In Drosophila, the transcription factor cAMP response element-binding protein 2 (dCREB2) has been reported to modulate the formation of long-term olfactory memory (LTM). Overexpression of a repressor isoform of CREB (dCREB2-b) under the control of a heat-shock promoter was reported to block LTM, whereas overexpression of an activator isoform (dCREB2-a) was reported to enhance LTM. A ratiometric model based on these results predicts that the balance of functional dCREB2-a and dCREB2-b provides a switch for memories to remain labile or to become enduring. We show here that the dCREB2-a transgene originally reported to enhance LTM carries a mutation that produces a translational reading-frame shift with the consequent formation of a stop codon at predicted amino acid position 79. Overexpression of this mutant dCREB2-a transgene or a corrected dCREB2-a transgene failed to show any enhancement of LTM. Overexpression of the dCREB2-b repressor transgene, in contrast, produced the anticipated block in LTM formation. We discuss the implications of these findings and propose an alternative model for the role of dCREB in Drosophila LTM.

Despite the success of imatinib mesylate (IM) in the early chronic phase of chronic myeloid leukemia (CML), patients are resistant to IM and other kinase inhibitors in the later stages of CML. Our findings indicate that inhibition of Janus kinase 2 (Jak2) in Bcr–Abl+ cells overcomes IM resistance although the precise mechanism of Jak2 action is unknown. Knocking down Jak2 in Bcr–Abl+ cells reduced levels of the Bcr–Abl protein and also the phosphorylation of Tyr177 of Bcr–Abl, and Jak2 overexpression rescued these knockdown effects. Treatment of Bcr–Abl+ cells with Jak2 inhibitors for 4–6 h but not with IM also reduced Bcr–Abl protein and pTyr177 levels. In vitro kinase experiments performed with recombinant Jak2 showed that Jak2 readily phosphorylated Tyr177 of Bcr–Abl (a Jak2 consensus site, YvnV) whereas c-Abl did not. Importantly, Jak2 inhibition decreased pTyr177 Bcr–Abl in immune complexes but did not reduce levels of Bcr–Abl, suggesting that the reduction of Bcr–Abl by Jak2 inhibition is a separate event from phosphorylation of Tyr177. Jak2 inhibition by chemical inhibitors (TG101209/WP1193) and Jak2 knockdown diminished the activation of Ras, PI-3 kinase pathways and reduced levels of pTyrSTAT5. These findings suggest that Bcr–Abl stability and oncogenic signaling in CML cells are under the control of Jak2.

The phototaxis receptor sensory rhodopsin I (SRI) transmits signals through a membrane-bound transducer protein, HtrI. The genes for the receptor and transducer, sopI and htrI, respectively, are normally cotranscribed; however, previous work has established that fully functional interacting proteins are produced when htrI is expressed from the chromosome and sopI is expressed from a different promoter on a plasmid. In this report we show that in the membrane, concentrations of SRI from plasmid expression of wild-type sopI are negligible in the absence of HtrI protein in the cell. This requirement for HtrI is eliminated when sopI is extended at the 5-end with 63 nucleotides of the bop gene, which encodes the N-terminal signal sequence of the bacteriorhodopsin protein. The signal is cleaved from the chimeric protein, and processed SRI is stable in the HtrI-free membrane. These results suggest a chaperone-like function for HtrI that facilitates membrane insertion or proper folding of the SRI protein. Six deletion constructs of HtrI were examined to localize the interaction sites for its putative chaperone function and for HtrI control of the SRI photocycle, a phenomenon described previously. The smallest HtrI fragment identified, which contained interaction sites for both SRI stability and photocycle control, consisted of the N-terminal 147 residues of the 536-residue HtrI protein. The active fragment is predicted to contain two transmembrane helices and the first ϳ20% of the cytoplasmic portion of the protein.The integral membrane protein HtrI (22) transmits signals from the membrane-embedded phototaxis receptor sensory rhodopsin I (SRI) (18) to a cytoplasmic signal transduction pathway (14) that controls the flagellar motors of Halobacterium salinarium cells (for reviews, see references 13, 16, and 19). The primary sequence of HtrI predicts two transmembrane helices (TM1 and TM2) within the N-terminal ϳ60 residues, and these are followed by a hydrophilic cytoplasmic domain of ϳ480 residues. The C-terminal ϳ260 residues are homologous to the adaptation and signaling regions of eubacterial chemotaxis receptors (2). SRI photoreactions involving proton transfer reactions in the SRI photoactive site are altered when HtrI is genetically deleted from the membrane (15,17). This effect indicates that SRI and HtrI are physically associated in the cell, which is strongly supported by the isolation of a molecular complex of purified SRI and HtrI from H. salinarium membranes (14a). In the present work we report an additional influence of HtrI on SRI, namely, the facilitation of SRI insertion in a stable form in the membrane.The effects of HtrI on the SRI photocycle are readily measured by flash spectroscopy, providing an assay for receptortransducer interaction. In particular, HtrI-free SRI exhibits a strongly pH-dependent photocycle, which becomes pH insensitive in the SRI-HtrI complex (15). The pH sensitivity of the photocycle and the newly reported stabilization effects are exploited in this communication to determine from...

