Differential activation of protein kinase C isoforms by endothelin-1 and phenylephrine and subsequent stimulation of p42 and p44 mitogen-activated protein kinases in ventricular myocytes cultured from neonatal rat hearts.

The translocation of protein kinase C (PKC) isoforms PKC-alpha, PKC-delta, PKC-epsilon, and PKC-zeta from soluble to particulate fractions was studied in ventricular cardiomyocytes cultured from neonatal rats. Endothelin-1 (ET-1) caused a rapid ETA receptor-mediated translocation of PKC-delta and PKC-epsilon (complete in 0.5-1 min). By 3-5 min, both isoforms were returning to the soluble fraction, but a greater proportion of PKC-epsilon remained associated with the particulate fraction. The EC50 of translocation for PKC-delta was 11-15 nM ET-1 whereas that for PKC-epsilon was 1.4-1.7 nM. Phenylephrine caused a rapid translocation of PKC-epsilon (EC50 = 0.9 microM) but the proportion lost from the soluble fraction was less than with ET-1. Translocation of PKC-delta was barely detectable with phenylephrine. Neither agonist caused any consistent translocation of PKC-alpha or PKC-zeta. Activation of p42 and p44 mitogen-activated protein kinase (MAPK) by ET-1 or phenylephrine followed more slowly (complete in 3-5 min). Phosphorylation of p42-MAPK occurred simultaneously with its activation. The proportion of the total p42-MAPK pool phosphorylated in response to ET-1 (50%) was greater than with phenylephrine (20%). In addition to activation of MAPK, an unidentified p85 protein kinase was activated by ET-1 in the soluble fraction whereas an unidentified p58 protein kinase was activated in the particulate fraction.

Stimulation of phosphatidylinositol hydrolysis, protein kinase C translocation, and mitogen-activated protein kinase activity by bradykinin in rat ventricular myocytes: dissociation from the hypertrophic response.

In ventricular myocytes cultured from neonatal rat hearts, bradykinin (BK), kallidin or BK(1-8) [(Des-Arg9)BK] stimulated PtdinsP2 hydrolysis by 3-4-fold. EC50 values were 6 nM (BK), 2 nM (kallidin), and 14 microM [BK(1-8)]. BK or kallidin stimulated the rapid (less than 30 s) translocation of more than 80% of the novel protein kinase C (PKC) isoforms nPKC-delta and nPKC-epsilon from the soluble to the particulate fraction. EC50 values for nPKC-delta translocation by BK or kallidin were 10 and 2 nM respectively. EC50 values for nPKC-epsilon translocation by BK or kallidin were 2 and 0.6 nM respectively. EC50 values for the translocation of nPKC-delta and nPKC-epsilon by BK(1-8) were more than 5 microM. The classical PKC, cPKC-alpha, and the atypical PKC, nPKC-zeta, did not translocate. BK caused activation and phosphorylation of p42-mitogen-activated protein kinase (MAPK) (maximal at 3-5 min, 30-35% of p42-MAPK phosphorylated). p44-MAPK was similarly activated. EC50 values for p42/p44-MAPK activation by BK were less than 1 nM whereas values for BK(1-8) were more than 10 microM. The order of potency [BK approximately equal to kallidin > BK (1-8)] for the stimulation of PtdInsP2 hydrolysis, nPKC-delta and nPKC-epsilon translocation, and p42/p44-MAPK activities suggests involvement of the B2 BK receptor subtype. In addition, stimulation of all three processes by BK was inhibited by the B2BK receptor-selective antagonist HOE140 but not by the B1-selective antagonist Leu8BK(1-8). Exposure of cells to phorbol 12-myristate 13-acetate for 24 h inhibited subsequent activation of p42/p44-MAPK by BK suggesting participation of nPKC (and possibly cPKC) isoforms in the activation process. Thus, like hypertrophic agents such as endothelin-1 (ET-1) and phenylephrine (PE), BK activates PtdInsP2 hydrolysis, translocates nPKC-delta, and nPKC-epsilon, and activates p42/p44-MAPK. However, in comparison with ET-1 and PE, BK was only weakly hypertrophic as assessed by cell morphology and patterns of gene expression. This difference could not be attributed to dissimilarities between the duration of activation of p42/p44-MAPK by BK or ET-1. Thus activation of these signalling pathways alone may be insufficient to induce a powerful hypertrophic response.

