Targeting of the Akt/PKB kinase to the actin skeleton.

Serine/threonine kinase Akt/PKB intracellular distribution undergoes rapid changes in response to agonists such as Platelet-derived growth factor (PDGF) or Insulin-like growth factor (IGF). The concept has recently emerged that Akt subcellular movements are facilitated by interaction with nonsubstrate ligands. Here we show that Akt is bound to the actin skeleton in in situ cytoskeletal matrix preparations from PDGF-treated Saos2 cells, suggesting an interaction between the two proteins. Indeed, by immunoprecipitation and subcellular fractioning, we demonstrate that endogenous Akt and actin physically interact. Using recombinant proteins in in vitro binding and overlay assays, we further demonstrate that Akt interacts with actin directly. Expression of Akt mutants strongly indicates that the N-terminal PH domain of Akt mediates this interaction. More important, we show that the partition between actin bound and unbound Akt is not constant, but is modulated by growth factor stimulation. In fact, PDGF treatment of serum-starved cells triggers an increase in the amount of Akt associated with the actin skeleton, concomitant with an increase in Akt phosphorylation. Conversely, expression of an Akt mutant in which both Ser473 and Thr308 have been mutated to alanine completely abrogates PDGF-induced binding. The small GTPases Rac1 and Cdc42 seem to facilitate actin binding, possibly increasing Akt phosphorylation.

Insulin activates protein kinases C-zeta and C-lambda by an autophosphorylation-dependent mechanism and stimulates their translocation to GLUT4 vesicles and other membrane fractions in rat adipocytes.

In rat adipocytes, insulin provoked rapid increases in (a) endogenous immunoprecipitable combined protein kinase C (PKC)-zeta/lambda activity in plasma membranes and microsomes and (b) immunoreactive PKC-zeta and PKC-lambda in GLUT4 vesicles. Activity and autophosphorylation of immunoprecipitable epitope-tagged PKC-zeta and PKC-lambda were also increased by insulin in situ and phosphatidylinositol 3,4,5-(PO(4))(3) (PIP(3)) in vitro. Because phosphoinositide-dependent kinase-1 (PDK-1) is required for phosphorylation of activation loops of PKC-zeta and protein kinase B, we compared their activation. Both RO 31-8220 and myristoylated PKC-zeta pseudosubstrate blocked insulin-induced activation and autophosphorylation of PKC-zeta/lambda but did not inhibit PDK-1-dependent (a) protein kinase B phosphorylation/activation or (b) threonine 410 phosphorylation in the activation loop of PKC-zeta. Also, insulin in situ and PIP(3) in vitro activated and stimulated autophosphorylation of a PKC-zeta mutant, in which threonine 410 is replaced by glutamate (but not by an inactivating alanine) and cannot be activated by PDK-1. Surprisingly, insulin activated a truncated PKC-zeta that lacks the regulatory (presumably PIP(3)-binding) domain; this may reflect PIP(3) effects on PDK-1 or transphosphorylation by endogenous full-length PKC-zeta. Our findings suggest that insulin activates both PKC-zeta and PKC-lambda in plasma membranes, microsomes, and GLUT4 vesicles by a mechanism requiring increases in PIP(3), PDK-1-dependent phosphorylation of activation loop sites in PKC-zeta and lambda, and subsequent autophosphorylation and/or transphosphorylation.

Phosphatidylinositol 3-kinase translocation to the nucleus is induced by interleukin 1 and prevented by mutation of interleukin 1 receptor in human osteosarcoma Saos-2 cells.

Although interleukin 1 (IL-1) functions have been extensively characterized, the mechanisms by which IL-1 signals are transduced from the plasma membrane to the nucleus are less known. Recent evidence indicates that phosphatidylinositol 3-kinase (PI3-kinase) could be activated by a direct association with the activated IL-1 receptor. In this study we analyzed the effects of IL-1 on the intracellular distribution of PI3-kinase in wild-type Saos-2 human osteosarcoma cells, and in cell clones overexpressing type I IL-1 receptor (IL-1RI). PI3-kinase intracellular distribution displays two distinct patterns. In quiescent cells, PI3-kinase is distributed through the cytoplasm, although a portion is present in the nucleus; following stimulation with IL-1, PI3-kinase is redistributed, increasing in the nuclear compartment. Both immunoblotting and immunofluorescence data indicate that IL-1 causes a rapid and transient translocation of PI3-kinase from the cytoplasm to the nucleus. This phenomenon is prevented by PI3-kinase inhibitors, suggesting that the maintenance of PI3-kinase activity is essential for IL-1-induced translocation. Indeed, in cell clones stably transfected with Y479F receptor mutant, in which the binding of the enzyme to the activated receptor is blocked, IL-1-induced PI3-kinase translocation to the nucleus is completely prevented. These data suggest that PI3-kinase translocation to the nucleus upon IL-1R activation is an early event in IL-1 signaling mechanism, and may be involved in transcriptional activation.

Phosphatidylinositol 3-kinase is recruited to a specific site in the activated IL-1 receptor I.

Interleukin 1 (IL-1) delivers a stimulatory signal which increases the expression of a set of genes by modulating the transcription factor NF-kappaB. The IL-1 receptors are transmembrane glycoproteins which lack a catalytic domain. The C-terminal portion of the type I IL-1 receptor (IL-IRI) is essential for IL-1 signalling and for IL-1 dependent activation of NF-kappaB. This portion contains a putative phosphatidylinositol 3-kinase (PI 3-kinase) binding domain (Tyr-E-X-Met), which is highly conserved between the human, mouse and chicken sequences, as well as the related cytoplasmic domain of the Drosophila receptor Toll. This observation prompted us to investigate the role of PI 3-kinase in IL-1 signalling. Here we report evidence that PI 3-kinase is recruited by the activated IL-IRI, causing rapid and transient activation of PI 3-kinase. We also show that the receptor is tyrosine phosphorylated in response to IL-1. Expression of a receptor mutant lacking the putative binding site for p85 demonstrates that Tyr479 in the receptor cytoplasmic domain is essential for PI 3-kinase activation by IL-1. Our results indicate that PI 3-kinase is likely to be an important mediator of some IL-1 effects, providing docking sites for additional signalling molecules.