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

Activation of the mitogen-activated protein kinase (MAP kinase) pathways in cultured porcine aortic vascular smooth muscle cells (VSMCs) was determine d following a 5-min stimulation with endothelin-l (ET-l), phorbol 12-myrist ate: 13-acetate (PMA), H2O2, or sodium arsenite. Extracellular signal-relat ed kinase (ERK1/2), p38, and c-Jun N-terminal kinase (JNK1/2) MAP kinase ac tivation was assessed using anti-phospho-MAPK kinase antibodies. The activa tion of these kinase cascades was also determined by resolving lysates on M ono Q using a fast protein liquid chromatography (FPLC) system and measurin g the phosphorylation of specific substrates ERK1, c-bun, and hsp27. The su bstrates were subsequently resolved from each other and the [gamma-P-32]ATP in the reaction mixture by SDS-polyacrylamide gel electrophoresis (SDS-PAG E) and the incorporation of P-32 was quantified by phosphor imaging. This t echnique revealed the presence of multiple peaks of activity phosphorylatin g 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 resp onse differed depending upon the stimulus. Each stimulus that activated the MAP kinase cascades did not result in equivalent 'profile' of activation o f kinase activities. These results suggest the presence of a mechanism of s tructural organization of the MAP kinase signaling molecules themselves res ulting in the compartmentalization of responses with respect to the various cellular stimuli. (C) 2001 Elsevier Science Inc. All rights reserved.