Combined elevation of TRIB2 and MAP3K1 indicates poor prognosis and chemoresistance to temozolomide in glioblastoma.

INTRODUCTION: Glioblastoma (GBM) is the most lethal primary malignant brain tumor in adults with poor survival due to acquired therapeutic resistance and rapid recurrence. Currently, the standard clinical strategy for glioma includes maximum surgical resection, radiotherapy, and temozolomide (TMZ) chemotherapy; however, the median survival of patients with GBM remains poor despite these comprehensive therapies. Therefore, the identification of new prognostic biomarkers is urgently needed to evaluate the malignancy and long-term outcome of glioma. AIMS: To further investigate prognostic biomarkers and potential therapeutic targets for GBM. RESULTS: In this study, we identified tribbles pseudokinase 2 (TRIB2) as one of the genes that is most correlated with pathological classification, radioresistance, and TMZ resistance in glioma. Additionally, the expression of mitogen-activated protein kinase kinase kinase 1 (MAP3K1) showed a strong correlation with TRIB2. Moreover, a combined increase in TRIB2 and MAP3K1 was observed in GBM and indicated a poor prognosis of patients with glioma. Finally, enriched TRIB2 expression and MAP3K1 expression were shown to be associated with resistance to TMZ and radiotherapy. CONCLUSION: Combined elevation of TRIB2 and MAP3K1 could be novel prognostic biomarkers and potential therapeutic targets to evaluate the malignancy and long-term outcomes of GBM.

Copper-induced activation of TRP channels promotes extracellular calcium entry and activation of CaMK, PKA, PKC, PKG and CBLPK leading to increased expression of antioxidant enzymes in Ectocarpus siliculosus.

The existence of functional Transient Receptor Potential (TRP) channels was analyzed in Ectocarpus siliculosus using agonists of human TRPs and specific antagonists of TRPA1, TRPC5, TRPM8 and TRPV; intracellular calcium was detected for 60 min. Increases in intracellular calcium were observed at 13, 29, 39 and 50-52 min, which appeared to be mediated by the activation of TRPM8/V1 at 13 min, TRPV1 at 29 min, TRPA1/V1 at 39 min and TRPA1/C5 at 50-52 min. In addition, intracellular calcium increases appear to be due to extracellular calcium entry, not requiring protein kinase activation. On the other hand, 2.5 µM copper exposure induced increased intracellular calcium at 13, 29, 39 and 51 min, likely due to the activation of a TRPA1/V1 at 13 min, TRPA1/C5/M8 at 29 min, TRPC5/M8 at 39 min, and a TRPC5/V1 at 51 min. The increases in intracellular calcium induced by copper were due to extracellular calcium entry and required protein kinase activation. Furthermore, from 3 to 24 h, copper exposure induced an increase in the level of transcripts encoding antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, glutathione reductase and peroxiredoxin. The described upregulation decreased with inhibitors of CaMK, PKA, PKC, PKG and CBLPK, as well as with a mixture of TRP inhibitors. Thus, copper induces the activation of TRP channels allowing extracellular calcium entry as well as the activation of CaMK, PKA, PKC, PKG and CBLPK leading to increased expression of genes encoding antioxidant enzymes in E. siliculosus.

Tribbles 2 mediates cisplatin sensitivity and DNA damage response in epithelial ovarian cancer.

Aim was to identify methylated genes with functional involvement in cisplatin-resistance development of epithelial ovarian cancer (EOC). Genome-wide analyses of hypermethylated CpG-islands in resistant cell lines in combination with qRT-PCR analyses were used to identify epigenetically silenced genes. EOC-Type-II tumors were analyzed for gene methylation and expression and TCGA data were interrogated in-silico. Experiments revealed 37 commonly hypermethylated genes in resistant cells of which Tribbles 2 (TRIB2) showed the most pronounced downregulation on mRNA level and was characterized further. TRIB2 showed a reactivation after 5'-Aza-Cytidine treatment in resistant cells but a cisplatin-dependent, prominent upregulation on mRNA level in sensitive cells, only. Re-expression in resistant A2780 cells increased the sensitivity to cisplatin and other DNA-damaging agents, but not taxanes. Contrary, knockdown of TRIB2 increased resistance to cisplatin in sensitive cells. TRIB2 was involved in the induction of a cisplatin-dependent cell cycle arrest and apoptosis by influencing p21 and survivin expression. An increased Pt-DNA-adduct formation in TRIB2 re-expressing cells did not translate in higher levels of dsDNA damage (yH2AX-foci). Thus, TRIB2 is potentially involved in the signal transduction from nucleotide excision repair of intrastrand cross links. Importantly, patient stratification of two homogenous cohorts of EOC-Type-II patients from Jena (n = 38) and the TCGA (n = 149) by TRIB2 mRNA expression consistently revealed a significantly decreased PFS for patients with low TRIB2 levels (log-rank p < 0.05). Tumors from resistant patients expressed the lowest levels of TRIB2. Downregulation of TRIB2 contributes to platin-resistance and TRIB2 expression should be validated as prognostic and predictive marker for EOC.

