Kinetics Of Kinase Activation Research Articles

Regulation of alveolar macrophage p40phox: hierarchy of activating kinases and their inhibition by PGE2.

PGE(2), produced in the lung during infection with microbes such as Klebsiella pneumoniae, inhibits alveolar macrophage (AM) antimicrobial functions by preventing H(2)O(2) production by NADPH oxidase (NADPHox). Activation of the NADPHox complex is poorly understood in AMs, although in neutrophils it is known to be mediated by kinases including PI3K/Akt, protein kinase C (PKC) δ, p21-activated protein kinase (PAK), casein kinase 2 (CK2), and MAPKs. The p40phox cytosolic subunit of NADPHox has been recently recognized to function as a carrier protein for other subunits and a positive regulator of oxidase activation, a role previously considered unique to another subunit, p47phox. The regulation of p40phox remains poorly understood, and the effect of PGE(2) on its activation is completely undefined. We addressed these issues in rat AMs activated with IgG-opsonized K. pneumoniae. The kinetics of kinase activation and the consequences of kinase inhibition and silencing revealed a critical role for a PKCδ-PAK-class I PI3K/Akt1 cascade in the regulation of p40phox activation upon bacterial challenge in AMs; PKCα, ERK, and CK2 were not involved. PGE(2) inhibited the activation of p40phox, and its effects were mediated by protein kinase A type II, were independent of interactions with anchoring proteins, and were directed at the distal class I PI3K/Akt1 activation step. Defining the kinases that control AM p40phox activation and that are the targets for inhibition by PGE(2) provides new insights into immunoregulation in the infected lung.

Histamine H4 receptor activation enhances LPS‐induced IL‐6 production in mast cells via ERK and PI3K activation

The histamine H(4) receptor (H(4)R) has been implicated in numerous inflammatory functions. Here it is shown that the receptor can mediate cytokine production from mast cells. Histamine and an H(4)R agonist, JNJ 28610244, induced the production of IL-6 in mouse bone marrow (BM)-derived mast cells. This effect was blocked by two different H(4)R antagonists and was not present in H(4)R-deficient cells. In addition, histamine acting via the H(4) R potentiated LPS-induced IL-6 production. Histamine-induced IL-6 production could be blocked by inhibitors of ERK and phosphoinositide 3-kinase γ (PI3Kγ) pathways. Furthermore, it was shown that H(4)R activation can induce phosphorylation of ERK, MEK and AKT. H(4)R activation led to a rapid and transient phosphorylation of these kinases, whereas with LPS the activation occurred at later time points. When both histamine and LPS were added, the phosphorylation was evident at 5 min and persisted for at least 60 min suggesting that changes in the kinetics of kinase activation may be one mechanism driving the signaling interaction between the H(4)R and toll-like receptors. These observations suggest that the H(4)R can synergize with other inflammatory signals to potentiate cytokine production and provides mechanistic information on the role of the H(4)R in inflammation.

Abstract 1941: TRAIL-induced kinase activation in Non small cell lung cancer cells

Abstract Non-small cell lung cancer (NSCLC) is a disease with poor prognosis and novel therapeutic approaches are greatly needed. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an interesting agent that is able to trigger apoptosis through interactions with TRAIL receptors. An important feature of TRAIL is its ability to induce apoptosis in a wide variety of tumors without harming normal cells, making it an attractive anti-cancer therapeutic compared to conventional anti-cancer agents. However, TRAIL is also able to activate signaling pathways that are involved in survival, proliferation and migration of tumor cells. Thus, TRAIL-based therapies combined with inhibitors of such pathways are expected to enhance therapeutic benefit. In this study, we aimed to identify and characterize kinases that are activated by recombinant TRAIL in NSCLC cells. We monitored the activation of a number of kinases known to be involved in TRAIL signaling by Western blotting, including p38 MAPK, JNK, ERK and Akt. In addition we employed PepChip kinase arrays. With these arrays 1024 peptide kinase substrates can be screened in one experiment, whereby a comparison of kinase patterns between untreated and treated cells can be obtained. NSCLC, H460 and A549 cell lines, which are sensitive and resistant for TRAIL, respectively, were exposed to 50 ng/ml TRAIL for different periods of time (5 to 240 minutes) to evaluate the kinetics of kinase activation. In H460 cells, TRAIL induced the phosphorylation of p38 MAPK after 2 hours and JNK1/2 after 3 hours. As the activation of these kinases can be both anti- and pro-apoptotic, kinase inhibitors were used to explore this further. In H460 cells the activation of JNK, ERK and Akt had anti-apoptotic activity. Inhibition of these kinases with SP600125, PD098059, and LY294002, respectively, showed a 2-fold increase in apoptosis when combined with TRAIL. The activation of MAPK p38 on the other hand was pro-apoptotic, since its inhibition with SB203580 resulted in a reduction of TRAIL-induced apoptosis in H460 cells. In resistant A549 cells, however, Akt, ERK, p38 MAPK, and JNK1/2 activation appeared to have anti-apoptotic activity. Furthermore, in these cells an increase in IκBα phosphorylation was observed that was not seen in H460 cells, where levels of phosphorylated IκBα decreased after 1 hour that correlated with cleavage of RIP. Thus suppression of NFκB activation could be associated with TRAIL sensitivity. PepChip kinase arrays, revealed the activation of kinases that are involved in cell migration, such as Rho/Rock in A549 cells, and further investigations are ongoing. In conclusion, we observed differential TRAIL-dependent activation of p38 MAPK, JNK1/2, ERK, Akt and IκBα in sensitive and resistant NSCLC cells as well as in pathways that regulate migration. The relationship with TRAIL antitumor activity is currently further explored. 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 1941. doi:10.1158/1538-7445.AM2011-1941

