptors, one possessing tyrosine kinase activity and the other activating GTP-binding proteins.

The eukaryotic polypeptide chain releasing factor (eRF3/GSPT) carrying the translation termination signal to the 3'-poly(A) tail of mRNA-Direct association of eRF3/GSPT with polyadenylate-binding protein

The mammalian GTP-binding protein GSPT, whose carboxyl-terminal sequence is homologous to the eukaryotic elongation factor EF1α, binds to the polypeptide chain releasing factor eRF1 to function as eRF3 in the translation termination. The amino-terminal domain of GSPT was, however, not required for the binding. Search for other GSPT-binding proteins in yeast two-hybrid screening system resulted in the identification of a cDNA encoding polyadenylate-binding protein (PABP), whose amino terminus is associating with the poly(A) tail of mRNAs presumably for their stabilization. The interaction appeared to be mediated through the carboxyl-terminal domain of PABP and the amino-terminal region of GSPT. Interestingly, multimerization of PABP with poly(A), which is ascribed to the action of its carboxyl-terminal domain, was completely inhibited by the interaction with the amino-terminal domain of GSPT. These results indicate that GSPT/eRF3 may play important roles not only in the termination of protein synthesis but also in the regulation of mRNA stability. Thus, the present study is the first report showing that GSPT/eRF3 carries the translation termination signal to 3′-poly(A) tail ubiquitously present in eukaryotic mRNAs.

Ski7p G protein interacts with the exosome and the Ski complex for 3′-to-5′ mRNA decay in yeast

Two cytoplasmic mRNA‐decay pathways have been characterized in yeast, and both are initiated by shortening of the 3′‐poly(A) tail. In the major 5′‐to‐3′ decay pathway, the deadenylation triggers removal of the 5′‐cap, exposing the transcript body for 5′‐to‐3′ degradation. An alternative 3′‐to‐5′ decay pathway also follows the deadenylation and requires two multi‐complexes: the exosome containing various 3′‐exonucleases and the Ski complex consisting of the RNA helicase Ski2p, Ski3p and Ski8p. In addition, Ski7p, which has an N‐terminal domain and a C‐terminal elongation factor 1α‐like GTP‐binding domain, is involved in the 3′‐to‐5′ decay. However, physical interaction between the exosome and the Ski complex, together with the function of Ski7p, has remained unknown. Here we report that the N domain of Ski7p is required and sufficient for the 3′‐to‐5′ decay. Furthermore, the exosome and the Ski complex interact with the different regions of Ski7p N domain, and both interactions are required for the 3′‐to‐5′ decay. Thus, Ski7p G protein appears to function as a signal‐coupling factor between the two multi‐complexes operating in the 3′‐to‐5′ mRNA‐decay pathway.

Translation termination factor eRF3 mediates mRNA decay through the regulation of deadenylation

Messenger RNA decay, which is a regulated process intimately linked to translation, begins with the deadenylation of the poly(A) tail at the 3′ end. However, the precise mechanism triggering the first step of mRNA decay and its relationship to translation have not been elucidated. Here, we show that the translation termination factor eRF3 mediates mRNA deadenylation and decay in the yeast Saccharomyces cerevisiae. The N-domain of eRF3, which is not necessarily required for translation termination, interacts with the poly(A)-binding protein PABP. When this interaction is blocked by means of deletion or overexpression of the N-domain of eRF3, half-lives of all mRNAs are prolonged. The eRF3 mutant lacking the N-domain is deficient in the poly(A) shortening. Furthermore, the eRF3-mediated mRNA decay requires translation to proceed, especially ribosomal transition through the termination codon. These results indicate that the N-domain of eRF3 mediates mRNA decay by regulating deadenylation in a manner coupled to translation.

A human homologue of the yeast GST1 gene codes for a GTP-binding protein and is expressed in a proliferation-dependent manner in mammalian cells.

A human homologue (GST1‐Hs) of the yeast GST1 gene that encodes a new GTP‐binding protein essential for the G1‐to‐S phase transition of the cell cycle was cloned from the cDNA library of human KB cells. The GST1‐Hs cDNA contained a 1497 bp open reading frame coding for a 499 amino acid protein with mol. wt 55,754 and with the amino acid sequence homologies of 52.3 and 37.8% to the GST1 protein and polypeptide chain elongation factor EF1 alpha respectively. The regions potentially responsible for GTP binding and GTP hydrolysis were conserved in the GST1‐Hs protein as well. When expressed in yeast cell, the GST1‐Hs gene could complement the ts phenotype of yeast gst1 mutant. GST1‐Hs and its mouse homologue were expressed in human fibroblasts and in various mouse cell types respectively, at relatively low levels in their quiescent states, and the level of those expressions increased rapidly, prior to the onset of DNA replication and the total RNA synthesis, when human or mouse fibroblasts were progressed out of the growth‐arrested state by the addition of serum. A possible role of GST1‐Hs in mammalian cell growth is discussed.

