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.Received 10 September 2003; accepted 25 September 2003     

Dephosphorylation and inactivation of Akt/PKB is counteracted by protein kinase CK2 in HEK 293T cells

Akt (PKB) is a critical kinase in cell-survival pathways. Its activity depends on the phosphorylation of Thr308 and Ser473, by PDK1 and mTORC2, respectively. We found that Akt can be further stimulated through phosphorylation of Ser129 by another kinase, CK2. Here we show that phosphorylation of Akt at Ser129 also facilitates its association with Hsp90 chaperone, thus preventing Thr308 dephosphorylation. This is supported by the following observations: (1) phospho-Thr308 decreases when Ser129 is mutated to alanine, (2) this decrease is abolished by cell treatment with okadaic acid (to inactivate PP2A) or geldanamycin (to inactivate Hsp90), (3) phosphorylation of Ser129 neither enhances the activity of PDK1 nor hampers the in vitro activity of PP2A on Thr308, but increases the Hsp90 association to Akt. These data support the view that the antiapoptotic potential of CK2 is at least in part mediated by its ability to maintain Akt in its active form.     

Isoform specific phosphorylation of p53 by protein kinase CK1

The ability of three isoforms of protein kinase CK1 (α, γ₁, and δ) to phosphorylate the N-terminal region of p53 has been assessed using either recombinant p53 or a synthetic peptide reproducing its 1–28 sequence. Both substrates are readily phosphoylated by CK1δ and CK1α, but not by the γ isoform. Affinity of full size p53 for CK1 is 3 orders of magnitude higher than that of its N-terminal peptide (K _m 0.82 μM vs 1.51 mM). The preferred target is S20, whose phosphorylation critically relies on E17, while S6 is unaffected despite displaying the same consensus (E-x-x-S). Our data support the concept that non-primed phosphorylation of p53 by CK1 is an isoform-specific reaction preferentially affecting S20 by a mechanism which is grounded both on a local consensus and on a remote docking site mapped to the K²²¹RQK²²⁴ loop according to modeling and mutational analysis.     

Hyperpolarization by cyclic AMP (activation of diphosphoinositide kinase)

Zusammenfassung In-vitro-und In-vivo-Versuche beweisen, dass ein molekulärer Mechanismus für das Zustandekommen der Membran-Hyperpolarisation verantwortlich ist.

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Presented in part at the Annual Meeting of the Biophysical Society, Toronto, Canada, February 1972.     

Covalent and noncovalent modifiers of the p53 protein

Despite the massive attention it has received, there is still much to learn about the p53 tumour suppressor protein. Given that it plays complex and multiple roles in cells, it is not surprising that p53 is subjected to an intricate array of regulatory processes. p53 receives signals from cells in multiple ways, leading to its stabilization and activation. The functions of the protein are altered by phosphorylation and other covalent modifications. However, a number of proteins can regulate p53 function dramatically by noncovalent means. p53 is thus subjected to numerous signalling and regulatory pathways which we have only begun to decipher.     

Conversion of mammalian cyclic GMP-dependent protein kinase into modulator-dependent protein kinase (type II) in vitro

The spontaneous conversion of mammalian cyclic GMP-dependent protein kinase (G-PK) into modulator-dependent protein kinase (type II) (M-PKII) in the absence of cGMP or histone was observed in vitro. The findings, together with similarity in substrate protein specificity, suggest that M-PKII is the catalytic subunit of mammalian G-PK.     

Separation of modulator-dependent protein kinase (type I) from cyclic GMP-dependent protein kinase in mouse testes

A new type of enzyme, modulator-dependent protein kinase (type I) (M-PKI), was successfully isolated from the cytosol fraction of mouse testes. It was eluted slightly after the peak of cyclic GMP-dependent protein kinase (G-PK) by Sephadex G-200 gel filtration. Unlike either cyclic AMP-dependent protein Kinase (A-PK) or G-PK, its maximal activity depended exclusively on the presence of crude protein kinase modulators (PKM) or partially purified stimulatory modulator (PKMs).     