Mechanism of CheA protein kinase activation in receptor signaling complexes.

The chemotaxis receptor for aspartate, Tar, generates responses by regulating the activity of an associated histidine kinase, CheA. Tar is composed of an extracellular sensory domain connected by a transmembrane sequence to a cytoplasmic signaling domain. The cytoplasmic domain fused to a leucine zipper dimerization domain forms soluble active ternary complexes with CheA and an adapter protein, CheW. The kinetics of kinase activity within these complexes compared to CheA alone indicate approximately a 50% decrease in the KM for ATP and a 100-fold increase in the Vmax. A truncated CheA construct that lacks the phosphoaccepting H-domain and the CheY/CheB-binding domain forms an activated ternary complex that is similar to the one formed by the full-length CheA protein. The Vmax of H-domain phosphorylation by this complex is enhanced approximately 60-fold, the KM for ATP decreased to 50%, and the KM for H-domain decreased to 20% of the values obtained with the same CheA construct in the absence of receptor and CheW. The kinetic data support a mechanism of CheA regulation that involves perturbation of an equilibrium between an inactive form where the H-domain is loosely bound and an active form where the H-domain is tightly associated with the CheA active site and properly positioned for phosphotransfer. The data are consistent with an asymmetric mechanism of CheA activation [Levit, M., Liu, I., Surette, M. G., and Stock, J. B. (1996) J. Biol. Chem. 271, 32057-32063] wherein only one phosphoaccepting domain of CheA at a time can interact with an active center within a CheA dimer.

The tobacco wounding-activated mitogen-activated protein kinase is encoded by SIPK.

It has been demonstrated that both salicylic acid and fungal elicitors activate a 48-kDa mitogen-activated protein kinase termed salicylic acid-induced protein kinase (SIPK) in tobacco suspension cells. Here, we show that infiltration of these agents into tobacco leaves also activates SIPK. Of particular interest, infiltration of water alone activated a kinase of the same size, possibly because of wounding and/or osmotic stresses. The kinetics of kinase activation, however, differ for these different treatments. Various mechanical stresses, including cutting and wounding by abrasion, also activated a 48-kDa kinase. By using an immune-complex kinase assay with antibodies specific for SIPK or wounding-induced protein kinase, we demonstrate that this wounding-activated 48-kDa kinase is SIPK, rather than wounding-induced protein kinase, as reported [Seo, S., Okamoto, M., Seto, H., Ishizuka, K., Sano, H. & Ohashi, Y. (1995) Science 270, 1988-1992]. Activation of SIPK after wounding was associated with tyrosine phosphorylation but not with increases in SIPK mRNA or protein levels. Thus, the same mitogen-activated protein kinase, SIPK, appears to facilitate signaling for two distinct pathways that lead to disease resistance responses and wounding responses.

Epidermal growth factor-mediated activation of an S6 kinase in Swiss mouse 3T3 cells.

Extracts from epidermal growth factor (EGF)-stimulated Swiss mouse 3T3 cells are up to 10 times more potent in phosphorylating ribosomal protein S6 than extracts from quiescent cells. Preparation of extracts in the absence of phosphatase inhibitors leads to a time-dependent loss of kinase activity. In order of potency, the most efficient phosphatase inhibitors in protecting the S6 kinase activity are phosphotyrosine followed by p-nitrophenyl phosphate, beta-glycerol phosphate, and phosphoserine. The kinetics of kinase activation following EGF treatment are rapid and transient. The maximum increase is observed between 15 and 30 min with only 20-30% of the activity remaining after 2 h. Phosphorylation of S6 in the intact cell follows a similar pattern of activation, reaching a maximum between 30 and 60 min and then slowly returning to basal levels by approximately 3 h. The activation of protein synthesis is also rapid; however, in contrast to the transient activation of the S6 kinase and S6 phosphorylation, it remains persistently high for at least 6 h following EGF treatment. Comparison of these events with EGF binding shows that about 50% of the cell surface binding sites are lost within 10 min of exposure to EGF, and about 25% remain after 2 h. Finally, sodium orthovanadate, which is known to mimic the mitogenic effect of EGF, also leads to activation of the S6 kinase, however, with distinct kinetics and by an apparent EGF receptor-independent pathway.