Phosphorylation of Notch1 by Pim kinases promotes oncogenic signaling in breast and prostate cancer cells

Tumorigenesis is a multistep process involving co-operation between several deregulated oncoproteins. In this study, we unravel previously unrecognized interactions and crosstalk between Pim kinases and the Notch signaling pathway, with implications for both breast and prostate cancer. We identify Notch1 and Notch3, but not Notch2, as novel Pim substrates and demonstrate that for Notch1, the serine residue 2152 is phosphorylated by all three Pim family kinases. This target site is located in the second nuclear localization sequence (NLS) of the Notch1 intracellular domain (N1ICD), and is shown to be important for both nuclear localization and transcriptional activity of N1ICD. Phosphorylation-dependent stimulation of Notch1 signaling promotes migration of prostate cancer cells, balances glucose metabolism in breast cancer cells, and supports in vivo growth of both types of cancer cells on chick embryo chorioallantoic membranes. Furthermore, Pim-induced growth of orthotopic prostate xenografts in mice is associated with enhanced nuclear Notch1 activity. Finally, simultaneous inhibition of Pim and Notch abrogates the cellular responses more efficiently than individual treatments, opening up new vistas for combinatorial cancer therapy.</p

Pyrimidinoceptor potentiation by ATP in NG108-15 cells

AbstractRegulation of inositol phospholipid hydrolysis by UTP and UDP in neuroblastoma×glioma hybrid cell line NG108-15 was potentiated in the presence of ATP. The effect of ATP was dose dependent and shifted the EC50 value for these uracil nucleotides up to three powers of magnitude, having no influence on the maximal value of the response. Adenine nucleotides (ADP, AMP, adenosine 5′-O-(3-thiotriphosphate) (ATPγS), β,γ-methyleneadenosine 5′-triphosphate (βγMeATP), 3′-O-(4-benzoyl)benzoyl ATP (BzATP) and 3′-deoxyadenosine 5′-O-(1-thio)triphosphate (dATPαS)) as well as adenosine, had no influence on the pyrimidinoceptor response. The potentiation effect was abolished by excess of EDTA. The results were in agreement with the hypothesis of pyrimidinoceptor affinity regulation via extracellular phosphorylation of the receptor protein, initiated by ATP. This mechanism may have physiological implication for functioning of uracil nucleotides as endogenous signaling molecules

Almost complete radiationless energy transfer from excited triplet state of a dim phosphor to a covalently linked adjacent fluorescent dye in purely organic tandem luminophores doped into PVA matrix

In solid PVA matrix some thiophene- and selenophene-comprising heteroaromatic compounds reveal moderate room temperature phosphorescence with emission peaks between 550 and 600 nm and possessing lifetime in millisecond range upon excitation with near-UV radiation. In tandem luminophores almost complete intramolecular interchromophore energy transfer takes place by FRET mechanism from the named phosphors in excited triplet state to adjacent covalently linked fluorescent dyes, leading to emission of light from the fluorophore. Variation of length of the linker connecting the chromophores enables tuning of luminescence lifetime in the range from 5 ms to 100 μs. The delayed emission spectrum of the tandem luminophores originates from the acceptor fluorophore. Triplet–singlet energy transfer outperforms other relaxation pathways of the excited triplet state even at 75 °C, making the luminescence lifetime temperature independent. Thus a universal strategy is provided for converting inherently dim organic phosphors into bright long-lifetime luminescence emitters. This discovery may open new avenues for construction of efficient purely organic light emitting devices and photoluminescent sensors for measurement of various analyte

Deactivatable Bisubstrate Inhibitors of Protein Kinases

Bivalent ligands, including bisubstrate inhibitors, are conjugates of pharmacophores, which simultaneously target two binding sites of the biomolecule. Such structures offer attainable means for the development of compounds whose ability to bind to the biological target could be modulated by an external trigger. In the present work, two deactivatable bisubstrate inhibitors of basophilic protein kinases (PKs) were constructed by conjugating the pharmacophores via linkers that could be cleaved in response to external stimuli. The inhibitor ARC-2121 incorporated a photocleavable nitrodibenzofuran-comprising &beta;-amino acid residue in the structure of the linker. The pharmacophores of the other deactivatable inhibitor ARC-2194 were conjugated via reduction-cleavable disulfide bond. The disassembly of the inhibitors was monitored by HPLC-MS. The affinity and inhibitory potency of the inhibitors toward cAMP-dependent PK (PKAc&alpha;) were established by an equilibrium competitive displacement assay and enzyme activity assay, respectively. The deactivatable inhibitors possessed remarkably high 1&ndash;2-picomolar affinity toward PKAc&alpha;. Irradiation of ARC-2121 with 365 nm UV radiation led to reaction products possessing a 30-fold reduced affinity. The chemical reduction of ARC-2194 resulted in the decrease of affinity of over four orders of magnitude. The deactivatable inhibitors of PKs are valuable tools for the temporal inhibition or capture of these pharmacologically important enzymes

Deoxygenation Increases Photoluminescence Lifetime of Protein-Responsive Organic Probes with Triplet–Singlet Resonant Energy Transfer

Cells and bodily fluids possess strong nanosecond-lifetime autofluorescence, therefore photoluminescent probes with microsecond-scale luminescence decay time would be useful for analysis of biological samples, as they allow the performance of measurements in time-resolved (TR) format in a time gate (time window) where the nonspecific background fluorescence has ceased. We have previously disclosed binding-responsive luminescent probes for protein kinases (PKs), ARC-Lum­(Fluo) probes. High brightness of the probes is achieved through intramolecular Förster-type resonant energy transfer (FRET) from excited triplet state of a thiophene- or selenophene-comprising phosphor (³D*) to singlet acceptor dye (¹A) leading to amplified emission from the dye. Here, we determined quantum yields (QYs) and oxygen sensitivity of separate phosphorescent donor and fluorescent acceptor and compared these with those of the corresponding ARC-Lum­(Fluo) probes both in nonbound and PK-bound states. The microsecond-scale luminescence of free and of PK-bound probes was quenched with different efficiency by molecular oxygen and the luminescence intensity of the probes was substantially increased upon deoxygenation. The brightness of an ARC-Lum­(Fluo) probe in PK-bound state was more than 50-fold higher than that of the phosphorescent donor alone. The findings of the study can be used for the construction of bright long-lifetime organic tandem probes

Thiazole- and selenazole-comprising high-affinity inhibitors possess bright microsecond-scale photoluminescence in complex with protein kinase CK2.

A previously disclosed protein kinase (PK) CK2-selective inhibitor 4-(2-amino-1,3-thiazol-5-yl)benzoic acid (ATB) and its selenium-containing counterpart (ASB) revealed remarkable room temperature phosphorescence when bound to the ATP pocket of the protein kinase CK2. Conjugation of these fragments with a mimic of CK2 substrate peptide resulted in bisubstrate inhibitors with increased affinity towards the kinase. Attachment of the fluorescent acceptor dye 5-TAMRA to the conjugates led to significant enhancement of intensity of long-lifetime (microsecond-scale) photoluminescence of both sulfur- and selenium-containing compounds. The developed photoluminescent probes make possible selective determination of the concentration of CK2 in cell lysates and characterization of CK2 inhibitors by means of time-gated measurement of photoluminescence