Calcium-dependent protein kinases responsible for the phosphorylation of a bZIP transcription factor FD crucial for the florigen complex formation.

Appropriate timing of flowering is critical for reproductive success and necessarily involves complex genetic regulatory networks. A mobile floral signal, called florigen, is a key molecule in this process, and flowering locus T (FT) protein is its major component in Arabidopsis. FT is produced in leaves, but promotes the floral transition in the shoot apex, where it forms a complex with a basic region/leucine-zipper (bZIP) transcription factor, FD. Formation of the florigen complex depends on the supposed phosphorylation of FD; hitherto, however, the responsible protein kinase(s) have not been identified. In this study, we prepared protein extracts from shoot apices of plants around the floral transition, and detected a protein kinase activity that phosphorylates a threonine residue at position 282 of FD (FD T282), which is a crucial residue for the complex formation with FT via 14-3-3. The kinase activity was calcium-dependent. Subsequent biochemical, cellular, and genetic analyses showed that three calcium-dependent protein kinases (CDPKs) efficiently phosphorylate FD T282. Two of them (CPK6 and CPK33) are expressed in shoot apical meristem and directly interact with FD, suggesting they have redundant functions. The loss of function of one CDPK (CPK33) resulted in a weak but significant late-flowering phenotype.

Negative feedback regulation of calcineurin-dependent Prz1 transcription factor by the CaMKK-CaMK1 axis in fission yeast.

Calcium signals trigger the translocation of the Prz1 transcription factor from the cytoplasm to the nucleus. The process is regulated by the calcium-activated phosphatase calcineurin, which activates Prz1 thereby maintaining active transcription during calcium signalling. When calcium signalling ceases, Prz1 is inactivated by phosphorylation and exported to the cytoplasm. In budding yeast and mammalian cells, different kinases have been reported to counter calcineurin activity and regulate nuclear export. Here, we show that the Ca(2+)/calmodulin-dependent kinase Cmk1 is first phosphorylated and activated by the newly identified kinase CaMKK2 homologue, Ckk2, in response to Ca(2+). Then, active Cmk1 binds, phosphorylates and inactivates Prz1 transcription activity whilst at the same time cmk1 expression is enhanced by Prz1 in response to Ca(2+). Furthermore, Cdc25 phosphatase is also phosphorylated by Cmk1, inducing cell cycle arrest in response to an increase in Ca(2+). Moreover, cmk1 deletion shows a high tolerance to chronic exposure to Ca(2+), due to the lack of cell cycle inhibition and elevated Prz1 activity. This work reveals that Cmk1 kinase activated by the newly identified Ckk2 counteracts calcineurin function by negatively regulating Prz1 activity which in turn is involved in activating cmk1 gene transcription. These results are the first insights into Cmk1 and Ckk2 function in Schizosaccharomyces pombe.

Characterization of the low pH/low nutrient-resistant LNCaP cell subline LNCaP-F10.

Intratumoral regions of low extracellular pH and low nutrition are common features of solid tumors. Although cancer cells normally die when they are removed from their environment, a small population of cells survive. In the present study, the subline LNCaP-F10, of the prostate cancer cell line LNCaP, was isolated and its low pH/low nutrient-resistant properties were examined. LNCaP-F10 cells were grown under low-pH/low-nutrient conditions, which caused cell death of the LNCaP cells. The cell death was associated with oligonucleosomal DNA fragmentation and poly (ADP-ribose) polymerase (PARP) cleavage, indicating that low-pH/low-nutrient induced apoptosis in these cells. Significant differences in the expression of BCL2, BIRC5 and DAPK1 were detected between LNCaP-F10 and LNCaP cells. Tumor growth caused by implantation of LNCaP-F10 cells into the renal subcapsular space of nude mice in the absence or presence of prostate stromal cell stimulation was greater than that caused by implantation of LNCaP cells. LNCaP-F10 cells were resistant to apoptosis induced by an environment of low-pH/low-nutrient in vitro, and displayed malignant potential in vivo.

