Journal of Pharmacological and Toxicological Methods
Original articleSimultaneous measurement of ERK, p38, and JNK MAP kinase cascades in vascular smooth muscle cells
Introduction
The three parallel well-characterized subfamilies of the mitogen-activated protein kinase (MAP kinase) family are the extracellular-signal regulated kinase (ERKs), the c-Jun N-terminal kinases (JNKs) that are also known as stress-activated protein kinases (SAPKs), and the p38 MAP kinases (Fig. 1). MAP kinases are proline-directed, serine/threonine protein kinases that play a central role in the intracellular signal transduction pathways in response to a variety of cellular stimuli. A MAP kinase cascade consists of three sequentially acting kinases, and this organization may function as a switch to provide a threshold-like input–output response to receptor activation Ferrell, 1996, Huang & Ferrell, 1996. The last member of the cascade, MAPK, is activated by dual phosphorylation at tyrosine and threonine residues in a T–X–Y motif, where X represents the amino acids glutamic acid, glycine, and proline for ERK, p38, and JNK, respectively. In each case, the activating kinase is the second kinase in the cascade, MAPKK. MAPKK is, in turn, activated by phosphorylation at serine and threonine residues by the first member of the cascade, MAPKKK. These cascades of kinase reactions occur in the cytosol with the activated MAPK phosphorylating targets in both the cytosol and nucleus.
Both ERK Adams & Hathaway, 1993, Childs & Mak, 1993, Childs et al., 1992, Gerthoffer et al., 1996 and p38 Hedges et al., 1998, Yamboliev et al., 2000 MAP kinases phosphorylate caldesmon, an actin-binding protein that inhibits actin-activated myosin ATPase activity in a phosphorylation-dependent manner (Ngai & Walsh, 1987). In addition, hsp27, a substrate for MAP kinase-activated protein kinase-2 (MAPKAP kinase-2) (Stokoe, Engel, Campbell, Cohen, & Gaestel, 1992), is an actin-capping protein that has been implicated in regulating smooth muscle contraction via interactions with the actin filaments (Bitar, Kaminski, Hailat, Cease, & Strahler, 1991). In both airway and colonic smooth muscle, muscarinic agonists induce activation of p38 MAP kinase and phosphorylation of hsp27 (Larsen, Yamboliev, Weber, & Gerthoffer, 1997). These effects are blocked by the specific inhibitor of p38 MAP kinase, SB203580 (Larsen et al., 1997). Inhibition of p38 (Yamboliev, Hedges, et al., 2000), but not ERK Gorenne et al., 1998, Yamboliev et al., 2000, reduces smooth muscle contraction, whereas both ERK and p38 activity are required for the regulation of chemotactic migration in colonic myocytes (Yamboliev, Wiesmann, Singer, Hedges, & Gerthoffer, 2000). Hence, the p38 and ERK MAP kinase cascades may play roles in regulating smooth muscle function.
We report here a means whereby the activation of the ERK, p38, and JNK MAP kinase cascades can be determined simultaneously. In addition, we demonstrate that different patterns of kinase activation, which were not apparent when the activation status of the MAP kinases were determined by their phosphorylation, may be observed using this methodology. These data suggest that, even within a given MAP kinase cascade, there exists a level of organization such that specific modules become activated in response to different agonists or forms of cellular stress.
Section snippets
Materials
[γ-32P]ATP was from Amersham Pharmacia Biotech (Baie d'Urfé, Québec). Membrane grade (reduced) Triton X-100, leupeptin, and phenylmethylsulfonyl fluoride (PMSF) were from Roche Molecular Biochemicals (Laval, Québec). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) reagents, nitrocellulose, and Bradford protein assay reagents were from Bio-Rad Laboratories (Canada) (Mississauga, Ontario). Microcystin LR and phorbol 12-myristate 13-acetate (PMA) were from Calbiochem-Novabiochem (San Diego, CA).
