Lijun Wang

Ras/Erk Signaling Is Essential for Activation of Protein Synthesis by Gq Protein-Coupled Receptor Agonists in Adult Cardiomyocytes

Abstract— The Gq protein-coupled receptor agonists phenylephrine (PE) and endothelin-1 (ET-1) induce cardiac hypertrophy and stimulate protein synthesis in cardiomyocytes. This study aims to investigate how they activate mRNA translation in adult cardiomyocytes. PE and ET-1 do not activate protein kinase B but stimulate Ras and Erk, and their ability to activate protein synthesis was blocked by inhibition of Ras or MEK and by rapamycin, which inhibits mTOR (mammalian target of rapamycin). These agonists activated ribosomal protein S6 kinase 1 (S6K1) and induced phosphorylation of eIF4E-binding protein-1 (4E-BP1) and its release from eIF4E. These effects were blocked by inhibitors of MEK. Furthermore, adenovirus-mediated expression of constitutively-active MEK1 caused activation of S6K1, phosphorylation of 4E-BP1, and activation of protein synthesis in a rapamycin-sensitive manner. Expression of N17Ras inhibited the regulation of S6K1 and protein synthesis by GqPCR agonists. These data point to a signaling pathway involving Ras and MEK that acts, with mTOR, to control regulatory translation factors and activate protein synthesis. This study provides new insights into the mechanisms underlying the stimulation of protein synthesis by hypertrophic agents in heart.

Protein Kinase C Phosphorylates Ribosomal Protein S6 Kinase βII and Regulates Its Subcellular Localization

ABSTRACT The ribosomal protein S6 kinase (S6K) belongs to the AGC family of Ser/Thr kinases and is known to be involved in the regulation of protein synthesis and the G1/S transition of the cell cycle. There are two forms of S6K, termed S6Kα and S6Kβ, which have cytoplasmic and nuclear splice variants. Nucleocytoplasmic shuttling has been recently proposed for S6Kα, based on the use of the nuclear export inhibitor, leptomycin B. However, the molecular mechanisms regulating subcellular localization of S6Ks in response to mitogenic stimuli remain to be elucidated. Here we present data on the in vitro and in vivo phosphorylation of S6Kβ, but not S6Kα, by protein kinase C (PKC). The site of phosphorylation was identified as S486, which is located within the C-terminal nuclear localization signal. Mutational analysis and the use of phosphospecific antibodies provided evidence that PKC-mediated phosphorylation at S486 does not affect S6K activity but eliminates the function of its nuclear localization signal and causes retention of an activated form of the kinase in the cytoplasm. Taken together, this study uncovers a novel mechanism for the regulation of nucleocytoplasmic shuttling of S6KβII by PKC-mediated phosphorylation.

Regulation of the phosphorylation of elongation factor 2 by MEK‐dependent signalling in adult rat cardiomyocytes

The Gq‐coupled agonists phenylephrine and endothelin‐1 each activate protein synthesis in cardiomyocytes as part of the programme that leads to cardiac hypertrophy. Here we show that they each induce the dephosphorylation of elongation factor (eEF) 2, a protein that in its dephosphorylated state mediates the translocation step of elongation. The ability of both agonists to induce dephosphorylation of eEF2 requires signalling via the mTOR and MEK/Erk signalling pathways, but is independent of phosphoinositide 3‐kinase. Expression of an activated form of MEK leads to dephosphorylation of eEF2, in an mTOR independent manner, indicating that signalling via MEK/Erk suffices to cause dephosphorylation of eEF2.

β-Adrenergic agonists increase phosphorylation of elongation factor 2 in cardiomyocytes without eliciting calcium-independent eEF2 kinase activity

The β‐adrenergic agonist isoproterenol increased the phosphorylation of elongation factor eEF2 in ventricular cardiomyocytes from adult rats (ARVC). Phosphorylation of eEF2 inhibits its activity, and protein synthesis was inhibited in ARVC concomitantly with increased eEF2 phosphorylation. eEF2 kinase activity in ARVC extracts was completely dependent upon Ca2+/calmodulin. In contrast to other cell types, however, treatments designed to raise intracellular cAMP failed to induce Ca2+/calmodulin‐independent activity. Instead, they increased maximal eEF2 kinase activity. Similar data were obtained when partially purified ARVC eEF2 kinase was treated with cAMP‐dependent protein kinase in vitro. These data suggest that ARVC possess a distinct isoform of eEF2 kinase.

