Phosphoinositide-dependent protein kinase-1 (PDK1) is a recently identified serine/threonine kinase that phosphorylates and activates Akt and p70 S6K, two downstream kinases of phosphatidylinositol 3-kinase. To further study the potential role of PDK1, we have screened a mouse liver cDNA library and identified a cDNA encoding the enzyme. The predicted mouse PDK1 (mPDK1) protein contained 559 amino acids and a COOH-terminal pleckstrin homology domain. A 7-kilobase mPDK1 mRNA was broadly expressed in mouse tissues and in embryonic cells. In the testis, a high level expression of a tissue-specific 2-kilobase transcript was also detected. Anti-mPDK1 antibody recognized multiple proteins in mouse tissues with molecular masses ranging from 60 to 180 kDa. mPDK1 phosphorylated the conserved threonine residue (Thr 402 ) in the activation loop of protein kinase C-and activated the enzyme in vitro and in cells. Our findings suggest that there may be different isoforms of mPDK1 and that the protein is an upstream kinase that activates divergent pathways downstream of phosphatidylinositol 3-kinase.

Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding-and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics.Raf kinase inhibitory protein (RKIP/PEBP1) is a signaling modulator that regulates key signal transduction cascades in mammalian cells (reviewed in reference 16). A negative regulator of mitogen-activated protein kinase (MAPK) signaling (42), RKIP inhibits Raf kinase by binding directly to Raf-1, thereby preventing the phosphorylation and activation of 38). RKIP functions as a regulator of the spindle checkpoint and promotes genomic stability by preventing MAPK from inhibiting Aurora B kinase (10). Consistent with this role, RKIP suppresses lung metastasis by prostate tumor cells in an orthotopic murine model (15). RKIP may be a general metastasis suppressor for solid tumors, since RKIP expression is low or undetectable in prostate and breast tumors, melanoma, hepatocellular carcinoma, and colorectal tumors (1,2,14,15,19,34). Finally, RKIP suppresses NF-B activation (43), inhibits G protein-coupled receptor (GPCR) kinase 2 (GRK2)-mediated downregulation of GPCRs (28), and potentiates the efficacy of chemotherapeutic agents (5). Thus, RKIP regulates three key mammalian signaling pathways involving MAPK, GPCR, and NF-B signaling.RKIP is a member of the phosphatidylethanolamine binding protein (PEBP) family, which extends from bacteria to humans and consists of more than 400 proteins (16, 33). X-ray crystallographic studies have demonstrated that highly conserved sequences cluster around a pocket capable of binding anions, including o-phosphorylethanolamine (PE), acetate, and cacodylate (3,35). This pocket is the only clearly identifiable feature for potent...

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Individuals with developmental language disabilities, including developmental dyslexia and specific language impairment (SLI), exhibit impairments in processing rapidly presented auditory stimuli. It has been hypothesized that these deficits are associated with concurrent deficits in speech perception and, in turn, impaired language development. Additionally, postmortem analyses of human dyslexic brains have revealed the presence of focal neocortical malformations such as cerebrocortical microgyria. In an initial study bridging these research domains, we found that male rats with induced microgyria were impaired in discriminating rapidly presented auditory stimuli. In order to further assess this anatomical- behavioral association, we designed two experiments using auditory-reflex modification. These studies were intended to assess whether auditory processing deficits in microgyric male rats would be seen in threshold detection of a silent gap in white noise, and in oddball detection of a two-tone stimulus of variable duration. Results showed no differences between sham and microgyric subjects on gap detection, but did show that microgyric subjects were impaired in the discrimination of two-tone stimuli presented in an oddball paradigm. This impairment was evident for stimuli with total duration of 64 msec or less, while both groups were able to discriminate stimuli with duration of 89 msec or greater. The current results further support the relationship between malformations of the cerebral cortex and deficits in rapid auditory processing. They also suggest that the parameters characterizing rapid auditory processing deficits for a specific task may be influenced by stimulus features and/or cognitive demand of that particular task.

BackgroundRaf Kinase Inhibitory Protein (RKIP, also PEBP1), a member of the Phosphatidylethanolamine Binding Protein family, negatively regulates growth factor signaling by the Raf/MAP kinase pathway. Since an organic compound, locostatin, was reported to bind RKIP and inhibit cell migration by a Raf-dependent mechanism, we addressed the role of RKIP in locostatin function.Methods/FindingsWe analyzed locostatin interaction with RKIP and examined the biological consequences of locostatin binding on RKIP function. NMR studies show that a locostatin precursor binds to the conserved phosphatidylethanolamine binding pocket of RKIP. However, drug binding to the pocket does not prevent RKIP association with its inhibitory target, Raf-1, nor affect RKIP phosphorylation by Protein Kinase C at a regulatory site. Similarly, exposure of wild type, RKIP-depleted HeLa cells or RKIP-deficient (RKIP−/−) mouse embryonic fibroblasts (MEFs) to locostatin has no effect on MAP kinase activation. Locostatin treatment of wild type MEFs causes inhibition of cell migration following wounding. RKIP deficiency impairs migration further, indicating that RKIP protects cells against locostatin-mediated inhibition of migration. Locostatin treatment of depleted or RKIP−/− MEFs reveals cytoskeletal disruption and microtubule abnormalities in the spindle.Conclusions/SignificanceThese results suggest that locostatin's effects on cytoskeletal structure and migration are caused through mechanisms independent of its binding to RKIP and Raf/MAP kinase signaling. The protective effect of RKIP against drug inhibition of migration suggests a new role for RKIP in potentially sequestering toxic compounds that may have deleterious effects on cells.