Results
We have examined the activation of the ERK, p38, and JNK MAP kinase cascades in cultured porcine aortic smooth muscle cells in response to mitogens and cellular stress. ET-1 and tumor-promoting phorbol esters are mitogens and activate the ERK pathway, whereas H2O2 and arsenite are activators of the stress-activated p38 and JNK MAP kinase pathways (Fig. 1). Kinase activities were determined both by using antisera for the dual-phosphorylated forms of ERK1/2, p38, and JNK1/2, and by chromatography
Discussion
We have examined the activation of the ERK, p38, and JNK MAP kinase cascades in cultured porcine aortic smooth muscle cells in response to mitogens and cellular stress. Weak responses were observed following treatment with arsenite and this could be due to the short treatment times employed in the present study. Arsenite is a potent activator of p38 MAP kinase and MAPKAP kinase-2 activity (Rouse et al., 1994); however, activation of p38 MAP kinase by arsenite is maximal at 60–120 min and ERK
Acknowledgements
This paper is supported by grants from the Medical Research Council of Canada (MT-14725), the Quebec Heart and Stroke Foundation, the Fonds de la Recherche en Santé du Québec (FRSQ), and the Montreal Heart Institute Research Center. BGA and ET are currently research scholars of the Heart and Stroke Foundation of Canada.
References (39)
- et al.
Hsp27 is a mediator of sustained smooth muscle contraction in response to bombesin
Biochemical and Biophysical Research Communications
(1991) - et al.
IB1 reduces cytokine-induced apoptosis of insulin-secreting cells
Journal of Biological Chemistry
(2000) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Analytical Biochemistry
(1976)- et al.
Phosphorylation of smooth muscle caldesmon by mitogen-activated protein (MAP) kinase and expression of MAP kinase in differentiated smooth muscle cells
Journal of Biological Chemistry
(1992) - et al.
A comparison of the substrate specificity of MAPKAP kinase-2 and MAPKAP kinase-3 and their activation by cytokines and cellular stress
FEBS Letters
(1996) Tripping the switch fantastic: how a protein kinase cascade can convert graded inputs into switch-like outputs
Trends in Biological Sciences
(1996)- et al.
Identification of the phosphorylation sites of the murine small heat shock protein hsp25
Journal of Biological Chemistry
(1991) - et al.
Molecular cloning of human p38 MAP kinase
Biochimica et Biophysica Acta
(1995) - et al.
Characterization of the structure and function of a new mitogen-activated protein kinase (p38beta)
Journal of Biological Chemistry
(1996) - et al.
Characterization of the structure of the fourth member of p38 group mitogen-activated protein kinases, p38δ
Journal of Biological Chemistry
(1997)
Kinase suppressor of ras inhibits the activation of extracellular ligand-regulated (ERK) mitogen-activated protein (MAP) kinase by growth factors, activated ras, and ras effectors
Journal of Biological Chemistry
Cloning and sequencing of a cDNA encoding the canine hsp27 protein
Gene
The primary structure of p38γ: a new member of the p38 group of MAP kinases
Biochemical and Biophysical Research Communications
The stress inducer arsenite activates mitogen-activated protein kinases extracellular signal-regulated kinases 1 and 2 via a MAPK kinase 6/p38-dependent pathway
Journal of Biological Chemistry
MAPKAPK5, a novel mitogen-activated protein kinase (MAPK)-activated protein kinase, is a substrate of the extracellular-regulated kinase (ERK) and p38 kinase
Biochemical and Biophysical Research Communications
A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins
Cell
Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins
FEBS Letters
Identification of mitogen-activated protein kinase phosphorylation sequences in mammalian h-caldesmon
FEBS Letters
Smooth-muscle mitogen-activated protein (MAP) kinase: purification and characterization, and the phosphorylation of caldesmon
Biochemical Journal
Cited by (16)
Regulation of membrane-bound PKC in adult cardiac ventricular myocytes
2003, Cellular SignallingInvolvement of p44/42 mitogen-activated protein kinases in regulating angiotensin II- and endothelin-1-induced contraction of rat thoracic aorta
2002, European Journal of PharmacologyValsartan ameliorates ageing-induced aorta degeneration via angiotensin II type 1 receptor-mediated ERK activity
2014, Journal of Cellular and Molecular MedicineCharacterization of hsp27 kinases activated by elevated aortic pressure in heart
2012, Molecular and Cellular BiochemistryEffect of a high dairy diet on serum antibody titers to heat shock protein 27 in overweight and obese children
2009, Iranian Journal of PediatricsComparison of gene expression profiles in HepG2 cells exposed to arsenic, cadmium, nickel, and three model carcinogens for investigating the mechanisms of metal carcinogenesis
2009, Environmental and Molecular Mutagenesis