Stimulation of the p38 mitogen-activated protein kinase pathway in neonatal rat ventricular myocytes by the G protein-coupled receptor agonists, endothelin-1 and phenylephrine: a role in cardiac myocyte hypertrophy?

We examined the activation of the p38 mitogen-activated protein kinase (p38-MAPK) pathway by the G protein-coupled receptor agonists, endothelin-1 and phenylephrine in primary cultures of cardiac myocytes from neonatal rat hearts. Both agonists increased the phosphorylation (activation) of p38-MAPK by approximately 12-fold. A p38-MAPK substrate, MAPK-activated protein kinase 2 (MAPKAPK2), was activated approximately fourfold and 10 microM SB203580, a p38-MAPK inhibitor, abolished this activation. Phosphorylation of the MAPKAPK2 substrate, heat shock protein 25/27, was also increased. Using selective inhibitors, activation of the p38-MAPK pathway by endothelin-1 was shown to involve protein kinase C but not Gi/Go nor the extracellularly responsive kinase (ERK) pathway. SB203580 failed to inhibit the morphological changes associated with cardiac myocyte hypertrophy induced by endothelin-1 or phenylephrine between 4 and 24 h. However, it decreased the myofibrillar organization and cell profile at 48 h. In contrast, inhibition of the ERK cascade with PD98059 prevented the increase in myofibrillar organization but not cell profile. These data are not consistent with a role for the p38-MAPK pathway in the immediate induction of the morphological changes of hypertrophy but suggest that it may be necessary over a longer period to maintain the response.

Regulation of mitogen-activated protein kinase cascades in the heart.

Using primary cultures of neonatal rat ventricular myocytes and isolated adult rat hearts as models, we have characterized extensively the regulation of MAPKs in the heart. The ERKs are activated primarily by GPCR agonists acting through PKC. These agonists can also activate the JNKs although the mechanism is unclear. Cellular stresses stimulate strong activation of the JNKs, but also cause some stimulation of ERKs. Activation of p38-MAPK has so far only been demonstrated in intact adult hearts subjected to stresses and probably leads to activation of MAPKAPK2. Both cellular stresses and GPCR agonists induce phosphorylation of c-Jun, but only the latter causes upregulation of c-Jun protein.

Regulation of protein kinase B and 4E-BP1 by oxidative stress in cardiac myocytes.

Stimulation of phosphatidylinositol 3'-kinase (PI3K) and protein kinase B (PKB) is implicated in the regulation of protein synthesis in various cells. One mechanism involves PI3K/PKB-dependent phosphorylation of 4E-BP1, which dissociates from eIF4E, allowing initiation of translation from the 7-methylGTP cap of mRNAs. We examined the effects of insulin and H(2)O(2) on this pathway in neonatal cardiac myocytes. Cardiac myocyte protein synthesis was increased by insulin, but was inhibited by H(2)O(2). PI3K inhibitors attenuated basal levels of protein synthesis and inhibited the insulin-induced increase in protein synthesis. Insulin or H(2)O(2) increased the phosphorylation (activation) of PKB through PI3K, but, whereas insulin induced a sustained response, the response to H(2)O(2) was transient. 4E-BP1 was phosphorylated in unstimulated cells, and 4E-BP1 phosphorylation was increased by insulin. H(2)O(2) stimulated dephosphorylation of 4E-BP1 by increasing protein phosphatase (PP1/PP2A) activity. This increased the association of 4E-BP1 with eIF4E, consistent with H(2)O(2) inhibition of protein synthesis. The effects of H(2)O(2) were sufficient to override the stimulation of protein synthesis and 4E-BP1 phosphorylation induced by insulin. These results indicate that PI3K and PKB are important regulators of protein synthesis in cardiac myocytes, but other factors, including phosphatase activity, modulate the overall response.

