Physical and functional interaction of the TPL2 kinase with nucleophosmin.

Tumor Progression Locus 2 (TPL2) is widely recognized as a cytoplasmic mitogen-activated protein 3 kinase with a prominent role in the regulation of inflammatory and oncogenic signal transduction. Herein we report that TPL2 may also operate in the nucleus as a physical and functional partner of nucleophosmin (NPM/B23), a major nucleolar phosphoprotein with diverse cellular activities linked to malignancy. We demonstrate that TPL2 mediates the phosphorylation of a fraction of NPM at threonine 199, an event required for its proteasomal degradation and maintenance of steady-state NPM levels. Upon exposure to ultraviolet C, Tpl2 is required for the translocation of de-phosphorylated NPM from the nucleolus to the nucleoplasm. NPM is an endogenous inhibitor of HDM2:p53 interaction and knockdown of TPL2 was found to result in reduced binding of NPM to HDM2, with concomitant defects in p53 accumulation following genotoxic or ribosomal stress. These findings expand our understanding of the function of TPL2 as a negative regulator of carcinogenesis by defining a nuclear role for this kinase in the topological sequestration of NPM, linking p53 signaling to the generation of threonine 199-phosphorylated NPM.

Phosphorylation of TPL-2 on serine 400 is essential for lipopolysaccharide activation of extracellular signal-regulated kinase in macrophages.

Tumor progression locus 2 (TPL-2) kinase is essential for Toll-like receptor 4 activation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) and for upregulation of the inflammatory cytokine tumor necrosis factor (TNF) in lipopolysaccharide (LPS)-stimulated macrophages. LPS activation of ERK requires TPL-2 release from associated NF-kappaB1 p105, which blocks TPL-2 access to its substrate, the ERK kinase MEK. Here we demonstrate that TPL-2 activity is also regulated independently of p105, since LPS stimulation was still needed for TPL-2-dependent activation of ERK in Nfkb1(-/-) macrophages. In wild-type macrophages, LPS induced the rapid phosphorylation of serine (S) 400 in the TPL-2 C-terminal tail. Mutation of this conserved residue to alanine (A) blocked the ability of retrovirally expressed TPL-2 to induce the activation of ERK in LPS-stimulated Nfkb1(-/-) macrophages. TPL-2(S400A) expression also failed to reconstitute LPS activation of ERK and induction of TNF in Map3k8(-/-) macrophages, which lack endogenous TPL-2. Consistently, the S400A mutation was found to block LPS stimulation of TPL-2 MEK kinase activity. Thus, induction of TPL-2 MEK kinase activity by LPS stimulation of macrophages requires TPL-2 phosphorylation on S400, in addition to its release from NF-kappaB1 p105. Oncogenic C-terminal truncations of TPL-2 that remove S400 could promote its transforming potential by eliminating this critical control step.

Adenoviral gene transfer of Akt enhances myocardial contractility and intracellular calcium handling.

The serine-threonine kinase Akt/PKB mediates stimuli from different classes of cardiomyocyte receptors, including the growth hormone/insulin like growth factor and the beta-adrenergic receptors. Whereas the growth-promoting and antiapoptotic properties of Akt activation are well established, little is known about the effects of Akt on myocardial contractility, intracellular calcium (Ca(2+)) handling, oxygen consumption, and beta-adrenergic pathway. To this aim, Sprague-Dawley rats were subjected to a wild-type Akt in vivo adenoviral gene transfer using a catheter-based technique combined with aortopulmonary crossclamping. Left ventricular (LV) contractility and intracellular Ca(2+) handling were evaluated in an isolated isovolumic buffer-perfused, aequorin-loaded whole heart preparations 10 days after the surgery. The Ca(2+)-force relationship was obtained under steady-state conditions in tetanized muscles. No significant hypertrophy was detected in adenovirus with wild-type Akt (Ad.Akt) versus controls rats (LV-to-body weight ratio 2.6+/-0.2 versus 2.7+/-0.1 mg/g, controls versus Ad.Akt, P, NS). LV contractility, measured as developed pressure, increased by 41% in Ad.Akt. This was accounted for by both more systolic Ca(2+) available to the contractile machinery (+19% versus controls) and by enhanced myofilament Ca(2+) responsiveness, documented by an increased maximal Ca(2+)-activated pressure (+19% versus controls) and a shift to the left of the Ca(2+)-force relationship. Such increased contractility was paralleled by a slight increase of myocardial oxygen consumption (14%), while titrated dose of dobutamine providing similar inotropic effect augmented oxygen consumption by 39% (P<0.01). Phospholamban, calsequestrin, and ryanodine receptor LV mRNA and protein content were not different among the study groups, while sarcoplasmic reticulum Ca(2+) ATPase protein levels were significantly increased in Ad.Akt rats. beta-Adrenergic receptor density, affinity, kinase-1 levels, and adenylyl cyclase activity were similar in the three animal groups. In conclusion, our results support an important role for Akt/PKB in the regulation of myocardial contractility and mechanoenergetics.

