TGF-beta(1) modulates NOS expression and phosphorylation of Akt/PKB in rat myocytes exposed to hypoxia-reoxygenation

Myocardial hypoxia-reoxygenation (H-R) is associated with upregulation of inducible nitric oxide synthase (iNOS), decrease in endothelial NOS (eNOS), and increase in protein kinase B (PKB). Previous work also shows that transforming growth factor-beta(1) (TGF-beta(1)) can attenuate myocardial injury induced by H-R. We examined the modulation of NOS and PKB expression in response to H-R by TGF- beta(1). Myocytes from Sprague-Dawley rat hearts were cultured and exposed to hypoxia (95% N(2)-5% CO(2), PO(2) ~30 mmHg) for 24 h and reoxygenation (95% air-5% CO(2)) for 3 h. Myocytes were then examined for lactate dehydrogenase (LDH) release, iNOS activity (conversion of L-[(3)H]arginine to L-[(3)H]citrulline), iNOS and eNOS expression, and PKB phosphorylation. H-R alone resulted in myocyte injury, upregulation of iNOS activity and expression, decrease in eNOS expression, and increase in PKB phosphorylation (all P < 0.05 vs. cells cultured in normoxic conditions). Treatment of myocytes with TGF-beta(1) (1 ng/ml) resulted in a reduction in LDH release, attenuation of the alterations in NOS expression (both iNOS and eNOS), and PKB phosphorylation in response to H-R (all P < 0.05 vs. H-R alone). These observations suggest that TGF-beta(1) decreases H-R injury and attenuates alterations in NOS and PKB phosphorylation in myocytes exposed to H-R.     

TGF-beta activates Erk MAP kinase signalling through direct phosphorylation of ShcA

Erk1/Erk2 MAP kinases are key regulators of cell behaviour and their activation is generally associated with tyrosine kinase signalling. However, TGF-beta stimulation also activates Erk MAP kinases through an undefined mechanism, albeit to a much lower level than receptor tyrosine kinase stimulation. We report that upon TGF-beta stimulation, the activated TGF-beta type I receptor (TbetaRI) recruits and directly phosphorylates ShcA proteins on tyrosine and serine. This dual phosphorylation results from an intrinsic TbetaRI tyrosine kinase activity that complements its well-defined serine-threonine kinase function. TGF-beta-induced ShcA phosphorylation induces ShcA association with Grb2 and Sos, thereby initiating the well-characterised pathway linking receptor tyrosine kinases with Erk MAP kinases. We also found that TbetaRI is tyrosine phosphorylated in response to TGF-beta. Thus, TbetaRI, like the TGF-beta type II receptor, is a dual-specificity kinase. Recruitment of tyrosine kinase signalling pathways may account for aspects of TGF-beta biology that are independent of Smad signalling.     

Progesterone potentiates IP(3)-mediated calcium signaling through Akt/PKB.

The activity of cells critically depends on the control of their cytosolic free calcium ion (Ca(2+)) concentration. The objective of the present study was to identify mechanisms of action underlying the control of the gain of intracellular Ca(2+) release by circulating gonadal steroid hormones. Acute stimulation of isolated neurons with progesterone led to IP(3)R-mediated Ca(2+) transients that depend on the activation of the PI3 kinase/Akt/PKB signaling pathway. These results were confirmed at the molecular level and phosphorylation of IP(3)R type 1 by Akt/PKB was identified as the mechanism of action. Hence, it is likely that circulating gonadal steroid hormones control neuronal activity including phosporylation status through receptor- and kinase-mediated signaling. With a direct control of the gain of the Ca(2+) second messenger system as a signaling gatekeeper for neuronal activity the present study identifies a novel pathway for interaction of the endocrine and central nervous system.     

Novel roles of Akt and mTOR in suppressing TGF-beta/ALK5-mediated Smad3 activation

Insulin-like growth factor-I inhibits transforming growth factor-beta (TGF-beta) signaling by blocking activation of Smad3 (S3), via a phosphatidylinositol 3-kinase (PI3K)/Akt-dependent pathway. Here we provide the first report that the kinase activity of Akt is necessary for its ability to suppress many TGF-beta responses, including S3 activation and induction of apoptosis. Wild-type and myristoylated Akts (Akt(WT) and Akt(Myr)) suppress TGF-beta-induced phospho-activation of S3 but not Smad2 (S2), whereas kinase-dead Akt1 (Akt1K179M) or dominant-negative PI3K enhances TGF-beta-induced phospho-activation of both S2 and S3. Using siRNA, rapamycin (Rap), and adenoviral expression for FKBP12-resistant and constitutively active TGF-beta type I receptor (ALK5), we demonstrate that mammalian target of Rap (mTOR) mediates Akt1 suppression of phospho-activation of S3. These and further data on Akt1-S3 binding do not support a recently proposed model that Akt blocks S3 activation through physical interaction and sequestration of S3 from TGF-beta receptors. We propose a novel model whereby Akt suppresses activation of S3 in an Akt kinase-dependent manner through mTOR, a likely route for loss of tumor suppression by TGF-beta in cancers.     

Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40

Insulin stimulates protein synthesis and cell growth by activation of the protein kinases Akt (also known as protein kinase B, PKB) and mammalian target of rapamycin (mTOR). It was reported that Akt activates mTOR by phosphorylation and inhibition of tuberous sclerosis complex 2 (TSC2). However, in recent studies the physiological requirement of Akt phosphorylation of TSC2 for mTOR activation has been questioned. Here, we identify PRAS40 (proline-rich Akt/PKB substrate 40 kDa) as a novel mTOR binding partner that mediates Akt signals to mTOR. PRAS40 binds the mTOR kinase domain and its interaction with mTOR is induced under conditions that inhibit mTOR signalling, such as nutrient or serum deprivation or mitochondrial metabolic inhibition. Binding of PRAS40 inhibits mTOR activity and suppresses constitutive activation of mTOR in cells lacking TSC2. PRAS40 silencing inactivates insulin-receptor substrate-1 (IRS-1) and Akt, and uncouples the response of mTOR to Akt signals. Furthermore, PRAS40 phosphorylation by Akt and association with 14-3-3, a cytosolic anchor protein, are crucial for insulin to stimulate mTOR. These findings identify PRAS40 as an important regulator of insulin sensitivity of the Akt-mTOR pathway and a potential target for the treatment of cancers, insulin resistance and hamartoma syndromes.     

PKB/Akt:一个具有多种功能的蛋白激酶

蛋白激酶B(protein kinaseB,PKB)是细胞信号传导过程中的一个重要的中间体。在过去lO多年的研究中,发现它不仅参与调节细胞糖代谢、细胞增殖、细胞凋亡,而且与糖尿病和癌症的发生也有关。目前PKB的相关调控机制还未得到完全阐明。在此,作者谨就PKB的一些研究进展作一介绍。     

Akt/PKB localisation and 3' phosphoinositide generation at sites of epithelial cell-matrix and cell-cell interaction

Protein kinase B (PKB or Akt) is a mitogen-regulated protein kinase involved in the protection of cells from apoptosis, the promotion of cell proliferation and diverse metabolic responses [1]. Its activation is initiated by the binding of 3' phosphorylated phosphoinositide lipids to its pleckstrin homology (PH) domain, resulting in the induction of activating phosphorylation at residues Thr308 and Ser473 by upstream kinases such as phosphoinositide-dependent protein kinase-1 (PDK1) [2]. Adhesion of epithelial cells to extracellular matrix leads to protection from apoptosis via the activation of phosphoinositide (PI) 3-kinase and Akt/PKB through an unknown mechanism [3] [4]. Here, we use the localisation of Akt/PKB within the cell to probe the sites of induction of PI 3-kinase activity. In fibroblasts, immunofluorescence microscopy showed that endogenous Akt/PKB localised to membrane ruffles at the outer edge of the cell following mitogen treatment as did green fluorescent protein (GFP) fusions with full-length Akt/PKB or its PH domain alone. In epithelial cells, the PH domain of Akt/PKB localised to sites of cell-cell and cell-matrix contact, distinct from focal contacts, even in the absence of serum. As this localisation was disrupted by PI 3-kinase inhibitory drugs and by mutations that inhibit interaction with phosphoinositides, it is likely to represent the sites of constitutive 3' phosphoinositide generation that provide a cellular survival signal. We propose that the attachment-induced, PI-3-kinase-mediated survival signal in epithelial cells is generated not only by cell-matrix interaction but also by cell-cell interaction.     

