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Concept: Protein kinases

170

Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) play key roles in physiological and pathological responses in cardiac myocytes. The mechanisms whereby H(2)O(2)-modulated phosphorylation pathways regulate the endothelial isoform of nitric oxide synthase (eNOS) in these cells are incompletely understood. We show here that H(2)O(2) treatment of adult mouse cardiac myocytes leads to increases in intracellular Ca(2+) ([Ca(2+)](i)), and document that activity of the L-type Ca(2+) channel is necessary for the H(2)O(2)-promoted increase in sarcomere shortening and of [Ca(2+)](i). Using the chemical NO sensor Cu(2)(FL2E), we discovered that the H(2)O(2)-promoted increase in cardiac myocyte NO synthesis requires activation of the L-type Ca(2+) channel, as well as phosphorylation of the AMP-activated protein kinase (AMPK), and mitogen-activated protein kinase kinase ½ (MEK1/2). Moreover, H(2)O(2)-stimulated phosphorylations of eNOS, AMPK, MEK1/2, and ERK1/2 all depend on both an increase in [Ca(2+)](i) as well as the activation of protein kinase C (PKC). We also found that H(2)O(2)-promoted cardiac myocyte eNOS translocation from peripheral membranes to internal sites is abrogated by the L-type Ca(2+) channel blocker nifedipine. We have previously shown that kinase Akt is also involved in H(2)O(2)-promoted eNOS phosphorylation. Here we present evidence documenting that H(2)O(2)-promoted Akt phosphorylation is dependent on activation of the L-type Ca(2+) channel, but is independent of PKC. These studies establish key roles for Ca(2+)- and PKC-dependent signaling pathways in the modulation of cardiac myocyte eNOS activation by H(2)O(2).

Concepts: Oxygen, Signal transduction, Heart, Cardiac muscle, Protein kinase, Nitric oxide, Protein kinases, Serine/threonine-specific protein kinase

167

LY2228820 dimesylate is a highly selective small molecule inhibitor of p38α and p38β mitogen activated protein kinases (MAPKs) that is currently under clinical investigation for human malignancies. p38 MAPK is implicated in a wide range of biological processes, in particular those that support tumorigenesis. One such process, angiogenesis, is required for tumor growth and metastasis, and many new cancer therapies are therefore directed against the tumor vasculature. Using an in vitro co-culture endothelial cord formation assay, a surrogate of angiogenesis, we investigated the role of p38 MAPK in growth factor and tumor-driven angiogenesis using LY2228820 dimesylate treatment and by shRNA gene knockdown. p38 MAPK was activated in endothelial cells upon growth factor stimulation with inhibition by LY2228820 dimesylate treatment causing a significant decrease in VEGF, bFGF, EGF, and IL-6 induced endothelial cord formation and an even more dramatic decrease in tumor-driven cord formation. In addition to involvement in downstream cytokine signaling, p38 MAPK was important for VEGF, bFGF, EGF, IL-6 and other proangiogenic cytokine secretion in stromal and tumor cells. LY2228820 dimesylate results were substantiated using p38α MAPK specific shRNA and shRNA against the downstream p38 MAPK effectors MAPKAPK-2 and HSP27. Using in vivo models of functional neoangiogenesis, LY2228820 dimesylate treatment reduced hemoglobin content in a plug assay and decreased VEGF-A stimulated vascularization in a mouse ear model. Thus, p38α MAPK is implicated in tumor angiogenesis through direct tumoral effects and through reduction of proangiogenic cytokine secretion via the microenvironment.

Concepts: Cancer, Signal transduction, Angiogenesis, Enzyme inhibitor, Mitogen-activated protein kinase, Protein kinases, P38 mitogen-activated protein kinases, Mitogen

166

The PACSIN (protein kinase C and casein kinase 2 substrate in neurons) adapter proteins couple components of the clathrin-mediated endocytosis machinery with regulators of actin polymerization and thereby regulate the surface expression of specific receptors. The brain-specific PACSIN 1 is enriched at synapses and has been proposed to affect neuromorphogenesis and the formation and maturation of dendritic spines. In studies of how phosphorylation of PACSIN 1 contributes to neuronal function, we identified serine 358 as a specific site used by casein kinase 2 (CK2) in vitro and in vivo. Phosphorylated PACSIN 1 was found in neuronal cytosol and membrane fractions. This localization could be modulated by trophic factors such as BDNF. We further show that expression of a phospho-negative PACSIN 1 mutant, S358A, or inhibition of CK2 drastically reduces spine formation in neurons. We identified a novel protein complex containing the spine regulator Rac1, its GAP neuron-associated developmentally-regulated protein (NADRIN) and PACSIN 1. CK2 phosphorylation of PACSIN 1 leads to a dissociation of the complex upon BDNF-treatment and induces Rac1-dependent spine formation in dendrites of hippocampal neurons. These findings suggest that upon BDNF signaling PACSIN 1 is phosphorylated by CK2 which is essential for spine formation.