Role of AMP-activated protein kinase in α-lipoic acid-induced vasodilatation in spontaneously hypertensive rats.

BACKGROUND: Adenosine monophosphate (AMP)-activated protein kinase (AMPK) has recently emerged as an attractive and novel target for the regulation of vascular smooth muscle contraction. The present study investigated the vasodilatory effects of α-lipoic acid (α-LA) and the possible mechanism of its action on aortic rings from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). METHODS: Aortae were removed from WKY and SHR, and contractile responses to acetylcholine and α-LA studied in organ chamber. Phosphorylated AMPK (pAMPK), phosphorylated Peutz-Jeghers syndrome kinase LKB1 (pLKB1) and calcium/calmodulin-dependent protein kinase (CaMKK) protein level were measured in SHR, WKY, and aortic smooth muscle (A10) cells. RESULTS: α-LA (1-500 µmol/l) produced a concentration-dependent relaxation of precontracted aortic rings from 8- and 16-week-old SHR, but not in those from WKY rats. This vasodilatory effect of α-LA did not change after preincubation with N(G)-nitro-L-arginine methyl ester (100 µmol/l), but significantly suppressed by an AMPK inhibitor, compound C (40 µmol/l). The expression of pAMPKα, pLKB1, and CaMKK were also significantly reduced in endothelium-denuded arteries from 16-week-old SHR compared with those from younger SHR or age-matched WKY rats. After incubation with α-LA (100 µmol/l), the expression of pAMPKα and pLKB1 was significantly increased in the endothelium-denuded aortas from 16-week-old SHR, the expression of CaMKK was more reduced in the endothelium-denuded aortas of 8-week-old SHR, but this was not observed in WKY rats. α-LA also activated AMPKα phosphorylation in A10 cells. CONCLUSIONS: The effects of α-LA on vascular relaxation in SHR result from the enhanced phosphorylation of LKB1-AMPK in aortic smooth muscle.

Genetic variation predicting cisplatin cytotoxicity associated with overall survival in lung cancer patients receiving platinum-based chemotherapy.

PURPOSE: Inherited variability in the prognosis of lung cancer patients treated with platinum-based chemotherapy has been widely investigated. However, the overall contribution of genetic variation to platinum response is not well established. To identify novel candidate single nucleotide polymorphisms (SNP)/genes, we carried out a genome-wide association study (GWAS) for cisplatin cytotoxicity by using lymphoblastoid cell lines (LCL), followed by an association study of selected SNPs from the GWAS with overall survival (OS) in lung cancer patients. EXPERIMENTAL DESIGN: A GWAS for cisplatin was conducted with 283 ethnically diverse LCLs. A total of 168 top SNPs were genotyped in 222 small cell lung cancer (SCLC) and 961 non-SCLC (NSCLC) patients treated with platinum-based therapy. Association of the SNPs with OS was determined by using the Cox regression model. Selected candidate genes were functionally validated by siRNA knockdown in human lung cancer cells. RESULTS: Among 157 successfully genotyped SNPs, 9 and 10 SNPs were top SNPs associated with OS for patients with NSCLC and SCLC, respectively, although they were not significant after adjusting for multiple testing. Fifteen genes, including 7 located within 200 kb up or downstream of the 4 top SNPs and 8 genes for which expression was correlated with 3 SNPs in LCLs were selected for siRNA screening. Knockdown of DAPK3 and METTL6, for which expression levels were correlated with the rs11169748 and rs2440915 SNPs, significantly decreased cisplatin sensitivity in lung cancer cells. CONCLUSIONS: This series of clinical and complementary laboratory-based functional studies identified several candidate genes/SNPs that might help predict treatment outcomes for platinum-based therapy of lung cancer.

An experimental study on the therapy of infantile hemangioma with recombinant interferon γ.

