No abstract

A synthetic peptide corresponding to the autophosphorylation site of Ca 2؉ /calmodulin-dependent protein kinase II (CaMKII) (residues 281-289) was conjugated to paramagnetic particles, and phosphorylated by a constitutively active CaMKII fragment. Using this phosphopeptide conjugate as a substrate, a calyculin A-insensitive, Mn 2؉ -dependent, and poly-L-lysine-stimulated protein phosphatase activity was detected in the crude extract of rat brain. The protein phosphatase (designated as CaMKII phosphatase) (CaMKIIPase) was purified to near homogeneity from rat brain. CaMKIIPase showed apparent molecular weights of 54,000 and 65,000, on SDS-polyacrylamide gel electrophoresis and gel-filtration analysis, respectively. It was not inhibited by 100 nM calyculin A or 10 M okadaic acid. Mn 2؉ , but not Mg 2؉ , was absolutely required for activity. CaMKIIPase was potently activated by polycations. Autophosphorylated CaMKII was dephosphorylated by CaMKIIPase, whereas phosphorylase kinase, mixed histones, myelin basic protein, and ␣-casein (which had been phosphorylated by cAMP-dependent protein kinase) and phosphorylase a (phosphorylated by phosphorylase kinase) were not significantly dephosphorylated. No other proteins than CaMKII in rat brain extract which had been phosphorylated by CaMKII were dephosphorylated. The stimulated Ca 2؉

Cyclic ADP-ribose (cADPR) is generated in pancreatic islets by glucose stimulation, serving as a second messenger for Ca 2؉ mobilization from the endoplasmic reticulum for insulin secretion (Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H. (1993) Science 259, 370 -373). In the present study, we observed that the addition of calmodulin (CaM) to rat islet microsomes sensitized and activated the cADPR-mediated Ca 2؉ release. Inhibitors for CaM-dependent protein kinase II (CaM kinase II) completely abolished the glucose-induced insulin secretion as well as the cADPR-mediated and CaM-activated Ca 2؉ mobilization. Western blot analysis revealed that the microsomes contain the ␣ isoform of CaM kinase II but do not contain CaM. When the active 30-kDa chymotryptic fragment of CaM kinase II was added to the microsomes, fully activated cADPR-mediated Ca 2؉ release was observed in the absence of CaM. These results along with available evidence strongly suggest that CaM kinase II is required to phosphorylate and activate the ryanodine-like receptor, a Ca 2؉ channel for cADPR as an endogenous activator, for the cADPR-mediated Ca 2؉ release.

The hippocampus is essentially involved in learning and memory processes. Its functions are affected by various neuromodulators, including 17beta-estradiol, testosterone, and retinoid. Brain-synthesized steroid hormones act as autocrine and paracrine modulators. The regulatory mechanism underlying brain steroidogenesis has not been fully elucidated. Synthesis of sex steroids in the gonads is stimulated by retinoic acids. Therefore, we examined the effects of retinoic acids on estradiol and testosterone biosynthesis in the rat hippocampus. We used cultured hippocampal slices from 10- to 12-d-old male rats to investigate de novo steroidogenesis. The infant rat hippocampus possesses mRNAs for steroidogenic enzymes and retinoid receptors. Slices were used after 24 h of preculture to obtain maximal steroidogenic activity because steroidogenesis in cultured slices decreases with time. The mRNA levels for P450(17alpha), P450 aromatase and estrogen receptor-beta in the slices were increased by treatment with 9-cis-retinoic acid but not by all-trans-isomer. The magnitude of stimulation and the shape of the dose-response curve for the mRNA level for P450(17alpha) were similar to those for cellular retinoid binding protein type 2, the transcription of which is activated by retinoid X receptor signaling. 9-cis-Retinoic acid also induced a 1.7-fold increase in the protein content of P450(17alpha) and a 2-fold increase in de novo synthesis of 17beta-estradiol and testosterone. These steroids may be synthesized from a steroid precursor(s), such as pregnenolone or other steroids, or from cholesterol, as so-called neurosteroids. The stimulation of estradiol and testosterone synthesis by 9-cis-retinoic acid might be caused by activation of P450(17alpha) transcription via retinoid X receptor signaling.