Concepts: Neuron, Signal transduction, Adenosine triphosphate, Protein kinase, Synapse, Dendrite, Protein kinases, Purkinje cell

46

Once melanomas have progressed with acquired resistance to mitogen-activated protein kinase (MAPK)-targeted therapy, mutational heterogeneity presents a major challenge. We therefore examined the therapy phase before acquired resistance had developed and discovered the melanoma survival oncogene MITF as a driver of an early non-mutational and reversible drug-tolerance state, which is induced by PAX3-mediated upregulation of MITF. A drug-repositioning screen identified the HIV1-protease inhibitor nelfinavir as potent suppressor of PAX3 and MITF expression. Nelfinavir profoundly sensitizes BRAF and NRAS mutant melanoma cells to MAPK-pathway inhibitors. Moreover, nelfinavir is effective in BRAF and NRAS mutant melanoma cells isolated from patients progressed on MAPK inhibitor (MAPKi) therapy and in BRAF/NRAS/PTEN mutant tumors. We demonstrate that inhibiting a driver of MAPKi-induced drug tolerance could improve current approaches of targeted melanoma therapy.

Concepts: Gene expression, Signal transduction, Melanoma, Enzyme inhibitor, Inhibitor, Mitogen-activated protein kinase, Extracellular signal-regulated kinases, Protein kinases

29

The causative agent of toxoplasmosis, the intracellular parasite Toxoplasma gondii, delivers a protein, GRA24, into the cells it infects that interacts with the mitogen-activated protein (MAP) kinase p38α (MAPK14), leading to activation and nuclear translocation of the host kinase and a subsequent inflammatory response that controls the progress of the parasite. The purification of a recombinant complex of GRA24 and human p38α has allowed the molecular basis of this activation to be determined. GRA24 is shown to be intrinsically disordered, binding two kinases that act independently, and is the only factor required to bypass the canonical mitogen-activated protein kinase activation pathway. An adapted kinase interaction motif (KIM) forms a highly stable complex that competes with cytoplasmic regulatory partners. In addition, the recombinant complex forms a powerful in vitro tool to evaluate the specificity and effectiveness of p38α inhibitors that have advanced to clinical trials, as it provides a hitherto unavailable stable and highly active form of p38α.

Concepts: Signal transduction, Adenosine triphosphate, Enzyme, Apicomplexa, Mitogen-activated protein kinase, Toxoplasmosis, Toxoplasma gondii, Protein kinases

28

STUDY DESIGN.: An experimental comparative study on moderate epidural hypothermia (MEH) versus moderate systemic hypothermia (MSH) after spinal cord injury (SCI). OBJECTIVE.: To compare neuroprotective effects of hypothermia between MEH and MSH after SCI in rats. SUMMARY OF BACKGROUND DATA.: Experimental MEH or MSH has been attempted for neuroprotection after ischemic or traumatic SCI. However, there is no comparative study on neuroprotective effect of MEH and MSH after SCI. If hypothermia is to be considered as 1 modality for treating SCI, further studies on the advantages and disadvantages of hypothermia will be mandatory. METHODS.: A spinal cord contusion was produced in all 32 rats, and these rats were randomly divided into 4 groups-8 rats in each group: (1) the control group (spinal cord contusion only), (2) the methylprednisolone group, (3) the MEH group (28°C for 48 hr), and (4) the MSH group (32°C for 48 hr). The functional recovery was assessed using Basso, Beattie, Bresnahan scale and antiapoptotic and anti-inflammatory effects were assessed. RESULTS.: The Basso, Beattie, Bresnahan scale scores in both the hypothermia groups were significantly higher than that in the control group at 6 weeks. The numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells and OX-42 positive cells were significantly lower in both the MEH and MSH groups than that in the control group. The p38 mitogen-activated protein kinases expression of the treated groups was significantly lower than that of the control group. The expression of caspase-8 and caspase-9 significantly decreased in the treated groups compared with that of the control group. However, in terms of caspase-3, only the MSH group has shown to be significantly lower than that of the control group. CONCLUSION.: This study presented that both systemic and epidural hypothermia demonstrated neuroprotective effects after SCI. Systemic hypothermia showed more neuroprotective effect by antiapoptotic and anti-inflammatory effects.

