Biochemical Society Transactions (2000) Volume 28, Part 5
1564 A new role of 5’ AMP-activated protein kinase in Ras/Raf/Erk pathway J.M.Kim, S.S. Kim, I.S. Bang. Department of Molecular Biology, Kyung Hee College of Medicine, Hoegi-dong I, Tongdaemun-gu, Seoul 130-701,Korea 5’AMP-activated protein kinase (AMPK) is activated by a decrease in the ATRAMP ratio within the cell in response to metabolic stress and regulates a number of key enzymes involved in anabolic and catabolic pathways. Thus, it is known as a metabolic sensor to maintain ATP homeostasis. Previously, it was reported that AMPK phosphorylated Ser621 of Raf-1 kinase in vitro; phosphorylation on this residue has been reported to inactivate Raf-1 kinase. In the present study, we investigated a putative role of AMPK in Ras/Raf/Erk pathway using 5 aminoimidazole4-carboxamide riboside (AICAR), a cell permeable activator of AMPK. AICAR treatment inhibited Erk activation induced by IGF-1 and H202, but not by EGF in various cell lines including NIH-3T3, H9c2 myotube, and COS-7. In accordance with Erk activity, IGF-induced JH thymidine incorporation into D N A was reduced by AICAR whereas EGF-induced D N A synthesis was not affected by AICAR. In order to identify a putative target molecule of AMPK in Ras/Raf/Erk pathway, NIH-3T3 cells were transfected with Ras and Raf mutant plasmids. Transfection results revealed that AICAR had no effect on Raf-1 kinase activity and phosphorylation of S621. However, the induced Ras activity by EGF, IGF and H 2 0 2 was inhibited by AICAR. In this case, EGF and IGF-1 receptor tyrosine kinase activity was unaffected. O u r results suggest that AMPK may regulate Ras/Raf/Erk pathway at upstream of Ras implying a new role of AMPK in mitogenic signaling.
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566 Peptide binding studies of GST and 6His-cmyc tagged forms of the Fas binding PDZ domain of the protein tyrosine phosphatase FAP-1 H.R. H a y , D.P. Blowers, I.P. Hampton, I.W. Taylor, C. Grundy, D.W. Tonge AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire SKlO 4 T G PDZ domains have been identified in a variety of proteins and are considered to be involved in protein clustering and signal transduction events at the plasma membrane. A typical PDZ domain consists of approximately 90 residues and forms a discrete fold with a peptide binding function. The protein tyrosine phosphatase FAP-I contains five PDZ domains, the second of which (PDZZ) exhibits a specific interaction with the C-terminal three residues (SLV) of Fas. The PDZ2 domain of FAP-I was expressed in E.coli with an N-terrninal GST tag or with either an N- or C- terminal 6His-cmyc tag. The proteins were purified using glutathione and Ni-NTA based affinity chromatography respectively, and characterised using surface plasmon resonance (SPR) based experiments. In this study we compare the peptide binding properties of both the GST and 6His-cmyc tagged proteins. The GST fusion protein exhibits apparent higher affinity for an immobilised SLV containing peptide than a 6His-cmyc tagged counterpart. This observation is possibly due to an avidity effect introduced by the dimeric nature of GST. More detailed binding studies reveal not only a difference in the affinity of binding but also marked differences in on and off rates. When immobilised via an an anti-GST antibody, the GST fusion protein failed to exhibit significant binding to the SLV containing peptide presented in the mobile phase. This may be indicative of the need for multivalency or coupled interactions of FAP-1 with Fas at the membrane surface.
567 Muscarinic receptor crosstalk in the regulation of ERK and 1565 Phosphorylation of Histone 3 (H3) in cardiac myocytes subjected to hyperosmotic shock
J.G. Harrison, A. Clerk Division of Biomedical Sciences (Molecular Patholgy), Imperial College School of Medicine, Sir Alexander Fleming Building, South Kensington, SW7 2AZ.
[email protected] The contracile cells of the heart (cardiac myocytes) are terminally differentiated, but undergo hypertrophy in response to an increased workload. An early event in hypertrophy is the induction of immediate early genes (1EGs)such as c-jun. Phosphorylation of H 3 (Ser-10) is involved in the nucleosomal response that accompanies IEG induction in many cells by a wide range of stimuli (e.g. growth factors, UV irradiation). This phosphorylation may be mediated through stimulation of the kinse, MSKl by the mitogen-activated kinases, ERKs and p38-MAPKs. We have studied H 3 phosphorylation in myocytes exposed to hypertrophic stimuli [endothelin-1 (ET-I) and phenylephrine (PE)] and cellular stresses (H202, hyperosmolarity, anisomycin). Nuclear extracts were immunoblotted with antibodies either to total H 3 or selective for phosphorylated (Ser-10) H3. Hyperosmotic shock (0.5M sorbitol) stimulted H3 phosphorylation (maximal at 60-90 min) with no change in total H 3 levels. However other cellular stresses (H202, anisomycin) and hypertrophic agonists (ET-1, PE), stimuli which all activate ERKs and p38MAPKs, had no effect on either H3 phosphorylation or total H3. Furthermore, inhibitors of the ERK subfamily of MAPKs (PD98059) or p38-MAPKs (SB203580) had no effect on H 3 phophorylation. These data suggest that, in cardiac myocytes, H3 phosphorylation is not mediated through ERKs and p38-MAPKs and other kinases are involved.
JNK activities in CHO-m2m3 cells D.C. Hornisold, J.L. Blank, R.M. Eglen+ and R.A.J. Challiss Department of Cell Physiology E. Pharmacology, University of Leicester, Leicester, LEI 9HN, UK and ‘Roche Centre for Biological Research, Palo Alto, C A 94304, USA Multiple subtypes of muscarinic acetylcholine(mACh) receptor are co-expressed in a variety of cell-types, but the consequences of receptor co-activation remain poorly defined in the majority of cases. In this study we have explored the interplay between M2 and M3mACh receptors co-expressed in a model cell-line in the regulation of extracellular-regulated protein kinase (ERK) and c-Jun N-terminal protein kinase UNK) activities. Agonist-mediated effects in CHO-m2m3 cells (expressing approx. 5.5 x lo4 M2- and 1.5 x lo5 M3-mACh receptors per cell) were compared to those elicited in CHO-m2 (7.0 x lo4 M2-) and CHO-m3 (4.2 x lo5 M3-mACh receptors per cell). Methacholine (MCh, 100 pM) evoked large (15-30 fold) and rapid (peak 5 min) increases in ERK activity in all cell-lines, with the response being more sustained in the co-expressing cell-line. Assessment of the concentrationdependencies of the ERK activation clearly demonstrated that much lower N C h ] are needed to evoke maximal activation in the coexpressing cell-line (-log EC50 (M), 7.17 2 0.07 (n=6) compared to 5.58 0.09 (4) and 5.69 2 0.27 (5) in CHO-m2 and -m3 cells, respectively). In contrast, mACh receptor agonist-stimulatedJNK activation was only observed in CHO-m3 and CHO-m2m3 cells and only modest differences were seen between these cell-lines (-log EC50 (M), 6.55 2 0.13 (6) and 6.11 2 0.11 (4) in CHO-m2m3 and -m3 cells, respectively). We conclude that ERK, but not JNK. activation is markedly altered in cells co-expressing M2 and M3-mACh receptors revealing a profound crosstalk between these Gila and Gq/l 1-coupled receptors.
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0 2000 Biochemical Society
