Modulation of the stathmin-like microtubule destabilizing activity of RB3, a neuron-specific member of the SCG10 family, by its N-terminal domain

RB3 is a neuron-specific homologue of the SCG10/stathmin family proteins, possessing a unique N-terminal membrane-associated domain and the stathmin-like domain at the C terminus, which promotes microtubule (MT) catastrophe and/or tubulin sequestering. We examined herein the contribution of the N-terminal subdomain of RB3 to the regulation of MT dynamics. To begin with, we determined the effects of full-length (RB3-f) and short truncated (RB3-s) forms of RB3 on the polymerization of MT in vitro. RB3-s had a deletion of amino acids 1-75 from the N terminus, leaving the so-called stathmin-like domain, consisting of residues 76-217. Although both RB3-f and RB3-s exhibited MT-depolymerizing activity, RB3-f was less effective. The binding affinity for tubulin was also lower in RB3-f. Direct observation of the dynamics of individual MTs using dark field microscopy revealed that RB3-s slowed MT elongation velocity, increased catastrophes, and reduced rescues. This effect is almost identical to that by stathmin/oncoprotein 18. On the other hand, the MT elongation rate increased at lower concentrations of RB3-f. In addition, RB3-f, indicated higher rescue frequency than control as well as the catastrophe in a dose-dependent manner. The functionality of RB3-f indicated that full-length RB3 has not only stathmin-like MT destabilizing activity but also MT-associated protein-like MT stabilizing activity. Possibly, the balance of these activities is altered in a concentration-dependent manner in vitro. This interesting regulatory role of the unique N-terminal domain of RB3 in MT dynamics would contribute to the physiological regulation of neuronal morphogenesis.     

A stand-alone adenylation domain forms amide bonds in streptothricin biosynthesis

The streptothricin (ST) antibiotics, produced by Streptomyces bacteria, contain L-β-lysine ((3S)-3,6-diaminohexanoic acid) oligopeptides as pendant chains. Here we describe three unusual nonribosomal peptide synthetases (NRPSs) involved in ST biosynthesis: ORF 5 (a stand-alone adenylation (A) domain), ORF 18 (containing thiolation (T) and condensation (C) domains) and ORF 19 (a stand-alone A domain). We demonstrate that ST biosynthesis begins with adenylation of L-β-lysine by ORF 5, followed by transfer to the T domain of ORF 18. In contrast, L-β-lysine molecules adenylated by ORF 19 are used to elongate an L-β-lysine peptide chain on ORF 18, a reaction unexpectedly catalyzed by ORF 19 itself. Finally, the C domain of ORF 18 catalyzes the condensation of L-β-lysine oligopeptides covalently bound to ORF 18 with a freely diffusible intermediate to release the ST products. These results highlight an unusual activity for an A domain and unique mechanisms of crosstalk within NRPS machinery.     

Sequence analyses of the ITS regions and the matK gene for determining phylogenetic relationships of Diospyros kaki (persimmon) with other wild Diospyros (Ebenaceae) species

To elucidate the relationships among Diospyros kaki and species closely related in previous studies, the nuclear ribosomal internal transcribed spacer (ITS) DNA sequence and the chloroplast matK gene were sequenced and compared with those of nine Diospyros species from Thailand, four species from temperate regions, and one species of southern Africa, D. lycioides. Maximum parsimony, maximum likelihood, and neighbor joining analyses of the matK and ITS data sets revealed that D. kaki is closely related to two diploid species, D. oleifera and D. glandulosa. D. kaki, D. glandulosa, and D. oleifera were placed differently in the trees obtained from ITS and matK data sets, suggesting that hybridization and/or introgression may have occurred during the development of these species. D. kaki was not found to be closely related to D. ehretioides, a diploid species from Thailand. These results differed from a prior analysis of this genus performed with chloroplast DNA (cpDNA) restriction site mutations in 3.2- and 2.1-kb amplified sequences. The results supported Ng’s hypothesis that D. glandulosa and D. kaki may share a common ancestor. D. oleifera was also closely associated with D. kaki.     

High frequency of mating without egg release in highly promiscuous nonparasitic lamprey <Emphasis Type="Italic">Lethenteron kessleri</Emphasis>

Nonparasitic lampreys are highly promiscuous: a single female can mate over several dozen times with multiple males. It remains unknown why females mate so frequently despite presumed costs from an elongated spawning period. This paper documents that female Siberian brook lampreys mate without egg release (termed “sham mating”) at remarkably high frequencies. Females mated 20–196 times during a breeding experiment, of which sham mating comprised 35–90%. The number of eggs released may be physically constrained in each mating by the lamprey’s elongated body and behavior. Female lampreys might also control egg release depending on surrounding males.     

