Original article
Simultaneous measurement of ERK, p38, and JNK MAP kinase cascades in vascular smooth muscle cells

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Abstract

Activation of the mitogen-activated protein kinase (MAP kinase) pathways in cultured porcine aortic vascular smooth muscle cells (VSMCs) was determined following a 5-min stimulation with endothelin-1 (ET-1), phorbol 12-myristate 13-acetate (PMA), H2O2, or sodium arsenite. Extracellular signal-related kinase (ERK1/2), p38, and c-Jun N-terminal kinase (JNK1/2) MAP kinase activation was assessed using anti-phospho-MAPK kinase antibodies. The activation of these kinase cascades was also determined by resolving lysates on Mono Q using a fast protein liquid chromatography (FPLC) system and measuring the phosphorylation of specific substrates ERK1, c-Jun, and hsp27. The substrates were subsequently resolved from each other and the [γ-32P]ATP in the reaction mixture by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and the incorporation of 32P was quantified by phosphor imaging. This technique revealed the presence of multiple peaks of activity phosphorylating ERK1 (5), c-Jun (7), and hsp27 (9). Differences in activation revealed by the chromatographic technique suggest that, although equivalent levels of activation may be detected by immunoblotting, the actual nature of the response differed depending upon the stimulus. Each stimulus that activated the MAP kinase cascades did not result in equivalent ‘profile’ of activation of kinase activities. These results suggest the presence of a mechanism of structural organization of the MAP kinase signaling molecules themselves resulting in the compartmentalization of responses with respect to the various cellular stimuli.

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.

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