Kinase Cell Signaling Research Articles

Regulation of phosphoglucomutase 1 phosphorylation and activity by a signaling kinase.

We have identified a novel mechanism of cross-talk between cell signaling and metabolic pathways, whereby the signaling kinase p21-activated kinase 1 (Pak1) binds to, phosphorylates and enhances the enzymatic activity of phosphoglucomutase 1 (PGM), an important regulatory enzyme in cellular glucose utilization and energy homeostasis. Pak1 and PGM were colocalized in model cell systems and showed functional interactions in a physiological setting. Strong direct interaction of PGM with Pak1 but not Pak2, Pak3, or Pak4 was observed. PGM binding was within 75-149 amino acids (aa) of Pak1, while Pak1 binding to PGM was in the N-terminal 96 aa. Pak1-mediated phosphorylation of PGM selectively on threonine 466 significantly increased PGM enzymatic activity and could be blocked by transfection with a dominant-negative Pak1 expression vector and by Pak1-specific small inhibitory RNA. Stable transfection of PGM into PGM-deficient K562 leukemia cells further demonstrated the role of Pak1 in regulating PGM activity. The results presented here provide new evidence that the cell signaling kinase Pak1 is a novel regulator of glucose metabolism through its phosphorylation and regulation of PGM activity. These findings suggest a new mechanism whereby growth factor signaling may coordinately integrate metabolic regulation with established signaling functions of cell cycle regulation and cell growth.

Aspects of the early moments of interaction between tachyzoites of Toxoplasma gondii with neutrophils

The interaction of the tachyzoite stage of Toxoplasma gondii with neutrophils was investigated. Morphological aspects of the initial moments of interaction were analyzed by transmission and scanning electron microscopy, revealing at least three types of reaction from the leukocytes to the parasite: the projection of filopodia, formation of a tunnel-like projection involving the parasite, and invagination of the leukocyte surface at the point of entry. The influence on infectivity of tyrosine kinase, phosphokinase C and phosphatidylinositol 3 kinase cell signaling pathways were studied with the aid of drugs affecting these enzymes in these cells.

Inhibition of UVB-Induced Oxidative Stress-Mediated Phosphorylation of Mitogen-Activated Protein Kinase Signaling Pathways in Cultured Human Epidermal Keratinocytes by Green Tea Polyphenol (−)-Epigallocatechin-3-gallate

Exposure of normal human epidermal keratinocytes (NHEK) to UVB radiation induces intracellular release of hydrogen peroxide (oxidative stress) and phosphorylation of mitogen-activated protein kinase cell signaling pathways. Here, we demonstrate that pretreatment of NHEK with (−)-epigallocatechin-3-gallate (EGCG), an antioxidant from green tea, inhibits UVB-induced hydrogen peroxide (H2O2) production and H2O2-mediated phosphorylation of MAPK signaling pathways. We found that treatment of EGCG (20 μg/ml of media) to NHEK before UVB (30 mJ/cm2) exposure inhibited UVB-induced H2O2 production (66–80%) concomitant with the inhibition of UVB-induced phosphorylation of ERK1/2 (57–80%), JNK (53–83%), and p38 (50–77%) proteins. To demonstrate whether UVB-induced phosphorylation of MAPK occurs via UVB-induced H2O2 (oxidative stress) production, NHEK were treated with the oxidant H2O2. Treatment of H2O2 to NHEK resulted in phosphorylation of ERK1/2, JNK, and p38. Using the same in vitro system, when these cells were pretreated with EGCG or with the known antioxidant ascorbic acid (as positive control), H2O2-induced phosphorylation of ERK1/2, JNK, and p38 was found to be significantly inhibited. These findings demonstrate that EGCG has the potential to inhibit UVB-induced oxidative stress-mediated phosphorylation of MAPK signaling pathways, suggesting that EGCG could be useful in attenuation of oxidative stress-mediated and MAPK-caused skin disorders in humans.

Novel drugs for treating asthma.