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.     

Epsilon-poly-L-lysine dispersity is controlled by a highly unusual nonribosomal peptide synthetase

Epsilon-Poly-L-lysine (epsilon-PL) consists of 25-35 L-lysine residues in isopeptide linkages and is one of only two amino acid homopolymers known in nature. Elucidating the biosynthetic mechanism of epsilon-PL should open new avenues for creating novel classes of biopolymers. Here we report the purification of an epsilon-PL synthetase (Pls; 130 kDa) and the cloning of its gene from an epsilon-PL-producing strain of Streptomyces albulus. Pls was found to be a membrane protein with adenylation and thiolation domains characteristic of the nonribosomal peptide synthetases (NRPSs). It had no traditional condensation or thioesterase domain; instead, it had six transmembrane domains surrounding three tandem soluble domains. These tandem domains iteratively catalyzed L-lysine polymerization using free L-lysine polymer (or monomer in the initial reaction) as acceptor and Pls-bound L-lysine as donor, directly yielding chains of diverse length. Thus, Pls is a new single-module NRPS having an amino acid ligase-like catalytic activity for peptide bond formation.     

Drug–target identification from total cellular lysate by drug-induced conformational changes

Identification of drug targets is a key step in the development of novel pharmaceuticals. To this end, chemical probes or affinity matrices are often used, requiring substantial structure–activity relationship (SAR) studies. Here we report on the development of a novel technique for drug–target identification from total cellular lysate conducted independently of SAR information. This technique relies on binding of a drug to its target inducing a conformational change in target protein, thereby altering its susceptibility to proteolysis and resulting in specific degradation in some cases or in protection of target protein in others. As proof of concept, three drugs with identified targets were used. First, incubation of cellular lysates with okadaic acid elicited a specific protective effect on its target, protein phosphatase 2A catalytic subunit. Second, specific protection from exogenous protease of FKBP12 by FK506 and Hsp90 fragments by radicicol were observed. We then used the method to validate the targets of UCS15A, an Src signaling inhibitor. UCS15A induced proteolysis of a number of proteins, one of which was identified as Sam68. These studies suggest that the technology may be generally useful for identification and validation of drug targets.     

A hormonal regulatory module that provides flexibility to tropic responses

Plants orient their growth depending on directional stimuli such as light and gravity, in a process known as tropic response. Tropisms result from asymmetrical accumulation of auxin across the responding organ relative to the direction of the stimulus, which causes differential growth rates on both sides of the organ. Here, we show that gibberellins (GAs) attenuate the gravitropic reorientation of stimulated hypocotyls of dark-grown Arabidopsis (Arabidopsis thaliana) seedlings. We show that the modulation occurs through induction of the expression of the negative regulator of auxin signaling INDOLE-3-ACETIC ACID INDUCIBLE19/MASSUGU2. The biological significance of this regulatory mechanism involving GAs and auxin seems to be the maintenance of a high degree of flexibility in tropic responses. This notion is further supported by observations that GA-deficient seedlings showed a much lower variance in the response to gravity compared to wild-type seedlings and that the attenuation of gravitropism by GAs resulted in an increased phototropic response. This suggests that the interplay between auxin and GAs may be particularly important for plant orientation under competing tropic stimuli.     

Fgf16 Is Required for Specification of GABAergic Neurons and Oligodendrocytes in the Zebrafish Forebrain

Fibroblast growth factor (Fgf) signaling plays crucial roles in various developmental processes including those in the brain. We examined the role of Fgf16 in the formation of the zebrafish brain. The knockdown of fgf16 decreased cell proliferation in the forebrain and midbrain. fgf16 was also essential for development of the ventral telencephalon and diencephalon, whereas fgf16 was not required for dorsoventral patterning in the midbrain. fgf16 was additionally required for the specification and differentiation of γ–aminobutyric acid (GABA)ergic interneurons and oligodendrocytes, but not for those of glutamatergic neurons in the forebrain. Cross talk between Fgf and Hedgehog (Hh) signaling was critical for the specification of GABAergic interneurons and oligodendrocytes. The expression of fgf16 in the forebrain was down-regulated by the inhibition of Hh and Fgf19 signaling, but not by that of Fgf3/Fgf8 signaling. The fgf16 morphant phenotype was similar to that of the fgf19 morphant and embryos blocked Hh signaling. The results of the present study indicate that Fgf16 signaling, which is regulated by the downstream pathways of Hh-Fgf19 in the forebrain, is involved in forebrain development.