Mitogen-activated protein (MAP) kinase stimulation by phorbol esters and external load in the isolated perfused heart.

Abstract

Mitogen-activated protein kinases are a family of serine/ threonine kinases that have been implicated in a wide variety of cellular processes [for review see 1,2]. These kinases are thought to participate in a protein kinase cascade leading to cell growth as they have been shown to phosphorylate ribosomal protein S6 and various transcription factors. In response to most growthpromoting and mitogenic factors tested, as well as to many other signals such as phorbol esters, MAP kinase phosphorylation is increased on both threonine and tyrosine residues, leading to stimulation of its kinase activity [3-51. Phosphorylation of MAP kinase involves an activator which is itself a kinase, with dual specificity for both tyrosine and serindthreonine [6]. Recent reports suggest that MAP kinase is involved in the signalling events leading to heart hypertrophy [7,8]. To evaluate this hypothesis further, we have investigated the effects of hypertrophic agents on the activity of MAP kinase in isolated perfused heart preparations. As hypertrophic agents, TPA and high load were used as they are both known to induce responses characteristic of hypertrophy in rat heart preparations. Hearts were perfused retrogradely with Krebs-Henseleit saline (1 19 mM NaCI, 25 mM NaHCO,, 4.7 mM KCl, 2.5 mM CaCI,, 1.2 mM MgSO,, 1.2 mM KH,PO, and 10 mM glucose, equilibrated with 95% 45% C02, pH 7.6 at 37°C). For control experiments, perfusions were carried out at for 15 min at 5.5 kPa. After that, hearts were either exposed to TPA or high aortic pressure for a further 5 min period. When present, TPA was added in the perfusion medium at a final concentration of 1pM. For the experiments at high aortic pressure, the perfusion pressure was increased to 16.3 kPa (120 mm Hg). Hearts were extracted with 20mM j3-glycerophosphate pH 7.5, containing 20mM NaF, 2mM EDTA, 0.2mM Na,VO,, lOmM benzamidine, 25 pg/ml leupeptin, 50 pglml PMSF and 0.3% (v/v)J?-mercaptoethanol and centrifuged for lOmin at 1oooOxg. The supernatant was applied to a Mono Q FPLC column equilibrated with 50 mM Tris-HC1 pH 7.5, containing 2 mM EDTA, 2 mM EGTA, 0.1% Jmercaptoethanol, 5% glycerol, 0.03% Brij 35, 0.3 mM Na,VO,, ImM benzamidine and 4 pg/ml leupeptin. Myelin basic protein (MBP) kinase activity was eluted with linear gradient of NaCl (00.3 M) and measured as described previously [7]. FPLC of the heart extracts revealed two peaks of MBP kinase activity that were stimulated with exposure to TPA. The first peak eluted at 0.16 M NaCl and the activity was stimulated %fold compared with control. The second peak eluted at 0.22 M NaCl and the activity was stimulated 2.3-fold (Fig. 1). Extracts of hearts exposed to high aortic pressures showed an identical elution profile but stimulation was lower (2-fold for the first peak and 1.6-fold for the second peak) (Fig. 1). Using a kinase renaturation assay method [9], we examined the peak fractions from FPLC of TPA-treated hearts for their ability to phosphorylate MBP in gels in siru. We found that the molecular masses were 42kDa and 44kDa for the first and second peak respectively (Fig. 2a). The same method was used to examine the crude extracts of control and TPA-treated hearts. MBP kinase