Hepatocyte growth factor rapidly induces the tyrosine phosphorylation of 41-kDa and 43-kDa proteins in mouse keratinocytes.

We have examined the hepatocyte growth factor (HGF)-mediated changes in protein-tyrosine phosphorylation in mouse keratinocytes (PAM-212) and canine kidney epithelial cells (MDCK). In PAM-212 cells HGF and epidermal growth factor, both of which stimulated the DNA synthesis, rapidly induced the tyrosine phosphorylation of two 41-kDa and two 43-kDa proteins: increased tyrosine phosphorylation of those proteins has been commonly observed when quiescent fibroblasts are stimulated with a variety of mitogenic agents. In contrast, HGF did not stimulate the DNA synthesis but induced cell dissociation in MDCK cells; under this condition, increased tyrosine phosphorylation of the 41-kDa and 43-kDa protein was not observed. A possible role of the increased tyrosine phosphorylation of 41-kDa and 43-kDa protein in the signaling pathway of HGF is discussed.     

Cisplatin differently affects amino terminal and carboxyl terminal domains of HSP90

The 90-kDa heat shock protein (HSP90) is a molecular chaperone that assists in the folding and assembly of proteins in the cytosol. We previously demonstrated that the antineoplastic reagent, cisplatin, inhibits the aggregation prevention activity of mammalian HSP90. We now show that cisplatin binds both the amino terminal and carboxyl terminal domains of the human HSP90 and differently affects these two domains. Cisplatin blocks the aggregation prevention activity of HSP90C, but not HSP90N. In contrast, cisplatin induces a conformational change in HSP90N, but not HSP90C. These results indicate that cisplatin modulates the HSP90 activities through two different mechanisms using the two distinct binding sites of the HSP90 molecule.     

Mutations causing DOK7 congenital myasthenia ablate functional motifs in Dok-7

Dok-7 is a cytoplasmic activator of muscle-specific receptor-tyrosine kinase (MuSK). Both Dok-7 and MuSK are required for neuromuscular synaptogenesis. Mutations in DOK7 underlie a congenital myasthenic syndrome (CMS) associated with small and simplified neuromuscular synapses likely due to impaired Dok-7/MuSK signaling. The overwhelming majority of patients with DOK7 CMS have at least one allele with a frameshift mutation that causes a truncation in the COOH-terminal region of Dok-7 and affects MuSK activation. Dok-7 has pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains in the NH2-terminal moiety, both of which are indispensable for MuSK activation in myotubes, but little is known about additional functional elements. Here, we identify a chromosome region maintenance 1-dependent nuclear export signal (NES) in the COOH-terminal moiety and demonstrate that the NES-mediated cytoplasmic location of Dok-7 is essential for regulating the interaction with MuSK in myotubes. The NH2-terminal PH domain is responsible for the nuclear import of Dok-7. We also show that the Src homology 2 target motifs in the COOH-terminal moiety of Dok-7 are active and crucial for MuSK activation in myotubes. In addition, CMS-associated missense mutations found in the PH or PTB domain inactivate Dok-7. Together, these findings demonstrate that, in addition to the NH2-terminal PH and PTB domains, the COOH-terminal NES and Src homology 2 target motifs play key roles in Dok-7/MuSK signaling for neuromuscular synaptogenesis. Ablation or disruption of these functional elements in Dok-7 probably underlies the neuromuscular junction synaptopathy observed in DOK7 CMS.     