Reactions of radical cations of eight stilbene derivatives (S •+) have been studied using pulse radiolysis and γ-ray radiolysis in 1,2-dichloroethane or butyl chloride. Unimolecular isomerization from cis-S •+ to trans-S •+ and bimolecular reactions with O2 (oxidation) and a neutral stilbene (dimerization) occur depending on the substituents. The unimolecular c−t isomerization and the oxidation proceed preferably in S •+ substituted with a p-methoxyl group (as an electron-donating substituent) with rate constants of k i = 4.5 × 106 to 1.4 × 107 s-1 and k O2 = (1.2−4.5) × 107 M-1 s-1, respectively. On the basis of transient absorption measurements, it is concluded that separation and localization of a positive charge and an unpaired electron play the most important role as the controlling factors in the reactivities of the unimolecular isomerization and the oxidation. The dimerization involves initial formation of a π-complex with overlapping of two benzene rings and is inhibited by steric hindrance of substituents on the benzene rings and olefinic carbons.

The active 30-kDa chymotryptic fragment of calmodulin-dependent protein kinase II (CaM kinase II), devoid of the autoinhibitory domain, and the enzyme, autothiophosphorylated at Thr286/Thr287, were much more labile than was the original native enzyme. They were markedly stabilized by synthetic peptides, designed after the sequence around the autophosphorylation site in the autoinhibitory domain, such as autocamtide-2 and CaMK-(281-309), but such marked stabilizations were not observed with the ordinary exogenous substrates, such as syntide-2. These results suggest that the autoinhibitory domain of CaM kinase II plays a crucial role in stabilizing the enzyme. A nonphosphorylatable analog of autocamtide-2, AIP, strongly inhibited the activity of the 30-kDa fragment. Kinetic analysis revealed that the inhibition by AIP was competitive with respect to autocamtide-2 and CaMK-(281-289) and noncompetitive with respect to syntide-2 and ATP/Mg2+, suggesting that CaM kinase II possesses at least two distinct substrate-binding sites; one for ordinary exogenous substrates such as syntide-2 and the other for an endogenous substrate, the autophosphorylation site (Thr286/Thr287) in the autoinhibitory domain. Fluorescence analysis of the binding of 7-nitrobenz-2-oxa-1,3-diazole-4-yl labeled AIP to the 30-kDa fragment also supported this contention. Thus, the autoinhibitory domain appears to play a crucial role in keeping the enzyme stable by binding to the substrate-binding site for the autophosphorylation site.

BackgroundAccumulating evidence suggests that some long noncoding RNAs (lncRNAs) are involved in certain diseases, such as cancer. The lncRNA, CCDC26, is related to childhood acute myeloid leukemia (AML) because its copy number is altered in AML patients.ResultsWe found that CCDC26 transcripts were abundant in the nuclear fraction of K562 human myeloid leukemia cells. To examine the function of CCDC26, gene knockdown (KD) was performed using short hairpin RNAs (shRNAs), and four KD clones, in which CCDC26 expression was suppressed to 1% of its normal level, were isolated. This down-regulation included suppression of CCDC26 intron-containing transcripts (the CCDC26 precursor mRNA), indicating that transcriptional gene suppression (TGS), not post-transcriptional suppression, was occurring. The shRNA targeting one of the two CCDC26 splice variants also suppressed the other splice variant, which is further evidence for TGS. Growth rates of KD clones were reduced compared with non-KD control cells in media containing normal or high serum concentrations. In contrast, enhanced growth rates in media containing much lower serum concentrations and increased survival periods after serum withdrawal were observed for KD clones. DNA microarray and quantitative polymerase chain reaction screening for differentially expressed genes between KD clones and non-KD control cells revealed significant up-regulation of the tyrosine kinase receptor, KIT, hyperactive mutations of which are often found in AML. Treatment of KD clones with ISCK03, a KIT-specific inhibitor, eliminated the increased survival of KD clones in the absence of serum.ConclusionsWe suggest that CCDC26 controls growth of myeloid leukemia cells through regulation of KIT expression. A KIT inhibitor might be an effective treatment against the forms of AML in which CCDC26 is altered.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0364-7) contains supplementary material, which is available to authorized users.

No abstract