Peptide growth factors signal differentially through protein kinase C to extracellular signal-regulated kinases in neonatal cardiomyocytes

The extracellular signal-regulated kinases 1/2 (ERK1/2) are activated in cardiomyocytes by Gq protein-coupled receptors and are associated with induction of hypertrophy. Here, we demonstrate that, in primary cardiomyocyte cultures, ERK1/2 were also significantly activated by platelet-derived growth factor (PDGF), epidermal growth factor (EGF) or fibroblast growth factor (FGF), but insulin, insulin-like growth factor 1 (IGF-1) and nerve growth factor (NGF) had relatively minor effects. PDGF, EGF or FGF increased cardiomyocyte size via ERK1/2, whereas insulin, IGF-1 or NGF had no effect suggesting minimum thresholds/durations of ERK1/2 signaling are required for the morphological changes associated with hypertrophy. Peptide growth factors are widely accepted to activate phospholipase C gamma1 (PLCgamma1) and protein kinase C (PKC). In cardiomyocytes, only PDGF stimulated tyrosine phosphorylation of PLCgamma1 and nPKCdelta. Furthermore, activation of ERK1/2 by PDGF, but not EGF, required PKC activity. In contrast, EGF substantially increased Ras.GTP with rapid activation of c-Raf, whereas stimulation of Ras.GTP loading by PDGF was minimal and activation of c-Raf was delayed. Our data provide clear evidence for differential coupling of PDGF and EGF receptors to the ERK1/2 cascade, and indicate that a minimum threshold/duration of ERK1/2 signaling is required for the development of cardiomyocyte hypertrophy.

Diacylglycerol-induced protection against injury during ischemia and reperfusion can be achieved without activation of protein kinase C in the rat heart: Comparative studies with ischemic preconditioning

The role of protein kinase C (PKC) activation in ischemic preconditioning remains controversial. Since diacylglycerol is the endogenous activator of PKC and as such might be expected cardioprotective, we have investigated whether: (i) the diacylglycerol analog 1,2-dioctanoyl-sn-glycerol (DOG) can protect against injury during ischemia and reperfusion; (ii) any effect is mediated via PKC activation; and (iii) the outcome is influenced by the time of administration. Isolated rat hearts were perfused with buffer at 37°C and paced at 400 bpm. In Study 1, hearts (n=6/group) were subjected to one of the following: (1) 36 min aerobic perfusion (controls); (2) 20 min aerobic perfusion plus ischemic preconditioning (3 min ischemia/3 min reperfusion+5 min ischemia/5 min reperfusion); (3) aerobic perfusion with buffer containing DOG (10 μM) given as a substitute for ischemic preconditioning; (4) aerobic perfusion with DOG (10 μM) during the last 2 min of aerobic perfusion. All hearts then were subjected to 35 min of global ischemia and 40 min reperfusion. A further group (5) were perfused with DOG (10 μM) for the first 2 min of reperfusion. Ischemic preconditioning improved postischemic recovery of LVDP from 24±3% in controls to 71±2% (P<0.05). Recovery of LVDP also was enhanced by DOG when given just before ischemia (54±4%), however, DOG had no effect on the recovery of LVDP when used as a substitute for ischemic preconditioning (22±5%) or when given during reperfusion (29±6%). In Study 2, the first four groups of study were repeated (n=4–5/group) without imposing the periods of ischemia and reperfusion, instead hearts were taken for the measurement of PKC activity (pmol/min/mg protein±SEM). PKC activity after 36 min in groups (1), (2), (3) and (4) was: 332±102, 299±63, 521±144, and 340±113 and the membrane:cytosolic PKC activity ratio was: 5.6±1.5, 5.3±1.8, 6.6±2.7, and 3.9±2.1 (P=NS in each instance). In conclusion, DOG is cardioprotective but under the conditions of the present study is less cardioprotective than ischemic preconditioning, furthermore the protection does not appear to necessitate PKC activation prior to ischemia.