BACKGROUND/PURPOSE: The purpose of this investigation was to study the effect of interferon γ (IFN-γ) in the treatment of infantile hemangioma. METHODS: Hemangioma tissue excised from a 4-month-old female infant who underwent surgery were separated into small nubs (4 × 4 × 5mm(3)) and implanted subcutaneously into nude mice (2 nubs per mouse). Thirty-two surviving hemangioma nubs were randomly divided into 2 groups, an IFN-γ-administered group (16) and a control group (16). Interferon γ or saline solution was injected subcutaneously, and the growth of hemangioma in the nude mice was monitored. Proliferation and apoptosis of hemangioma were tested by immunohistochemistry and real-time polymerase chain reaction. RESULTS: Seven days after IFN-γ injection, the hemangiomas in the IFN-γ-administered group were significantly smaller than that in the control group (P < .01). The proliferation cytokine Ki-67 mRNA in the IFN-γ group was significantly lower than that in the control group (P < .05). DAPK-1 mRNA in the IFN-γ group was significantly higher than that in the control group (P < .05). Cell apoptosis expression in the IFN-γ group was significantly more than that in controls (P < .05). CONCLUSIONS: Exogenous IFN-γ can treat hemangioma effectively in a nude mice model. Its mechanism was closely related to both inhibition of hemangioma proliferation and acceleration of its apoptosis.

Tissue Factor/ FVIIa prevents the extrinsic pathway of apoptosis by regulation of the tumor suppressor Death-Associated Protein Kinase 1 (DAPK1).

INTRODUCTION: This study determines the impact of tissue factor (TF)-signaling on the extrinsic pathway of apoptosis in cancer cells and propose death associated protein kinase-1 (DAPK1) as a novel key regulator. MATERIALS AND METHODS: In MDA-MB-231 breast and PC3 prostate cancer cells, mRNA levels were analyzed by real-time PCR and protein expressions were assessed by flow cytometry or western blot. Caspase-8 and -3 levels, cell size, and changes in nuclear morphology were recorded using the ArrayScan microscope and 84 apoptosis-related genes were screened with the RT2 Profiler™ PCR Array. RESULTS: In serum starved MDA-MB-231 cells, a TF/FVIIa-sensitive upregulation of apoptosis markers was recorded. Similarly, TRAIL-induced apoptosis was negatively regulated by TF/FVIIa (10 and 100 nM) and TF/FVIIa/FXa but not by active-site inhibited FVIIa. FVIIa, moreover, decreased the transcription of DAPK1 and thereby diminished the association between DAPK1 and FADD in the caspase-8 activating death-inducing signaling complex (DISC). TF/FVIIa regulation of caspase-8 and DAPK1 was dependent on PI3-kinase/AKT and independent of the protease activated receptors (PAR) 1 and 2. Despite of receptor expression and functional signaling, both PAR-agonist treatment and PAR-blocking antibodies in combination with FVIIa failed to influence the anti-apoptotic signal. CONCLUSIONS: We hereby report that TF/FVIIa-induced signaling governs the extrinsic pathway of apoptosis by reducing the levels of DAPK1 in the DISC independently of PAR1 and PAR2. This implies the conceivable involvement of cell surface components other than the PARs and entails the search for TF-dependent regulators of DAPK1 transcription.

Calcium and secondary CPK signaling in plants in response to herbivore attack.

Plant Ca(2+) signals are involved in a sizable array of intracellular signaling pathways after pest invasion. Upon herbivore feeding there is a dramatic Ca(2+) influx, followed by the activation of Ca(2+)-dependent signal transduction pathways that include interacting downstream networks of kinases for defense responses. Notably, Ca(2+)-binding sensory proteins such as Ca(2+)-dependent protein kinases (CPKs) have recently been documented to mediate the signaling following Ca(2+) influx after herbivory, in phytohormone-independent manners. Here, we review the sequence of signal transductions triggered by herbivory-evoked Ca(2+) signaling leading to CPK actions for defense responses, and discuss in a comparative way the involvement of CPKs in the signal transduction of a variety of other biotic and abiotic stresses.

Suppressed protein expression of the death-associated protein kinase enhances 5-fluorouracil-sensitivity but not etoposide-sensitivity in human endometrial adenocarcinoma cells.