Calcium-mediated activation of PI3K and p53 leads to apoptosis in thyroid carcinoma cells

The molecular mechanism responsible for cadmium-induced cell death in thyroid cancer cells (FRO) is unknown. We demonstrated that apoptosis of FRO cells induced by cadmium was concentration and time dependent. Cadmium caused the rapid elevation of intracellular calcium and induced phosphorylation of Akt, p53, JNK, ERK and p38. Inhibition of PI3K/Akt attenuated the cadmium-induced apoptosis, but the inhibition of JNK inhibitor, ERK or p38 aggravated it, indicating that activation of PI3K/Akt was a pro-apoptosis signal in response to cadmium treatment, whereas the activation of stress-activated protein kinase JNK, ERK and p38 functioned as survival signals to counteract the cadmium-induced apoptosis. Buffering of the calcium response attenuated mitochondrial impairment, recovered the cadmium-activated Akt, p53, JNK, ERK and p38, and subsequently blocked the apoptosis. These results suggested that apoptosis induced by cadmium in FRO cells was initiated by the rapid elevation of intracellular calcium, followed by calcium-mediated activation of PI3K/Akt and mitochondrial impairment. Received 28 February 2007; received after revision 2 April 2007; accepted 23 April 2007     

Membrane shaping by the Bin/amphiphysin/Rvs (BAR) domain protein superfamily

BAR domain superfamily proteins have emerged as central regulators of dynamic membrane remodeling, thereby playing important roles in a wide variety of cellular processes, such as organelle biogenesis, cell division, cell migration, secretion, and endocytosis. Here, we review the mechanistic and structural basis for the membrane curvature-sensing and deforming properties of BAR domain superfamily proteins. Moreover, we summarize the present state of knowledge with respect to their regulation by autoinhibitory mechanisms or posttranslational modifications, and their interactions with other proteins, in particular with GTPases, and with membrane lipids. We postulate that BAR superfamily proteins act as membrane-deforming scaffolds that spatiotemporally orchestrate membrane remodeling.     

Phosphorylation of p27BBP/eIF6 and its association with the cytoskeleton are developmentally regulated in Xenopus oogenesis

p27^BBP/eIF6 is an evolutionarily conserved regulator of ribosomal function. It is necessary for 60S biogenesis and impedes improper joining of 40S and 60S subunits, regulated by protein kinase C or Efl1p. No data on p27^BBP/eIF6 during early development of Metazoa are available. We studied the distribution, post-translational changes and association with the cytoskeleton of p27^BBP/ eIF6 during Xenopus oogenesis and early development. Results indicate that p27^BBP/eIF6 is present throughout oogenesis, partly associated with 60S subunits, partly free and with little cytoskeleton bound. During prophase I, p27^BBP/eIF6 is detected as a single band of 27-kDa. Upon maturation induced by progesterone or protein kinase C, a serine-phosphorylated 29 kDa isoform appears and is kept throughout development to the neurula stage. Confocal microscopy showed that the distribution of p27^BBP/eIF6 and its association with the cytoskeleton varies according to oogenesis stages. Briefly, in stage 6 oocytes, p27^BBP/eIF6 has a limited dot-like distribution, and does not co-localize with cytokeratin, whereas upon maturation it spreads throughout the cytoplasm. After fertilization, a large fraction coalesces around cytomembranes and a cytochalasin B-sensitive co-localization with cytokeratin occurs. RNAse removes p27^BBP/eIF6 from the cytokeratin fibres. Developmental data suggest a role of p27^BBP/eIF6 in controlling ribosomal availability or regulating cross-talk between ribosomes and the cytoskeleton.Received 7 April 2005; received after revision 11 May 2005; accepted 25 May 2005R. Carotenuto and N. De Marco contributed equally to the paper     

A rapid assay for the localization of protein kinase modulator (inhibitor) in complete polyacrylamide gels

Summary A gel assay system for determination of inhibitory proteins for protein kinase(s) on isoelectric focusing gels has been developed. Preparations of heat-stable inhibitors were applied, focused, and the complete gels incubated with protein kinase in the presence of substrate protein. At the position where inhibitory protein had focused, the phosphorylation reaction was blocked selectively.This investigation was supported by the Deutsche Forschungsgemeinschaft.     

Effect of protein kinase C activation and depletion on insulin stimulation of glycogen synthesis in cultured hepatoma cells

Summary Insulin stimulation of glycogen synthesis was nearly abolished in hepatoma cells shortly treated with 4 ß-phorbol 12 \-myristate, 13 -acetate (protein kinase C activation) but remained unmodified in cells chronically treated with the phorbol ester (protein kinase C depletion). Thus, although exogenous activation of protein kinase C results in an inhibition of insulin action, protein kinase C depletion has no influence on this process. The results suggest that, in hepatoma cells, no endogenous activation of protein kinase C may occur in response to the signal triggered by insulin.