Growth of melanocytic cells is associated with down-regulation of protein kinase C α,δ and ε isoforms: Possible role of diacylglycerol

Protein kinase C (PKC) down-regulation has been shown to correlate with the growth of murine melanocytic cells in culture (Brooks, G., Wilson, R. E., Dooley, T. P., Goss, M. W., and Hart, I. R. (1991) Cancer Res. 51, 3281-3288). We now show that PKC alpha, delta, epsilon, and zeta isoforms are present at the protein level in quiescent, non-transformed Mel-ab melanocytes, maintained in the absence of phorbol ester. Proliferation of Mel-ab cells, achieved by incubation in the continual presence of phorbol 12,13-dibutyrate, was associated with a down-regulation of the PKC alpha, delta, and epsilon isozymes. Examination of two transformed syngeneic lines (the B16 murine melanoma and the long terminal repeat Ras.2 line), that grew in the absence of exogenous phorbol esters, showed that PKC alpha protein levels were either partially down-regulated or unaffected, the PKC delta and epsilon isoforms were down-regulated completely, and the levels of PKC zeta protein remained unaltered relative to quiescent Mel-ab cells. Basal levels of total diacylglycerol were elevated 5-fold in B16 melanoma cells compared with levels found in quiescent or proliferating Mel-ab melanocytes and appear to arise largely from the breakdown of phosphatidylinositol phospholipids accompanied by a significant rise in phospholipase C activity. Hourly treatments of quiescent Mel-ab melanocytes with the synthetic diacylglycerol analogue, 1,2-dioctanoyl-sn-glycerol, for 24 h, resulted in an induction of DNA synthesis which was associated with a significant down-regulation of PKC levels mediated largely via post-translational rather than transcriptional mechanisms. These results show for the first time that specific isoforms of PKC are down-regulated at the protein level during proliferation of murine melanocytic cells and suggest that the constitutive down-regulation of PKC in transformed melanoma cells may arise as a consequence of elevated endogenous phosphatidylinositol-derived diacylglycerol levels.

The effects of phorbol esters with different activities on protein kinase C

The sapintoxins are a series of naturally occurring fluorescent phorbol esters with a range of selective biological activities (e.g. pro-inflammatory but non-tumour promoting). Their ability to activate protein kinase C (PKC) in vitro has been studied. Both tumour promoting and non-promoting phorbol derivatives activate the enzyme in vitro at low concentrations. 12-deoxyphorbol-13-phenylacetate-20 acetate (DOPPA) acts as a partial agonist in the activation of protein kinase C. Structurally distinct phorbol esters may therefore preferentially activate different forms of protein kinase C. -sapinine, a biologically inactive compound, binds to protein kinase C without stimulating the enzyme and prevents subsequent activation by phorbol esters such as 12-O-tetradecanoyl phorbol-13-acetate (TPA).

The role of 80K/MARCKS, a specific substrate of protein kinase C, in cell growth and tumour progression

Since its discovery more than a decade ago [Wu et al., 1982; Rozengurt et al., 1983], the 80-87 kDa myristoylated a lanine-rich C-kinase substrate (80K/MARCKS) protein has attracted a great deal of attention from researchers interested in cell growth and tumour progression. However, despite its ubiquitous distribution, a definitive functional role for 80K/MARCKS has not been found. The purpose of this review is to describe the properties, distribution and regulation of 80K/MARCKS and to discuss some of the most recent findings, both from our laboratory and from others, that have suggested a functional role for this protein in modulating cell growth and tumour progression. Furthermore, I will present data from our laboratory that implicates 80K/MARCKS as a novel tumour suppressor in cells of melanocyte origin.

GTP binding and intramolecular regulation by the ROC domain of Death Associated Protein Kinase 1

The ROCO proteins are a family of large, multidomain proteins characterised by the presence of a Ras of complex proteins (ROC) domain followed by a COR, or C-terminal of ROC, domain. It has previously been shown that the ROC domain of the human ROCO protein Leucine Rich Repeat Kinase 2 (LRRK2) controls its kinase activity. Here, the ability of the ROC domain of another human ROCO protein, Death Associated Protein Kinase 1 (DAPK1), to bind GTP and control its kinase activity has been evaluated. In contrast to LRRK2, loss of GTP binding by DAPK1 does not result in loss of kinase activity, instead acting to modulate this activity. These data highlight the ROC domain of DAPK1 as a target for modifiers of this proteins function, and casts light on the role of ROC domains as intramolecular regulators in complex proteins with implications for a broad range of human diseases.