Molecular cloning of a novel member of the eukaryotic polypeptide chain-releasing factors (eRF)-Its identification as eRF3 interacting with eRF1

Yeast GST1 gene, whose product is a GTP-binding protein structurally related to polypeptide chain elongation factor-1α (EF1α), was first described to be essential for the G 1 to S phasetransition (GSPT) of the cell cycle, and the product was recently reported to function as a polypeptide chain release factor 3 (eRF3) in yeast. Although we previously cloned a human homologue (renamed as GSPT1) of the yeast gene, it has remained to be determined whether GSPT1 also functions as eRF3 or if another GSPT may have such a function in mammalian cells. In the present study, we isolated two mouse GSPT genes, the counterpart of human GSPT1 and a novel member of the GSPT gene family, GSPT2. Both the mouse GSPTs had a two-domain structure characterized as an amino-terminal no-homologous region (approximately 200 amino acids) and a carboxyl-terminal conserved eukaryotic elongation factor-1α-like domain (428 amino acids). Messenger RNAs of the two GSPTs could be detected in all mouse tissues surveyed, although the level of GSPT2 message appeared to be relatively abundant in the brain. The mouse GSPT1 was expressed in a proliferation-dependent manner in Swiss 3T3 cells, whereas the expression of GSPT2 was constant during the cell-cycle progression. Immunoprecipitation assays in COS-7 cells expressing flag epitope-tagged proteins demonstrated that not only GSPT1 but also GSPT2 was capable of interacting with eRF1. Such interaction between GSPT2 and eRF1 was also confirmed by yeast two-hybrid analysis. Taken together, these data indicated that the novel GSPT2 may interact with eRF1 to function as eRF3 in mammalian cells.

The Polypeptide Chain-releasing Factor GSPT1/eRF3 Is Proteolytically Processed into an IAP-binding Protein

Smac/Diablo and HtrA2/Omi are inhibitors of apoptosis (IAP)-binding proteins released from the mitochondria of human cells during apoptosis and regulate apoptosis by liberating caspases from IAP inhibition. Here we describe the identification of a proteolytically processed isoform of the polypeptide chain-releasing factor GSPT1/eRF3 protein, which functions in translation, as a new IAP-binding protein. In common with other IAP-binding proteins, the processed GSPT1 protein harbors a conserved N-terminal IAP-binding motif (AKPF). Additionally, processed GSPT1 interacts biochemically with IAPs and could promote caspase activation, IAP ubiquitination and apoptosis. The IAP-binding motif of the processed GSPT1 is absolutely required for these activities. Our findings are consistent with a model whereby processing of GSPT1 into the IAP-binding isoform could potentiate apoptosis by liberating caspases from IAP inhibition, or target IAPs and the processed GSPT1 for proteasome-mediated degradation.

ACCUMULATION OF CYCLIC ADP-RIBOSE MEASURED BY A SPECIFIC RADIOIMMUNOASSAY IN DIFFERENTIATED HUMAN LEUKEMIC HL-60 CELLS WITH ALL-TRANS-RETINOIC ACID

Cyclic adenosine diphosphoribose (cADPR) is a novel candidate for the mediator of Ca2+ release from intracellular Ca2+ stores. The formation of this cyclic nucleotide is catalyzed by not only Aplysia ADP‐ribosyl cyclase but also an ecto‐form enzyme of NAD+ glycohydrolase (NADase), which was previously identified as all‐trans‐retinoic acid (RA)‐inducible CD38 in human leukemic HL‐60 cells. In the present study, we developed a radioimmunoassay specific for cADPR, by which more than 100 fmol of cADPR could be detected without any interference by other nucleotides. The possible involvement of CD38 in the formation of cellular cADPR was investigated with the radioimmunoassay method. A marked increase in cellular cADPR was accompanied by all‐trans‐RA‐induced differentiation of HL‐60 cells. Moreover, a high level of cellular cADPR was observed in other leukemic cell lines, in which CD38 mRNA was expressed. Thus, CD38, which was initially identified as an NADase, appeared to be responsible for the formation of cellular cADPR.

LARK activates posttranscriptional expression of an essential mammalian clock protein, PERIOD1.

The mammalian molecular clock is composed of feedback loops to keep circadian 24-h rhythms. Although much focus has been on transcriptional regulation, it is clear that posttranscriptional controls also play important roles in molecular circadian clocks. In this study, we found that mouse LARK (mLARK), an RNA binding protein, activates the posttranscriptional expression of the mouse Period1 (mPer1) mRNA. A strong circadian cycling of the mLARK protein is observed in the suprachiasmatic nuclei with a phase similar to that of mPER1, although the level of the Lark transcripts are not rhythmic. We demonstrate that LARK causes increased mPER1 protein levels, most likely through translational regulation and that the LARK1 protein binds directly to a cis element in the 3′ UTR of the mPer1 mRNA. Alterations of mLark expression in cycling cells caused significant changes in circadian period, with mLark knockdown by siRNA resulting in a shorter circadian period, and the overexpression of mLARK1 resulting in a lengthened period. These data indicate that mLARKs are novel posttranscriptional regulators of mammalian circadian clocks.

TYROSINE PHOSPHORYLATION OF THE C-CBL PROTO-ONCOGENE PRODUCT MEDIATED BY CELL SURFACE ANTIGEN CD38 IN HL-60 CELLS

The human cell surface antigen CD38 is a 46-kDa type II transmembrane glycoprotein with a short N-terminal cytoplasmic domain and a long Cys-rich C-terminal extracellular one. We demonstrated previously that the extracellular domain of CD38 has NAD glycohydrolase (NADase) activity and that the ecto-form NADase activity induced in HL-60 cells during cell differentiation by retinoic acid is due to CD38. In the present study, we investigated the intracellular signaling mediated by CD38 in retinoic acid-differentiated HL-60 cells with an anti-CD38 monoclonal antibody. The addition of anti-CD38 monoclonal antibody to the cells induced rapid tyrosine phosphorylation of the cellular proteins with molecular weights of 120,000, 87,000, and 77,000. An increase in tyrosine kinase activity in the anti-phosphotyrosine immunoprecipitates of the cells was also observed after the addition of anti-CD38 monoclonal antibody. Moreover, one of the prominent tyrosine-phosphorylated proteins stimulated by the anti-CD38 monoclonal antibody was identified as the c-cbl proto-oncogene product, p120. These results indicated that tyrosine phosphorylation of cellular proteins, including p120, is possibly involved in transmembrane signaling mediated by CD38.