Muscarinic receptor-mediated activation of p70 S6 kinase 1 (S6K1) in 1321N1 astrocytoma cells: permissive role of phosphoinositide 3-kinase

In 1321N1 astrocytoma cells, carbachol stimulation of M3 muscarinic cholinergic receptors, coupled to phospholipase C, evoked a persistent 10-20-fold activation of p70 S6 kinase (S6K1). This response was abolished by chelation of cytosolic Ca2+ and reproduced by the Ca2+ ionophore ionomycin, but was not prevented by down-regulation or inhibition of protein kinase C. Carbachol-stimulated activation and phosphorylation of S6K1 at Thr389 were prevented by rapamycin, an inhibitor of mTOR (mammalian target of rapamycin), or by wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor. Carbachol also stimulated the phosphorylation of eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), a second mTOR-dependent event, with similar potency to its effect on S6K1. This response was blocked by rapamycin, but was not markedly affected by 100 nM wortmannin, implying separate roles for mTOR and PI3K in S6K1 activation. Wortmannin abolished the carbachol-stimulated rise in PtdIns(3,4,5)P3 and greatly reduced unstimulated levels of this lipid. By contrast, an inhibitor of epidermal growth factor receptor kinase, AG1478, which prevents carbachol-stimulated ErbB3 transactivation, PI3K recruitment and protein kinase B activation in 1321N1 cells, reduced activation of S6K1 by no more than 30%. This effect was overcome by 10 nM insulin, which on its own did not stimulate S6K1, but increased cellular PtdIns(3,4,5)P3 concentrations comparably with carbachol alone. These observations distinguish obligatory roles for mTOR and PI3K in regulating S6K1, but imply that minimal PI3K activity is sufficient to permit stimulation of S6K1 by other activating factors such as increased cytosolic Ca2+ concentrations, which are essential to the muscarinic receptor-mediated response. Moreover, 4E-BP1 and hence, presumably, mTOR can be regulated independently of PI3K activation through these mechanisms.

Ca2+-independent protein kinase C activity is required for α1-adrenergic-receptor-mediated regulation of ribosomal protein S6 kinases in adult cardiomyocytes

The alpha(1)-adrenergic agonist, phenylephrine (PE), exerts hypertrophic effects in the myocardium and activates protein synthesis. Both Ca(2+)-dependent protein kinase C (PKC, PKCalpha) and Ca(2+)-independent PKC isoforms (PKCdelta and epsilon ) are detectably expressed in adult rat cardiomyocytes. Stimulation of the alpha(1)-adrenergic receptor by PE results in activation of Ca(2+)-independent PKCs, as demonstrated by translocation of the delta and epsilon isoenzymes from cytosol to membrane fractions. PE also induces activation of p70 ribosomal protein S6 kinases (S6K1 and 2) in adult cardiomyocytes. We have studied the role of Ca(2+)-independent PKCs in the regulation of S6K activity by PE. Activation of S6K1/2 by PE was blocked by the broad-spectrum PKC inhibitor bisindolylmaleimide (BIM) I, whereas Gö6976, a compound that only inhibits Ca(2+)-dependent PKCs, did not inhibit S6K activation. Rottlerin, which selectively inhibits PKCdelta, also prevented PE-induced S6K activation. The isoform-specific PKC inhibitors had similar effects on the phosphorylation of eukaryotic initiation factor 4E (eIF4E)-binding protein 1, a translation repressor that, like the S6Ks, lies downstream of the mammalian target of rapamycin (mTOR). Infection of cells with adenoviruses encoding dominant-negative PKCdelta or epsilon inhibited the activation of extracellular-signal-regulated kinase (ERK) by PE, and also inhibited the activation and/or phosphorylation of S6Ks 1 and 2. The PE-induced activation of protein synthesis was abolished by BIM I and markedly attenuated by rottlerin. Our data thus suggest that Ca(2+)-independent PKC isoforms play an important role in coupling the alpha(1)-adrenergic receptor to mTOR signalling and protein synthesis in adult cardiomyocytes.