Targeted knockdown of the death-associated protein kinase (DAPK) expression in the endometrial adenocarcinoma HHUA cells reportedly induces cell death by enhancing the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in an autocrine/paracrine manner. This suggests that endogenous DAPK is a potential candidate for a molecularly targeted anticancer therapy for patients with endometrial adenocarcinoma. To investigate the role of endogenous DAPK in anticancer drug sensitivity, we examined effects on cellular anticancer drug sensitivities of transfections with 5 different specific DAPK small-interfering RNAs (siRNAs) into HHUA cells. DAPK siRNA transfections strongly enhanced 5-fluorouracil (5FU)-sensitivity, but not etoposide-sensitivity, of HHUA cells compared with control siRNA-transfected cells. These results indicate that etoposide-stimulated cell death signals may share or include TRAIL-mediated apoptotic signals, and that 5FU-stimulated cell death signals may be independent from TRAIL-mediated apoptotic signals induced by DAPK siRNA transfections. Moreover, 5FU-combined chemotherapy with DAPK siRNA transfection may show stronger anticancer effects on patients with endometrial adenocarcinoma than does chemotherapy alone.

Death-associated protein kinase is underexpressed in high-grade oral squamous cell carcinoma / La expresión de la proteína quinasa asociada a muerte celular (DAP quinasa) está reducida en carcinoma de células escamosas oral de alto grado

An immunohistochemical analysis of 40 cases of oral squamous cell carcinoma was performed to evaluate the relationship between the expression pattern of death-associated protein kinase (DAPk) positive cells with the histological malignancy grading of these lesions. According to our results, eleven cases (27.5 percent) were high-grade malignancy tumours and 29 (72.5 percent) were low-grade ones. We found that 92.86 percent of the low-grade tumours were positive to anti-DAP kinase antibody whereas only 7.14 percent of the high-grade tumours presented positivity, and this difference was statistically significant (p<0.01). Sixteen (55.2 percent) of the low-grade carcinomas exhibited moderate immunoreactivity whereas ten cases (34.5 percent) showed weak staining and three cases (10.3 percent) were negative tumours. Immunostaining was lacking in nine (81.8 percent) of the high-grade carcinomas and "weak" in the two (18.2 percent) remaining cases. Thus, DAPk expression is significantly decreased in high-grade oral carcinomas, and evidences indicate that it might be related to the severity of cytological atypia.

Fue realizado un análisis inmunohistoquímico de 40 casos de carcinoma oral de células escamosas para evaluar la relación entre el patrón de expresión de la proteína quinasa (DAPK) asociada a la muerte celular y la clasificación histológica de malignidad de estas lesiones. Según nuestros resultados, 11 casos (27,5 por ciento) eran tumores de alto grado de malignidad y 29 (72,5 por ciento) de bajo grado. Se encontró que 92,86 por ciento de los tumores de bajo grado de malignidad fueron eran positivos para anticuerpos anti-DAP-quinasa, mientras que sólo 7,14 por ciento de los tumores de alto grado presentaron positividad, esta diferencia fue estadísticamente significativa (p <0,01). En 16 casos (55,2 por ciento) los carcinomas de bajo grado de malignidad mostraron inmunorreactividad moderada mientras que 10 casos (34,5 por ciento) mostraron una tinción débil y 3 casos (10,3 por ciento) fueron negativos. La inmunotinción estuvo ausente en 9 (81,8 por ciento) de los carcinomas de alto grado y "débil" grado de malignidad en los 2 (18,2 por ciento) casos restantes. Así, la expresión DAPK se redujo significativamente en los carcinomas orales de alto grado y las evidencias indican que podría estar relacionado con la severidad de la atipia citológica.

Establishment and characterization of multidrug-resistant gastric cancer cell lines.

AIM: The aim of this study was to establish drug-resistant cell lines and to elucidate mechanisms leading to multi-drug resistance in gastric cancer. MATERIALS AND METHODS: Five cancer cell lines resistant to 5-fluorouracil, paclitaxel, oxaliplatin, irinotecan, or gemcitabine, were respectively established from a parent gastric cancer cell line, OCUM-2M, by stepwise exposure to each chemotherapeutical agent. RESULTS: Cell death by apoptosis induced by anti-cancer drugs was low in 5 chemo-resistant cell lines. Percentage of cells in S and G(0)/G(1) phase was low in cell lines resistant to oxaliplatin or irinotecan. Cell lines resistant to paclitaxel, oxaliplatin, and gemcitabine showed multi-drug resistance. Alterations in MRP, DAPK1, or DAPK2 expression were found in multi-drug resistant cell lines. CONCLUSION: The cell-cycle distribution and alterations of MRP, DAPK1, and DAPK2 genes may be integral part of mechanisms responsible for chemo-resistance. These cell lines might be useful to study molecular mechanisms leading to multi-drug resistance.

