The immunosuppressant rapamycin inhibited proliferation of the H4IIEC hepatoma cell line. Rapamycin, but not its structural analog FK506, also inhibited the basal and insulin-stimulated activity of the p70 ribosomal protein S6 kinase. By contrast, insulin stimulation of the p85 Rsk S6 kinase and mitogen-activated protein (MAP) kinase activity were unaffected by drug. Rapamycin treatment of COS cells transfected with recombinant p70 S6 kinase completely inhibited the appearance of the hyperphosphorylated form of p70 S6 kinase concomitant with the inhibition of enzyme activity toward 40S subunits. Thus, rapamycin inhibits a signal transduction element that is necessary for the activation of p70 S6 kinase and mitogenesis but unnecessary for activation of p85 Rsk S6 kinase or MAP kinase.

Despite the success of protein kinase inhibitors as approved therapeutics, drug discovery has focused on a small subset of kinase targets. Here we provide a thorough characterization of the Published Kinase Inhibitor Set (PKIS), a set of 367 small-molecule ATP-competitive kinase inhibitors that was recently made freely available with the aim of expanding research in this field and as an experiment in open-source target validation. We screen the set in activity assays with 224 recombinant kinases and 24 G protein-coupled receptors and in cellular assays of cancer cell proliferation and angiogenesis. We identify chemical starting points for designing new chemical probes of orphan kinases and illustrate the utility of these leads by developing a selective inhibitor for the previously untargeted kinases LOK and SLK. Our cellular screens reveal compounds that modulate cancer cell growth and angiogenesis in vitro. These reagents and associated data illustrate an efficient way forward to increasing understanding of the historically untargeted kinome.

Two classes of human cDNA encoding the insulin/mitogen-activated p70 S6 kinase have been isolated; the two classes differ only in the 5' region, such that the longer polypeptide (p70 S6 kinase aI; calculated Mr 58,946) consists of 525 amino acids, of which the last 502 residues are identical in sequence to the entire polypeptide encoded by the second cDNA (p70 S6 kinase all; calculated Mr 56,153). Both p70 S6 kinase polypeptides predicted by these cDNAs are present in p70 S6 kinase purified from rat liver, and each is thus expressed in vivo. Moreover, both (6,33). This family will be referred to henceforth as the p85 S6 kinases.Despite fairly ubiquitous expression (1), the p85 kinases are not the quantitatively dominant mitogen-stimulated kinases toward 40S S6 in mammalian cells. Purification of 5541

The molecular structure of a rat hepatoma 70-kDa insulin/mitogen-stimulated S6 protein kinase, obtained by molecular cloning, is compared to that of a rat homolog of the 85-kDa Xenopus S6 protein kinase a; both kinases were cloned from H4 hepatoma cDNA libraries Most efforts at purification of mitogen-stimulated S6 kinase activity from avian and mammalian sources have recovered 65-to 70-kDa polypeptides (10-12). We purified an hepatic 70-kDa S6 kinase, activated by cycloheximide treatment of the rat; this very low abundance enzyme cochromatographed with the major hepatic S6 kinase activated during liver regeneration and after insulin treatment of H4 hepatoma cells (13). The latter enzyme was also purified extensively from 32P-labeled H4 hepatoma cells and found to correspond to a 70-kDa 32P-labeled polypeptide, which is phosphorylated on serine residues in response to insulin, concomitant with activation (5). Like the Xenopus S6 kinase II, the hepatic p70 S6 kinase is also deactivated by protein phosphatase 2A; these two S6 kinases, however, are immunochemically distinct (D.J.P., J.A., E. Erikson, and J.