Monooxygenase activity of Octopus vulgaris hemocyanin

Octopus vulgaris hemocyanin ( Ov-Hc) and one of its minimal functional units ( Ov-g) have been purified, and their spectroscopic features and monooxygenase (phenolase) activity have been examined in detail. The oxy forms of both Ov-Hc and Ov-g are stable in 0.5 M borate buffer (pH 9.0) even in the presence of a high concentration of urea at 25 degrees C; approximately 90 and approximately 75% of the (mu-eta (2):eta (2)-peroxo)dicopper(II) species of Ov-Hc and Ov-g, respectively, remained unchanged after argon (Ar) gas flushing of the sample solutions for 1 h. The catalytic activity of Ov-g in the oxygenation reaction (multiturnover reaction) of 4-methylphenol ( p-cresol) to 4-methyl-1,2-dihydroxybenzene (4-methylcatechol) was higher than that of Ov-Hc, and its catalytic activity was further accelerated by the addition of urea. Kinetic deuterium isotope effect analysis and Hammett analysis using a series of phenol derivatives under anaerobic conditions (single-turnover reaction) have indicated that the monooxygenation reaction of phenols to catechols by the peroxo species of oxyhemocyanin proceeds via electrophilic aromatic substitution mechanism as in the case of tyrosinase. The effect of urea on the redox functions of oxyhemocyanin is discussed on the basis of the spectroscopic analysis and reactivity studies.     

A parallel affinity purification method for selective isolation of polyubiquitinated proteins

We developed a parallel affinity purification (PAP) procedure, in which ubiquitinated proteins are purified from the cells that coexpress two affinity-tagged ubiquitins by sequential use of affinity chromatography specific to each tag. In contrast with previous procedures using a single affinity-tagged ubiquitin, the PAP eliminates highly abundant ubiquitin monomers and monoubiquitinated proteins to selectively enrich proteins bearing both affinity-tags, or poly- and multiubiquitinated proteins. Accordingly, it would serve as a powerful method to facilitate mass-spectrometric identification of ubiquitinated proteins.     

In silico identification of short nucleotide sequences associated with gene expression of pollen development in rice

Microarray analysis of tiny amounts of RNA extracted from plant section samples prepared by laser microdissection (LM) can provide high-quality information on gene expression in specified plant cells at various stages of development. Having joined the LM-microarray analysis project, we utilized such genome-wide gene expression data from developing rice pollen cells to identify candidates for cis-regulatory elements for specific gene expression in these cells. We first found a few clusters of gene expression patterns based on the data from LM-microarrays. On one gene cluster in which the members were specifically expressed at the bicellular and mature pollen mitotic stages, we identified gene cluster fingerprints (GCFs), each of which consists of a short nucleotide representing the gene cluster. We expected that these GCFs would contain cis-regulatory elements for stage- and tissue-specific gene expression, and we further identified groups of GCFs with common core sequences. Some criteria, such as frequency of occurrence in the gene cluster in contrast to the total tested gene set, flanking sequence preference and distribution of combined GCF sets in the gene regions, allowed us to limit candidates for cis-regulatory sequences for specific gene expression in rice pollen cells to at least 20 sets of combined GCFs. This approach should provide a general purpose algorithm for identifying short nucleotides associated with specific gene expression.     

Eupalinin A isolated from Eupatorium chinense L. induces autophagocytosis in human leukemia HL60 cells

Eupalinin A, a natural phytoalexin included in Eupatorium chinense L., exhibited a marked inhibitory effect on cell growth in HL60 cells. The morphological aspects of eupalinin A-treated cells evaluated by Hoechst 33342 nuclear staining indicated cell death, only a small part of which showed a typical apoptosis with nuclear fragmentation and condensation. To determine what type of cell death is caused by eupalinin A, we examined the contribution of caspases, Bcl-2 family proteins, MAP kinase, and PI3K/Akt, and mitochondrial membrane potential to this cell death. As a result, most part of the cell death was not associated with apoptosis because of caspase independence and no death factor released from mitochondria. Electron microscopic study indicated a characteristic finding of autophagy such as the formation of autophagosomes. Furthermore, the level of microctubule-associated-protein light chain 3 (LC3) II protein and monodancylcanaverin (MDC) incorporation were gradually increased with reduction of mitochondrial membrane potential by the accumulation of intracellular ROS after eupalinin A treatment. From these results, we can conclude that eupalinin A-induced cell death was mainly due to autophagy, which was initiated by increased ROS, resulting in the perturbation of mitochondrial membrane potential. Since the class III PI3K inhibitor such as 3-MA or LY294002 did not inhibit the eupalinin A-induced type II programmed cell death (PCD II), it was suggested that the PCD II was executed by Beclin-1 independent pathway of damage-induced mitochondrial autophagy (mitophagy).