X-linked inhibitor of apoptosis protein mediates neddylation by itself but does not function as a NEDD8–E3 ligase for caspase-7

AbstractX-linked inhibitor of apoptosis protein (XIAP) is a potent antagonist of caspases, and functions as a ubiquitin–E3 ligase by itself and for caspases. Recently, NEDD8, a ubiquitin-like modifier, has been suggested to be used for modification of caspase-7 mediated by XIAP. However, it is not clear whether caspase-7 is a bona fide target for NEDD8. Here we showed that no neddylation of caspase-7 but that of XIAP itself was observed under the conditions in which caspase-7 was modified with ubiquitin. These results reveal that XIAP does not function as a NEDD8–E3 ligase for caspase-7 in vivo.Structured summary of protein interactionsNEDD8 physically interacts with Caspase-7 by pull down (View interaction)XIAP physically interacts with NEDD8 by anti-bait coimmunoprecipitation (View interaction

Expression of protein kinase C subspecies in human leukemia- lymphoma cell lines

AbstractExpression of protein kinase C (PKC) subspecies was studied in various human leukemia-lymphoma cell lines. The PKC in most cell lines examined was resolved into two major fractions corresponding to type II (β-sequence) and type III (α-sequence) PKC of the rat brain. The amounts of these two subspecies greatly varied among the cell lines. Type I PKC (γ-sequence) was expressed in none of the cell lines tested, but PKCs with undefined structures were frequently detected. The differential co-expression of several PKC subspecies is presumably related to the state of cell differentiation

Identification of the catalytic subunit of cAMP-dependent protein kinase from the photosynthetic flagellate, Euglena gracilis Z11The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession number AB021126.

AbstractA gene named epk2 that encodes the amino acid sequence of a protein kinase was identified from the photosynthetic flagellate, Euglena gracilis Z. Homology search and phylogenetic analysis revealed that the deduced amino acid sequence of epk2 is most similar to that of the catalytic subunit of cAMP-dependent protein kinase (PKA). Northern blot analysis showed that Euglena cells express a 1.4-kb transcript of this gene. When the EPK2 protein was coexpressed with the rat regulatory subunit of PKA in cultured mammalian cells, these two proteins were coimmunoprecipitated. The association of EPK2 and the rat regulatory subunit of PKA was not detected in the cell lysate incubated with cAMP. EPK2 immunoprecipitated from the transfected cells phosphorylated Kemptide, a synthetic peptide substrate for PKA, and the phosphorylation was inhibited by PKI, a PKA-selective protein kinase inhibitor. These results indicate that EPK2 is a PKA homologue in the photosynthetic flagellate, and this is the first evidence for the occurrence of the PKA catalytic subunit in photosynthetic organisms

Activation of Protein Kinase B Induced by H2O2 and Heat Shock through Distinct Mechanisms Dependent and Independent of Phosphatidylinositol 3-Kinase

Protein kinase B (PKB) is a downstream target of phosphatidylinositol (PI) 3-kinase in the signaling pathway of growth factors, and is activated by cellular stress such as H2O2 and heat shock. To study the mechanism of the stress-induced activation of PKB, PI 3-kinase products were measured in stress-stimulated cells. Both PI 3, 4-bisphosphate and PI 3, 4, 5-trisphosphate increased in H2O2 -treated cells, and the elevation of these phospholipids and activation of PKB were concurrently blocked by wortmannin, a potent inhibitor of PI 3-kinase. In heat-shocked cells, the level of PI 3, 4-bisphosphate did not change while that of PI 3, 4, 5-trisphosphate increased slightly, and an association between PKB molecules was observed. Two active PKB fractions, presumably monomeric and oligomeric forms, were resolved from heat-shocked cells by gel filtration column chromatography. Activation of the former was suppressed by pretreatment with wortmannin, whereas the generation and activation of the latter were not blocked by the PI 3-kinase inhibitor. Only the monomeric form, but not the oligomeric form, was recovered from H2O2 -treated cells, and its activation was prevented by wortmannin. These results indicate that PKB is activated by two distinct mechanisms that are dependent and independent of PI 3-kinase in stress-stimulated cell

Identification of regions critical for the integrity of the TSC1-TSC2-TBC1D7 complex

The TSC1-TSC2-TBC1D7 complex is an important negative regulator of the mechanistic target of rapamycin complex 1 that controls cell growth in response to environmental cues. Inactivating TSC1 and TSC2 mutations cause tuberous sclerosis complex (TSC), an autosomal dominant disorder characterised by the occurrence of benign tumours in various organs and tissues, notably the brain, skin and kidneys. TBC1D7 mutations have not been reported in TSC patients but homozygous inactivation of TBC1D7 causes megaencephaly and intellectual disability. Here, using an exon-specific deletion strategy, we demonstrate that some regions of TSC1 are not necessary for the core function of the TSC1-TSC2 complex. Furthermore, we show that the TBC1D7 binding site is encoded by TSC1 exon 22 and identify amino acid residues involved in the TSC1-TBC1D7 interaction

Contribution of Caspase(s) to the Cell Cycle Regulation at Mitotic Phase

Caspases have been suggested to contribute to not only apoptosis regulation but also non-apoptotic cellular phenomena. Recently, we have reported the involvement of caspase-7 to the cell cycle progression at mitotic phase by knockdown of caspase-7 using small interfering RNAs and short hairpin RNA. Here we showed that chemically synthesized broad-spectrum caspase inhibitors, which have been used to suppress apoptosis, prevented the cell proliferation in a dose-dependent manner, and that the subtype-specific peptide-based caspase inhibitor for caspase-3 and -7, but not for caspase-9, inhibited cell proliferation. It was also indicated that the BIR2 domain of X-linked inhibitor of apoptosis protein, functioning as an inhibitor for caspase-3 and -7, but not the BIR3 domain which plays as a caspase-9 inhibitor, induced cell cycle arrest. Furthermore, flow cytometry revealed that the cells treated with caspase inhibitors arrested at G2/M phase. By using HeLa.S-Fucci (fluorescent ubiquitination-based cell cycle indicator) cells, the prevention of the cell proliferation by caspase inhibitors induced cell cycle arrest at mitotic phase accompanying the accumulation of the substrates for APC/C, suggesting the impairment of the APC/C activity at the transition from M to G1 phases. These results indicate that caspase(s) contribute to the cell cycle regulation at mitotic phase

The Yeast Tor Signaling Pathway Is Involved in G2/M Transition via Polo-Kinase

The target of rapamycin (Tor) protein plays central roles in cell growth. Rapamycin inhibits cell growth and promotes cell cycle arrest at G1 (G0). However, little is known about whether Tor is involved in other stages of the cell division cycle. Here we report that the rapamycin-sensitive Tor complex 1 (TORC1) is involved in G2/M transition in S. cerevisiae. Strains carrying a temperature-sensitive allele of KOG1 (kog1-105) encoding an essential component of TORC1, as well as yeast cell treated with rapamycin show mitotic delay with prolonged G2. Overexpression of Cdc5, the yeast polo-like kinase, rescues the growth defect of kog1-105, and in turn, Cdc5 activity is attenuated in kog1-105 cells. The TORC1-Type2A phosphatase pathway mediates nucleocytoplasmic transport of Cdc5, which is prerequisite for its proper localization and function. The C-terminal polo-box domain of Cdc5 has an inhibitory role in nuclear translocation. Taken together, our results indicate a novel function of Tor in the regulation of cell cycle and proliferation