Death-associated protein kinase (DAPK1) in cerebral cortex of late-onset Alzheimer's disease patients and aged controls.

AIMS: Here our objective was to detect the pro-apoptotic serine/threonine kinase death-associated protein kinase (DAPK1) in aged human cerebral cortex and to test the hypothesis that DAPK1 abundance is associated with late-onset Alzheimer's disease (AD). METHODS: Using Western analysis and immunohistochemistry we evaluated post mortem frontal cerebral cortex from patients with severe AD (mean age 76 years, range 66-91, n = 11, all male), and from control cases without serious central nervous system illness (mean age 77 years, range 61-95, n = 12, all male). We also examined brains of Tg2576 transgenic mice (males, aged 16-21 months), a model for chronic amyloid-induced brain injury. RESULTS: Immunohistochemical labelling showed DAPK1 expression in cortical neurones of human cortex and axonal tracts within subcortical white matter, both in AD and in control brains. Western analysis confirmed DAPK1 expression in all samples, although expression was very low in some control cases. DAPK1 abundance in the AD group was not significantly different from that in controls (P = 0.07, Mann-Whitney test). In brains of Tg2576 mice DAPK1 abundance was very similar to that in wild-type littermates (P = 0.96, Mann-Whitney test). CONCLUSION: We found that DAPK1 was expressed in neurones of aged human frontal cortex, both in AD and in control cases.

Novel Ca2+/calmodulin-dependent protein kinase expressed in actively growing mycelia of the basidiomycetous mushroom Coprinus cinereus.

We isolated cDNA clones for novel protein kinases by expression screening of a cDNA library from the basidiomycetous mushroom Coprinus cinereus. One of the isolated clones was found to encode a calmodulin (CaM)-binding protein consisting of 488 amino acid residues with a predicted molecular weight of 53,906, which we designated CoPK12. The amino acid sequence of the catalytic domain of CoPK12 showed 46% identity with those of rat Ca2+/CaM-dependent protein kinase (CaMK) I and CaMKIV. However, a striking difference between these kinases is that the critical Thr residue in the activating phosphorylation site of CaMKI/IV is replaced by a Glu residue at the identical position in CoPK12. As predicted from its primary sequence, CoPK12 was found to behave like an activated form of CaMKI phosphorylated by an upstream CaMK kinase, indicating that CoPK12 is a unique CaMK with different properties from those of the well-characterized CaMKI, II, and IV. CoPK12 was abundantly expressed in actively growing mycelia and phosphorylated various proteins, including endogenous substrates, in the presence of Ca2+/CaM. Treatment of mycelia of C. cinereus with KN-93, which was found to inhibit CoPK12, resulted in a significant reduction in growth rate of mycelia. These results suggest that CoPK12 is a new type of multifunctional CaMK expressed in C. cinereus, and that it may play an important role in the mycelial growth.

Distinct developmental expression of two isoforms of Ca2+/calmodulin-dependent protein kinase kinases and their involvement in hippocampal dendritic formation.

Ca(2+)/calmodulin-dependent protein kinase kinases (CaMKKs) are upstream protein kinases that phosphorylate and activate CaMKI and CaMKIV, both of which are involved in a variety of neuronal functions. Here, we first demonstrated that the two isoforms of CaMKK were differentially expressed during neural development by in situ hybridization. We also demonstrated that both dominant negative and pharmacological interference with CaMKK inhibitor, STO-609 resulted in a significant decrease in the number of primary dendrites of cultured hippocampal neurons. Our present findings provide the detailed anatomical information on the developmental expression of CaMKKs and the functional involvement of CaMKK in the formation of primary dendrites.

Tubulin is a neuronal target of autoantibodies in Sydenham's chorea.