The two transducers in the phototaxis system of the archaeonHalobacterium salinarum, HtrI and HtrII, are methyl-accepting proteins homologous to the chemotaxis transducers in eubacteria. Consensus sequences predict three glutamate pairs containing potential methylation sites in HtrI and one in HtrII. Mutagenic substitution of an alanine pair for one of these, Glu265-Glu266, in HtrI and for the homologous Glu513-Glu514 in HtrII eliminated methylation of these two transducers, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autofluorography. Photostimulation of the repellent receptor sensory rhodopsin II (SRII) induced reversible demethylation of HtrII, while no detectable change in the extent of methylation of HtrI was observed in response to stimulation of its cognate sensory rhodopsin, the attractant receptor SRI. Cells containing HtrI or HtrII with all consensus sites replaced by alanine still exhibited phototaxis responses and behavioral adaptation, and methanol release assays showed that methyl group turnover was still induced in response to photostimulation of SRI or SRII. By pulse-chase experiments with in vivol-[methyl-3H]methionine-labeled cells, we found that repetitive photostimulation of SRI complexed with wild-type (or nonmethylatable) HtrI induced methyl group turnover in transducers other than HtrI to the same extent as in wild-type HtrI. Both attractant and repellent stimuli cause a transient increase in the turnover rate of methyl groups in wild-type H. salinarum cells. This result is unlike that obtained withEscherichia coli, in which attractant stimuli decrease and repellent stimuli increase turnover rate, and is similar to that obtained with Bacillus subtilis, which also shows turnover rate increases regardless of the nature of the stimulus. We found that a CheY deletion mutant of H. salinarum exhibited theE. coli-like asymmetric pattern, as has recently also been observed in B. subtilis. Further, we demonstrate that the CheY-dependent feedback effect does not require the stimulated transducer to be methylatable and operates globally on other transducers present in the cell.

Background: The success of tyrosine kinase inhibitors (TKIs) depends on the addiction of Philadelphia-positive (Ph+) CML progenitors to BCR-ABL1 kinase activity. However, CML quiescent hematopoietic stem cells (HSC) are TKI-resistant and represent an active disease reservoir. We hypothesize that this innate drug-resistance depends on inhibition of the tumor suppressor protein phosphatase 2A (PP2A). PP2A can be reactivated by FTY720, a drug that targets CML but not normal progenitors. Here we investigated the mechanism controlling survival/self-renewal of quiescent leukemic HSCs and their sensitivity to PP2A-activating drugs. Methods: HSCs from CML (n=68) and healthy (n=12) donors were FACS-isolated, and the biologic importance of PP2A inhibition and pharmacologic PP2A activation on their survival/self-renewal was assessed by BM serial transplantation; CFSE and Annexin-V staining; LTC-IC and CFC/replating assays; lentiviral shRNA/cDNA-transduction; LEF/TCF and proximity-ligation assays; Western blot, confocal microscopy and FACS analyses. Results: We observed increased BCR-ABL1 expression with impaired kinase activity in quiescent CML HSCs, in which BCR-ABL1 per se is required for induction of JAK2 that subsequently activated β-catenin and inhibited PP2A. In fact, PP2A was suppressed in CML but not normal CD34+/CD38−/CD90+ HSCs. FTY720 and/or its non-immunosuppressive (S)-FTY720-OMe derivative markedly reduced survival and self-renewal of CML but not normal quiescent HSCs through BCR-ABL1 kinase-independent and PP2A-mediated JAK2 and β-catenin inhibition. Importantly, FTY720 also strongly diminished BCR-ABL1+ LT-HSC frequency in serial BM transplantation assays. Conclusions: The pharmacologic targeting of the newly-identified BCR-ABL1 kinase-independent JAK2/β-catenin interplay in quiescent HSCs with FTY720 and its derivatives, might lead to cessation of lifelong patient dependence on TKIs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-109. doi:10.1158/1538-7445.AM2011-LB-109