Given the central position of the focal adhesion complex, both physically in coupling integrins to the interstitium and biochemically in providing an upstream site for anabolic signal generation, we asked whether the recruitment of non-receptor tyrosine kinases to the cytoskeleton might be a mechanism whereby cellular loading could activate growth regulatory signals responsible for cardiac hypertrophy. Analysis revealed cytoskeletal association of c-Src, FAK, and ␤3-integrin, but no Fyn, in the pressure-overloaded right ventricle. This association was seen as early as 4 h after right ventricular pressure overloading, increased through 48 h, and reverted to normal in 1 week. Cytoskeletal binding of nonreceptor tyrosine kinases was synchronous with tyrosine phosphorylation of several cytoskeletal proteins, including c-Src. Examination of cytoskeleton-bound cSrc revealed that a significant portion of the tyrosine phosphorylation was not at the Tyr-527 site and therefore presumably was at the Tyr-416 site. Thus, these studies strongly suggest that non-receptor tyrosine kinases, in particular c-Src, may play a critical role in hypertrophic growth regulation by their association with cytoskeletal structures, possibly via load activation of integrin-mediated signaling.

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...

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