kn-62 and rottlerin

Chronic exposure of pancreatic beta-cells to saturated fatty acids leads to loss of viability, an effect that has been implicated in the process of beta-cell 'lipotoxicity' associated with the progression of type 2 diabetes. The mechanisms involved are unknown but recent evidence has implicated the delta isoform of protein kinase C (PKCdelta) in mediating fatty acid toxicity. We have investigated this proposition in the clonal insulin-secreting cell line, BRIN-BD11. BRIN-BD11 cells were found to undergo apoptosis when exposed to palmitate and this response was attenuated by the purportedly selective inhibitor of PKCdelta, rottlerin. However, activation of PKCdelta with the phorbol ester, phorbol-12-myristate-13-acetate (PMA), failed to promote cell death and down-regulation of PKCdelta did not prevent the cytotoxic effects of palmitate. Moreover, rottlerin remained effective as a blocker of the palmitate response in cells depleted of PKCdelta. Since rottlerin can inhibit various other kinases in addition to PKCdelta, a range of additional kinase inhibitors was also tested. Of these, only the putative Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) inhibitor, KN-62, was found to inhibit palmitate-induced cell death. However, this effect was not reproduced by a more selective pseudo-substrate inhibitor of CaM kinase II. Therefore, the present results reveal that palmitate induces cell death in BRIN-BD11 cells and suggest that this may involve the activation of a rottlerin (and KN-62)-sensitive kinase. However, it is clear that PKCdelta is not required for this response.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Acetophenones; Animals; Apoptosis; Benzopyrans; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cell Survival; Cells, Cultured; Down-Regulation; Enzyme Inhibitors; Insulin; Insulin Secretion; Islets of Langerhans; Palmitates; Phorbol Esters; Phosphorylation; Protein Kinase C; Protein Kinase C-delta; Signal Transduction

Brain-derived neurotrophic factor (BDNF) plays fundamental roles in synaptic plasticity in rat hippocampus. Recently, using rat hippocampal slices, we found that BDNF induces activation of calcium/calmodulin-dependent protein kinase 2 (CaMKII), a critical mediator of synaptic plasticity. CaMKII in turn activates the p38 subfamily of mitogen-activated protein kinases (MAPK) and its downstream effector, MAPK-activated protein kinase 2 (MAPKAPK-2). Herein, we determined whether some kinases of this pathway connect BDNF to the cyclic AMP response element -binding protein (CREB), a transcription factor also involved in plasticity and survival. Crude cytosolic and nuclear fractions were prepared from hippocampal slices of adult rat, and then kinase involvement in CREB phosphorylation was studied with a combination of pharmacologic inhibition and antibody depletion. In addition, the regional localization of this signaling pathway was immunohistochemically investigated. We show that: (i). the BDNF-stimulated CaMKII cascade phosphorylates the key positive regulatory site of CREB via its end MAPKAPK-2 component; (ii). this process appears to be highly localized in the outermost cell layer of the dentate gyrus. The present findings suggest that CaMKII is involved in neurotrophic-dependent activation of CREB in the dentate gyrus. Such a signaling process could be important for controlling synaptic plasticity in this major area for the afferent inputs to the hippocampal formation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Acetophenones; Alkaloids; Androstadienes; Animals; Antibodies; Benzophenanthridines; Benzopyrans; Blotting, Western; Brain-Derived Neurotrophic Factor; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Carbazoles; Cell Nucleus; Chromones; Cyclic AMP Response Element-Binding Protein; Cytosol; Enzyme Inhibitors; Flavonoids; Hippocampus; Imidazoles; Immunohistochemistry; In Vitro Techniques; Indoles; Intracellular Signaling Peptides and Proteins; Male; Morpholines; Naphthalenes; Phenanthridines; Phosphorylation; Precipitin Tests; Protein Serine-Threonine Kinases; Pyridines; Pyrroles; Rats; Signal Transduction; Time Factors; Tyrphostins; Wortmannin

The specificities of 28 commercially available compounds reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases have been examined against a large panel of protein kinases. The compounds KT 5720, Rottlerin and quercetin were found to inhibit many protein kinases, sometimes much more potently than their presumed targets, and conclusions drawn from their use in cell-based experiments are likely to be erroneous. Ro 318220 and related bisindoylmaleimides, as well as H89, HA1077 and Y 27632, were more selective inhibitors, but still inhibited two or more protein kinases with similar potency. LY 294002 was found to inhibit casein kinase-2 with similar potency to phosphoinositide (phosphatidylinositol) 3-kinase. The compounds with the most impressive selectivity profiles were KN62, PD 98059, U0126, PD 184352, rapamycin, wortmannin, SB 203580 and SB 202190. U0126 and PD 184352, like PD 98059, were found to block the mitogen-activated protein kinase (MAPK) cascade in cell-based assays by preventing the activation of MAPK kinase (MKK1), and not by inhibiting MKK1 activity directly. Apart from rapamycin and PD 184352, even the most selective inhibitors affected at least one additional protein kinase. Our results demonstrate that the specificities of protein kinase inhibitors cannot be assessed simply by studying their effect on kinases that are closely related in primary structure. We propose guidelines for the use of protein kinase inhibitors in cell-based assays.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Acetophenones; Alkaloids; Amides; Animals; Benzamides; Benzophenanthridines; Benzopyrans; Butadienes; Cell Line; Enzyme Inhibitors; Flavonoids; Humans; Imidazoles; Indoles; Inhibitory Concentration 50; Isoquinolines; Lithium; Magnesium; Nitriles; Phenanthridines; Phosphorylation; Potassium Chloride; Protein Kinase Inhibitors; Protein Kinases; Pyridines; Sirolimus; Substrate Specificity; Sulfonamides