The health burden of asthma is increasing globally at an alarming rate, providing a strong impetus for the development of new therapeutics, particularly drugs that may prevent development of the disease. Currently available inhaled bronchodilators and anti-inflammatory drugs are effective in most asthmatic patients, but this palliative therapy requires long-term daily administration. Despite considerable efforts by the pharmaceutical industry, it has been difficult to develop novel therapeutic agents, the leukotriene antagonists being the only new class of asthma treatments to be licensed in the past 30 years. It is clearly important to understand more about the underlying mechanisms of asthma and about how currently used drugs work before rational improvements in therapy can be expected. There are numerous therapies in clinical development that combat the inflammation found in asthma, specifically targeting eosinophils, IgE, adhesion molecules, cytokines (interleukin-4, -5, -13) and chemokines, inflammatory mediators, and cell signaling (kinase inhibitors). In particular, there is the obvious need for new therapy for severe asthma that is poorly controlled by high-dose corticosteroids as well as agents to counter acute emergency asthma. A long-term goal is to develop disease-modifying immunotherapy that could be introduced in childhood to alter the natural history of asthma. Thanks to the extensive efforts of the pharmaceutical industry, we can expect the introduction of a range of novel therapies for asthma in the near future.

Tyrosine Kinase Cell Signaling Pathways of Rat Mesangial Cells in 3-Dimensional Cultures: Response to Fetal Bovine Serum and Platelet-Derived Growth Factor-BB

Cells grown in 3-dimensional collagen gels adopt a nonproliferative, contractile phenotype which is more characteristic of cellsin vivothan cells grown in 2-dimensional culture. The floating collagen gel contraction assay is a well-defined system used to study cell–extracellular matrix interactions grown in 3-dimensional culture. Although the cell biology of this system is well defined, the cell signaling associated with gel contraction has not been well characterized. In this study we demonstrate that fetal bovine (FBS) and platelet-derived growth factor (PDGF)-induced mesangial cell-collagen gel contraction is associated with increased tyrosine phosphorylation of a number of proteins including focal adhesion kinase (FAK) and the 42-kDa isoform of MAPK (ERK2). FBS-induced gel contraction is not affected by the presence of the MEK inhibitor PD098059. Low concentrations of PDGF-BB (10 ng/ml) induce gel contraction; however, at higher PDGF-BB concentrations (80 ng/ml) gel contraction is not observed. PDGF-BB-induced gel contraction as well as tyrosine phosphorylation of FAK are inhibited in the presence of the PI-3 kinase inhibitor wortmanin. Minimal autophosphorylation of the PDGF-β receptor is observed under 3-dimensional culture conditions following PDGF-BB stimulation; however, when mesangial cells grown in 2-dimensional culture are exposed to PDGF-BB, the PDGF-β receptor was prominently phosphorylated. We conclude that induction of collagen gel contraction by FBS and PDGF-BB is associated with tyrosine kinase phosphorylation and that these responses differ substantially from what occurs in 2-dimensional cultures in the presence of the same agonists.

A novel chloride channel in Vicia faba guard cell vacuoles activated by the serine/threonine kinase, CDPK.

Calcium-Dependent Protein Kinases (CDPKs) in higher plants contain a C-terminal calmodulin-like regulatory domain. Little is known regarding physiological CDPK targets. Both kinase activity and multiple Ca2+-dependent signaling pathways have been implicated in the control of stomatal guard cell movements. To determine whether CDPK or other protein kinases could have a role in guard cell signaling, purified and recombinant kinases were applied to Vicia faba guard cell vacuoles during patch-clamp experiments. CDPK activated novel vacuolar chloride (VCL) and malate conductances in guard cells. Activation was dependent on both Ca2+ and ATP. Furthermore, VCL activation occurred in the absence of Ca2+ using a Ca2+-independent, constitutively active, CDPK* mutant. Protein kinase A showed weaker activation (22% as compared with CDPK). Current reversals in whole vacuole recordings shifted with the Nernst potential for Cl-and vanished in glutamate. Single channel recordings showed a CDPK-activated 34 +/- 5 pS Cl- channel. VCL channels were activated at physiological potentials enabling Cl- uptake into vacuoles. VCL channels may provide a previously unidentified, but necessary, pathway for anion uptake into vacuoles required for stomatal opening. CDPK-activated VCL currents were also observed in red beet vacuoles suggesting that these channels may provide a more general mechanism for kinase-dependent anion uptake.