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 (alpha, gamma, and epsilon) 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.
Highlights
An adult feline right ventricular pressure overload (RVPO) model was used to examine the two S6 kinase (S6K) isoforms, p70S6K and p85S6K, that are involved in translational and transcriptional activation
Differential S6 Kinase Activation in Cardiac Hypertrophy ferent approaches were utilized to study the changes associated with S6K isoforms: (i) Western blot analysis with either regular S6K antibody or with phosphospecific S6K antibodies for measuring phosphorylation, (ii) immune complex assays for kinase activity, and (iii) confocal immunostaining studies for determining phosphorylation occurring at the level of cardiocytes in the pseudosubstrate domain of cytoplasmic (p70S6K) as well as nuclear (p85S6K) isoforms of S6K
To demonstrate whether S6K isoforms are activated in pressure-overloaded myocardium, pressure-overloaded right ventricles and normally loaded same-animal control left ventricles were used to obtain total lysates by extracting with Triton X-100 lysis buffer as described under “Experimental Procedures.”
Summary
S6K, p70 and p85 S6 kinase isoforms; LV, left ventricle; RV, right ventricle; RVPO, right ventricular pressure appear to be involved in phosphorylation of the S6 protein [2, 6, 7]. In addition to playing a role in protein translational control, nuclear p85S6K has been shown to be important for transcriptional activation [13], increased DNA synthesis, and G1 to S phase transition [14] Both isoforms of S6K undergo phosphorylation on multiple sites, which are largely classified into two groups ( the same numbering is used for both p70S6K and p85S6K, 23 residues should be added to convert to the numbering of p85S6K). The second set of phosphorylation sites consists of four Ser/Thr residues (Ser411, Ser-418, Thr-421, and Ser-424) in the pseudosubstrate region of S6K ( known as autoinhibitory domain), and their phosphorylation, except Ser-411, is independent of the rapamycin-sensitive pathway [15] Phosphorylation of these residues in the pseudosubstrate domain has been shown to play a role in regulating kinase function in vivo, it is not essential for catalytic activity [17, 18]. Our studies indicate that this mechanically induced signaling pathway is mediated by PKC and is independent of PI 3-kinase
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