GST pi modulates JNK activity through a direct interaction with JNK substrate, ATF2

Human GSTpi, an important detoxification enzyme, has been shown to modulate the activity of JNKs by inhibiting apoptosis and by causing cell proliferation and tumor growth. In this work, we describe a detailed analysis of the interaction in vitro between GSTpi and JNK isoforms (both in their inactive and active, phosphorylated forms). The ability of active JNK1 or JNK2 to phosphorylate their substrate, ATF2, is inhibited by two naturally occurring GSTpi haplotypes (Ile105/Ala114, WT or haplotype A, and Val105/Val114, haplotype C). Haplotype C of GSTpi is a more potent inhibitor of JNK activity than haplotype A, yielding 75-80% and 25-45% inhibition, respectively. We show that GSTpi is not a substrate of JNK, as was earlier suggested by others. Through binding studies, we demonstrate that the interaction between GSTpi and phosphorylated, active JNKs is isoform specific, with JNK1 being the preferred isoform. In contrast, GSTpi does not interact with unphosphorylated, inactive JNKs unless a JNK substrate, ATF2, is present. We also demonstrate, for the first time, a direct interaction: between GSTpi and ATF2. GSTpi binds with similar affinity to active JNK + ATF2 and to ATF2 alone. Direct binding experiments between ATF2 and GSTpi, either alone or in the presence of glutathione analogs or phosphorylated ATF2, indicate that the xenobiotic portion of the GSTpi active site and the JNK binding domain of ATF2 are involved in this interaction. Competition between GSTpi and active JNK for the substrate ATF2 may be responsible for the inhibition of JNK catalysis by GSTpi.     

PKB/Akt interacts with inosine-5' monophosphate dehydrogenase through its pleckstrin homology domain

The pleckstrin homology (PH) domain of the protooncogenic serine/threonine protein kinase PKB/Akt can bind phosphoinositides. A yeast-based two-hybrid system was employed which identified inosine-5' monophosphate dehydrogenase (IMPDH) type II as specifically interacting with PKB/Akts PH domain. IMPDH catalyzes the rate-limiting step of de novo guanosine-triphosphate (GTP) biosynthesis. Using purified fusion proteins, PKB/Akts PH domain and IMPDH associated in vitro and this association moderately activated IMPDH. Purified PKB/Akt also associated with IMPDH in vitro. We could specifically pull-down PKB/Akt or IMPDH from mammalian cell lysates using glutathione-S-transferase (GST)-IMPDH or GST-PH domain fusion proteins, respectively. Additionally, PKB/Akt and IMPDH could be co-immunoprecipitated from COS cell lysates and active PKB/Akt could phosphorylate IMPDH in vitro. These results implicate PKB/Akt in the regulation of GTP biosynthesis through its interaction with IMPDH, which is involved in providing the GTP pool used by signal transducing G-proteins.     

PKB/Akt partners with Dab2 in albumin endocytosis

Albumin in the glomerular filtrate is normally retrieved by concerted efforts of clathrin, LDL-type receptor megalin- and clathrin-associated sorting proteins. In glomerular diseases, albumin overload triggers a proapoptotic and inflammatory response contributing to tubulointerstitial fibrosis and tubular atrophy. The relationship between albumin overload-induced proximal tubule injury and albumin endocytosis remains to be discovered. We investigated presence of a possible overlap between endocytosis and cell survival. We showed a novel interaction between prosurvival protein, protein kinase B (PKB/Akt), and adaptor protein, disabled 2 (Dab2), with coimmunoprecipitation. Further delineation of this interaction by GST pull-down experiments utilizing different Dab2 constructs identified proline-rich domain as the interacting partner. Expression of Dab2 and PKB/Akt was downregulated at high concentrations of albumin associated with apoptosis. We then examined the physiological relevance of this interaction with functional studies. Overexpression of PKB/Akt increased albumin uptake in human proximal tubule cells. Conversely, inhibition of PKB/Akt with a nonselective Akt/PKB signaling inhibitor-2 and a dominant negative construct of PKB/Akt resulted in a decrease in albumin uptake. Inhibition of Dab2 by silencing RNA abolished PKB/Akt-induced albumin uptake demonstrating the physiological importance of this novel interaction. We concluded that PKB/Akt is part of an endocytic machinery and it mediates albumin uptake through its interaction with Dab2. The role that PKB/Akt plays in the endocytic cascade may dictate its decreased expression in proteinuric states in an attempt to limit albumin endocytosis that may tilt the balance between cell survival and apoptosis toward cell death.     

Regulation of Akt/PKB Ser473 phosphorylation

For years the Akt/PKB research field has been in turmoil, trying to understand how the activating phosphorylation of Akt/PKB at Ser473 is regulated. In the past month, papers in a recent issue of Molecular Cell (Gao et al., 2005) and in Science (Sarbassov et al., 2005) may have identified the phosphatase and kinase acting on this residue.