Sydenham's chorea is a CNS disorder and sequela of group A streptococcal infection where deposition of Abs in brain may result in movement and neuropsychiatric abnormalities. We studied human mAbs 24.3.1, 31.1.1, and 37.2.1 derived from chorea and selected for cross-reactivity with group A streptococci and brain Ags. Our novel findings reveal that Sydenham's chorea mAbs target a 55-kDa brain protein with an N-terminal amino acid sequence of MREIVHLQ corresponding to beta-tubulin. Chorea mAb specificity for purified brain tubulin was confirmed in ELISA and Western immunoblot, and significant levels of anti-tubulin IgG were found in acute chorea sera and cerebrospinal fluid. Lysoganglioside G(M1) inhibited binding of chorea mAbs to tubulin and mAb reactivity with human caudate and putamen brain sections was blocked by anti-tubulin mAb. The chorea mAbs labeled both intra- and extracellular Ags of a neuronal cell line providing evidence suggesting mimicry between intracellular brain protein tubulin and extracellular lysoganglioside. In addition, chorea mAb 24.3.1 and acute chorea sera induced calcium/calmodulin-dependent protein kinase II activity in human neuronal cells. Nucleotide sequence analysis of the chorea mAb V(H) genes revealed that mAb 24.3.1 V(H) gene was encoded by the V(H)1 germline gene family which encodes other anti-ganglioside V(H) genes associated with motor neuropathies. mAb recognition of tubulin and the neuronal cell surface with initiation of cell signaling and dopamine release supports an emerging theme in autoimmunity whereby cross-reactive or polyreactive autoantibodies against intracellular Ags recognize cell surface epitopes potentially leading to disease.

CaMKII regulates retinoic acid receptor transcriptional activity and the differentiation of myeloid leukemia cells.

Retinoic acid receptors (RARs) are members of the nuclear hormone receptor family and regulate the proliferation and differentiation of multiple different cell types, including promyelocytic leukemia cells. Here we describe a biochemical/functional interaction between the Ca(2+)/calmodulin-dependent protein kinases (CaMKs) and RARs that modulates the differentiation of myeloid leukemia cells. We observe that CaMKIIgamma is the CaMK that is predominantly expressed in myeloid cells. CaMKII inhibits RAR transcriptional activity, and this enzyme directly interacts with RAR through a CaMKII LxxLL binding motif. CaMKIIgamma phosphorylates RARalpha both in vitro and in vivo, and this phosphorylation inhibits RARalpha activity by enhancing its interaction with transcriptional corepressors. In myeloid cell lines, CaMKIIgamma localizes to RAR target sites within myeloid gene promoters but dissociates from the promoter upon retinoic acid-induced myeloid cell differentiation. KN62, a pharmacological inhibitor of the CaMKs, enhances the terminal differentiation of myeloid leukemia cell lines, and this is associated with a reduction in activated (autophosphorylated) CaMKII in the terminally differentiating cells. These observations reveal a significant cross-talk between Ca(2+) and retinoic acid signaling pathways that regulates the differentiation of myeloid leukemia cells, and they suggest that CaMKIIgamma may provide a new therapeutic target for the treatment of certain human myeloid leukemias.

The death-associated protein kinase 2 is up-regulated during normal myeloid differentiation and enhances neutrophil maturation in myeloid leukemic cells.

The death-associated protein kinase 2 (DAPK2) belongs to a family of Ca(2+)/calmodulin-regulated serine/threonine kinases involved in apoptosis. During investigation of candidate genes operative in granulopoiesis, we identified DAPK2 as highly expressed. Subsequent investigations demonstrated particularly high DAPK2 expression in normal granulocytes compared with monocytes/macrophages and CD34(+) progenitor cells. Moreover, significantly increased DAPK2 mRNA levels were seen when cord blood CD34(+) cells were induced to differentiate toward neutrophils in tissue culture. In addition, all-trans retinoic acid (ATRA)-induced neutrophil differentiation of two leukemic cell lines, NB4 and U937, revealed significantly higher DAPK2 mRNA expression paralleled by protein induction. In contrast, during differentiation of CD34(+) and U937 cells toward monocytes/macrophages, DAPK2 mRNA levels remained low. In primary leukemia, low expression of DAPK2 was seen in acute myeloid leukemia samples, whereas chronic myeloid leukemia samples in chronic phase showed intermediate expression levels. Lentiviral vector-mediated expression of DAPK2 in NB4 cells enhanced, whereas small interfering RNA-mediated DAPK2 knockdown reduced ATRA-induced granulocytic differentiation, as evidenced by morphology and neutrophil stage-specific maturation genes, such as CD11b, G-CSF receptor, C/EBPepsilon, and lactoferrin. In summary, our findings implicate a role for DAPK2 in granulocyte maturation.