Concepts: Scientific method, Signal transduction, Therapeutic hypothermia, Mitogen-activated protein kinase, Protein kinases, Neuroprotection

28

AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. In pancreatic beta cells, glucose induces the dephosphorylation of Thr172 within the catalytic subunit and the inactivation of the AMPK complex. Here we demonstrate that glucose also activates protein kinase A (PKA), leading to the phosphorylation of AMPKα at Ser485 and Ser497. However, these modifications do not impair the phosphorylation of Thr172 by upstream kinases, and phosphorylation of Thr172 does not affect the phosphorylation of AMPKα by PKA either. Thus, although phosphorylation of Thr172 and Ser485/Ser497 are inversely correlated in response to glucose, they follow an independent regulation.

Concepts: Signal transduction, Adenosine triphosphate, Enzyme, Insulin, Kinase, Protein kinase, Protein kinases, Protein kinase A

28

Casein Kinase 2 (CK2) is a ubiquitous kinase protein currently targeted for the treatment of some cancers. Recently, the series of indeno[1,2-b]indoles has revealed great interest as potent and selective CK(2) ATP-competitive inhibitors. Among them, 1-amino-5-isopropyl-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione (CM1) was selected for an encapsulation study in order to improve its biodisponibility. Its complexation was evaluated at the molecular scale, with a series of fluorinated or hydrocarbonated amphiphilic cyclodextrins (CDs). Then the encapsulation of CM1 within CD nanoparticles at the supramolecular level was achieved. Nanoparticles formed between CM1 and hexakis[6-deoxy-6-(3-perfluorohexylpropanethio)-2,3-di-O-methyl]-α-cyclodextrin, a fluorinated amphiphilic α-cyclodextrin, gave the best results in terms of encapsulation rate, stability and drug release. These nanospheres showed an encapsulation efficiency of 65% and a sustained release of the entrapped drug over 3h. Based on these results, encapsulation within fluorinated amphiphilic CD nanoparticles could be considered as a potential drug delivery system for indenoindole-type CK2 inhibitors, allowing better biodisponibility and offering perspectives for tumour targeting development.

Concepts: Protein, Molecular biology, Signal transduction, Supramolecular chemistry, Protein kinases, Cyclodextrin, Casein kinase 1, Casein kinase 2

28

Dendritic cells are a sentinel in defending against pathogens and tick saliva facilitates transmission of tick-borne pathogens by modulating the host immune response. The maturation of dendritic cells is inhibited by tick saliva. To elucidate the mechanism of this inhibition, we tested the impact of Ixodes ricinus tick saliva on signalling pathways activated by Toll-like receptor (TLR-2) ligand and Borrelia afzelii in spleen dendritic cells. The activation of nuclear factor-κB (NF-κB) p65 and phosphatidylinositol-3 kinase (PI3K)/Akt pathways was decreased by tick saliva upon both TLR-2 and Borrelia stimulation. Among the mitogen-activated protein kinases (MAPK), the activation of extracellular matrix-regulated kinase (Erk1/2) was suppressed by tick saliva, but not p38. In response to spirochaetes, the amount of TNF-α decreased in the presence of tick saliva which was mediated by selective suppression of Erk1/2, NF-κB and Akt as tick saliva mimicked the effect of their specific inhibitors, UO126, IKK-IV and LY294002, respectively. Saliva-induced enhancement of IL-10 was not observed in the presence of specific inhibitor of Protein Kinase A (PKA), H-89, suggesting the involvement of PKA pathway in IL-10 production. Our cumulative data show that tick saliva interferes with several signalling pathways, thus modulating the immune functions of dendritic cells.

Concepts: Immune system, Signal transduction, Lyme disease, Protein kinase, Enzyme inhibitor, Dendritic cell, Protein kinases, Serine/threonine-specific protein kinase

27

Transforming growth factor-β-activated kinase 1 (TAK1) is a mitogen-activated protein 3-kinase and an AMP-activated protein kinase (AMPK) kinase in some cell types. Although TAK1(-/-) mice display defects in developmental vasculogenesis, the role of TAK1 in endothelial cells has not been investigated in detail.

Concepts: Gene, Signal transduction, Adenosine triphosphate, Enzyme, Angiogenesis, Endothelium, Protein kinases, Protein kinase A