The mitogen-activated protein kinases (MAPK) (p42"' "pk, p44"' "P' and others [l]) are a family of Ser/Thr protein kinases that are activated in response to a variety of physiological stimuli (for a review see [2]). The activation of MAPKs results from the phosphorylation of both Tyr and Thr residues and this prompted the suggestion that MAPK may integrate the pathways from Ser/Thr protein kinase cascades and Tyr protein kinase cascades [3]. More recently a MAPK activator, now termed M A P kinase kinase (MAPKK), has been identified as a dual-specificity kinase that phosphorylates both the regulatory residues [4]. In the present study we investigated the activation of MAPK cascade by two agonists, endothelin-1 (ET-1) and acidic fibroblast growth factor (aFGF), that act via apparently different signalling pathways but produce responses in cultured cardiac myocytes that are typical of hypertrophy IS].Cardiac myocytes were isolated from the hearts of 1-to 3-day-old rats, maintained in culture for 7 d and exposed to ET-1 or aFGF in serum-free medium [6]. Where indicated, the cells were cultured in the presence of 1 pM phorbol ester for 24 h prior to exposure to either aFGF or ET-1 to downregulate cellular protein kinase C (PKC) isotypes.cytosolic extracts were prepared and the protein b a s e activity against myelin basic protein (MBP) was assayed [6]. ET-1 and aFGF stimulated MBPK activity in these extracts with maximal activation achieved at 5 min (2.9-and 2.4-fold, respectively). Activity then fell rapidly and returned to control values within 90 min. Using immunoblot analysis with antisera specific for each PKC isotype (71, we showed that cultured neonatal cardiomyocytes express PKC-a, -6 , -E and -<. After pretreatment with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) for 24 h, levels of PKC-a, -6 and -E fell and could not be detected. In contrast, levels of PKC-C remained unchanged. This TPA pretreatment prevented MBPK activation by TPA (3.6-fold stimulation decreased to 1.2-fold) but only partially decreased the response to ET-1 or aFGF (decreased to 1.8and 2.1-fold, respectively).To further characterize the MBPK activities, extracts from ET-1-or aFGF-stimulated cardiac myocytes were applied to a Mono Q column equilibrated with 50 mM TrisHCI, 2 mM EDTA, 2 mM EGTA, 0.1% (v/v) mercaptoethanol, 5% (v/v) glycerol, 0.05% (w/v) Brij 35, 0.3 mM Na,VO,, 1 mM benzamidine, and 4 r.cg/ml leupeptin (pH 7.3) at a flow rate of 1 ml/min. After washing with 5 ml of this buffer, protein was eluted with a linear gradient of NaCl (0 -0.3 M). Fractions (0.5 ml) were collected and assayed for MAPK and MAPKK [8]. F~G U R E 1 shows that both (a) ET-1-or (b) aFGFstimulated MBPK could be resolved as 2 peaks (K1 and K2) under these conditions. Renatured kinase activities in MBPcontaining gels and immunoblot analysis showed one peak was a p42 MAPK and the second peak a p44 MAPK. In addition, two peaks of MAPKK (KK1 and KK2) were identified in these cells. Immunoblot analysis using an antibody generated against recombinant MAPKK recognized...