Differential Activation of p70 and p85 S6 Kinase Isoforms during Cardiac Hypertrophy in the Adult Mammal

Abstract

An adult feline right ventricular pressure overload (RVPO) model was used to examine the two S6 kinase (S6K) isoforms, p70 S6K and p85 S6K , that are involved in translational and transcriptional activation. Biochemical and confocal microscopy analyses at the level of the cardiocyte revealed that p70 S6K is present predominantly in the cytosol, substantially activated in 1-h RVPO (>12 fold), and phosphorylated in the pseudosubstrate domain at the Ser-411, Thr-421, and Ser-424 sites. p85 S6K , which was localized exclusively in the nucleus, showed activation subsequent to p70 S6K , with a sustained increase in phosphorylation for up to 48 h of RVPO at equivalent sites of p70 S6K , Thr-421 and Ser-424, but not at Ser-411. Neither isoform translocated between the cytosol and the nucleus. Further studies to determine potential upstream elements of S6K activation revealed: (i) similar time course of activation for protein kinase C isoforms (␣, ␥, and ⑀) and c-Raf, (ii) absence of accompanying phosphatidylinositol 3-kinase activation, (iii) activation of c-Src subsequent to p70 S6K , and (iv) similar changes in adult cardiocytes after treatment with 12-O-tetradecanoylphorbol-13-acetate. Thus, these studies suggest that a protein kinase C-mediated pathway couples pressure overload to growth induction via differential activation of S6K isoforms in cardiac hypertrophy.Hypertrophic cardiac growth occurs by an increase in cellular mass via a relatively coordinate increase in cellular protein content (1). In the terminally differentiated adult cardiac muscle cell, translational mechanisms have a key role in regulating the rate of protein synthesis during hypertrophic growth. Phosphorylation of the carboxyl terminus of 40 S ribosomal S6 protein (S6 protein) has been shown to regulate protein synthesis rate by mediating protein translational initiation and elongation (2), and a growing number of studies show strong correlation between translational efficiency and S6 protein phosphorylation (3-5). Two distinct families of S6 kinases, p90Rsk (ribosomal S6 kinase) and p70/85 S6 kinase (S6K), 1 appear to be involved in phosphorylation of the S6 protein (2, 6, 7). However, S6K is suggested to be primarily responsible for the S6 protein phosphorylation upon stimulation of cells with insulin and a variety of mitogenic stimuli (8 -10). S6K consists of two isoforms transcribed by a single gene due to alternative splicing: a 70-kDa cytoplasmic isoform (p70 S6K ) and a 85-kDa nuclear isoform (p85 S6K ) with an additional 23 residues at the amino terminus consisting of a putative nuclear targeting sequence (11). Both isoforms are known to phosphorylate the S6 protein and mediate the translation of polypyrimidine tract mRNA, including transcripts of ribosomal proteins and translational elongation factors (12). In addition to playing a role in protein translational control, nuclear p85 S6K has been shown to be important for transcriptional activation (13), increased DNA synthesis, and G 1 to S phase transition (14).For activation, both is...

c-Raf/MEK/ERK Pathway Controls Protein Kinase C-mediated p70S6K Activation in Adult Cardiac Muscle Cells

“…In the case of the p85 isoform, Thr-389 phosphorylation was significant in 4-and 24-h pressure-overloaded myocardium, whereas for the Thr-421/Ser-424 sites, significant phosphorylation was observed as early as 1 h and persisted up to at least 48 h of pressure overloading. The pattern of S6K1 activation matches precisely with our earlier studies performed under similar conditions (42). Overall, these data demonstrate once again that S6K1 is activated to a substantial level as early as 1 h of pressure overloading of the myocardium, and the activation is sustained for at least 24 h. Interestingly, the S6K1 activation is also accompanied by an increased mTOR phosphorylation at the Ser-2448 site.…”

“…Several independent signaling pathways have been identified for S6K1 activation depending upon cell types and the nature of the stimulants (30 -36), and these pathways are activated subsequent to at least three independent agonists' stimulation: (i) TPA that activates a PKC-dependent pathway, (ii) insulin that activates a PI3K-dependent pathway, and (iii) forskolin that activates a protein kinase A (PKA)-dependent pathway. Activation of all these pathways has been demonstrated in pressure-overloaded myocardium by several research groups (58,61,(65)(66)(67), although our earlier study (42) demonstrated that the S6K1 activation was accompanied by the activation of PKC but not PI3K pathway in 1-to 4-h pressure overload myocardium. Therefore, we used cultured adult feline cardiocytes to explore the importance of potential downstream players of PKC, namely, c-Raf, MEK, and MAPK family members for S6K1 activation.…”

“…The link between S6K1 activation and cell growth has been well documented in other cell types using rapamycin (10,11,25,57) and through microinjection of neutralizing antibodies into cells (15,17) both of which selectively suppressed S6K1 activation and impeded cell growth. S6K1 activation has been demonstrated in hypertrophying myocardium (42,58) and in cardiocytes stimulated with hypertrophic agents (39, 59 -61). However, the major signaling pathway for S6K1 activation and its importance in the context of hypertrophic growth has not been studied in detail.…”

“…In 1-to 4-h pressure-overloaded feline myocardium, our recent study (42) showed that the PKC, but not the PI3K-dependent pathway, contributes significantly to S6K activation. In the present study, we observed that this activation was also accompanied by the activation of the c-Raf/MAPK pathway.…”

Rapamycin Attenuates Load-Induced Cardiac Hypertrophy in Mice

“…12,13 Furthermore, S6K1 was activated in a cat model of right ventricular pressure overload, suggesting that mTOR or its target(s) might be involved in load-induced cardiac hypertrophy. 14 To determine the role of mTOR or its target(s) in load-induced cardiac hypertrophy, we administered a clinically relevant dose of rapamycin to mice subjected to ascending aortic constriction.…”