Tumor induction by an Lck-MyrAkt transgene is delayed by mechanisms controlling the size of the thymus.

Transgenic mice expressing MyrAkt from a proximal Lck promoter construct develop thymomas at an early age, whereas transgenic mice expressing constitutively active Lck-AktE40K develop primarily tumors of the peripheral lymphoid organs later in life. The thymus of 6- to 8-week-old MyrAkt transgenic mice is normal in size but contains fewer, larger cells than the thymus of nontransgenic control and AktE40K transgenic mice. Earlier studies had shown that cell size and cell cycle are coordinately regulated. On the basis of this finding, and our observations that the oncogenic potential of Akt correlates with its effect on cell size, we hypothesized that mechanisms aimed at maintaining the size of the thymus dissociate cell size and cell cycle regulation by blocking MyrAkt-promoted G(1) progression and that failure of these mechanisms may promote cell proliferation resulting in an enlarged neoplastic thymus. To address this hypothesis, we examined the cell cycle distribution of freshly isolated and cultured thymocytes from transgenic and nontransgenic control mice. The results showed that although neither transgene alters cell cycle distribution in situ, the MyrAkt transgene promotes G(1) progression in culture. Freshly isolated MyrAkt thymocytes express high levels of cyclins D2 and E and cdk4 but lower than normal levels of cyclin D3 and cdk2. Cultured thymocytes from MyrAkt transgenic mice, on the other hand, express high levels of cyclin D3, suggesting that the hypothesized organ size control mechanisms may down-regulate the expression of this molecule. Primary tumor cells, similar to MyrAkt thymocytes in culture, express high levels of cyclin D3. These findings support the hypothesis that tumor induction is caused by the failure of organ size control mechanisms to down-regulate cyclin D3 and to block MyrAkt-promoted G(1) progression.

PDK2: a complex tail in one Akt.

The kinase Akt contains two phosphatidylinositol-3 kinase (PI3K)-dependent phosphorylation sites, one in the activation loop (Thr(308)) and one in the carboxyl-terminal tail (Ser(473)), both of which are conserved among the members of the AGC kinase family. Under physiological conditions, the phosphorylation of Thr(308) appears to be coordinately regulated with the phosphorylation of Ser(473). Under experimental conditions, however, the two sites can be uncoupled, suggesting that their phosphorylation is controlled by different kinases and phosphatases. Phosphoinositide-dependent kinase 1 (PDK1), the kinase that phosphorylates the activation loop site, has been unambiguously identified. However, PDK2, a kinase that is hypothesized to phosphorylate the hydrophobic carboxyl-terminal site, remains elusive. This Perspective examines the regulation and biological significance of Akt phosphorylation at Ser(473). The authors propose that Ser(473) undergoes both autophosphorylation and phosphorylation by other kinases. Both events may be promoted by interactions between PDK1 and phosphorylated or phosphomimetically altered hydrophobic phosphorylation motifs in kinases associated with Akt. These interactions may induce conformational changes in Akt that make Ser(473) accessible to phosphorylation.

AKT1/PKBalpha kinase is frequently elevated in human cancers and its constitutive activation is required for oncogenic transformation in NIH3T3 cells.

Extensive studies have demonstrated that the Akt/AKT1 pathway is essential for cell survival and inhibition of apoptosis; however, alterations of Akt/AKT1 in human primary tumors have not been well documented. In this report, significantly increased AKT1 kinase activity was detected in primary carcinomas of prostate (16 of 30), breast (19 of 50), and ovary (11 of 28). The results were confirmed by Western blot and immunohistochemical staining analyses with phospho-Ser473 Akt antibody. The majority of AKT1-activated tumors are high grade and stage III/lV (13 of 16 prostate, 15 of 19 breast, and 8 of 11 ovarian carcinomas). Previous studies showed that wild-type AKT1 was unable to transform NIH3T3 cells. To demonstrate the biological significance of AKT1 activation in human cancer, constitutively activated AKT1 (Myr-Akt) was introduced into NIH3T3 cells. Overexpression of Myr-Akt in the stably transfected cells resulted in malignant phenotype, as determined by growth in soft agar and tumor formation in nude mice. These data indicate that AKT1 kinase, which is frequently activated in human cancer, is a determinant in oncogenesis and a potential target for cancer intervention.

Oncogenic transformation induced by membrane-targeted Akt2 and Akt3.

The kinases Akt2, Akt3 and their myristylated variants, Myr-Akt2 and Myr-Akt3 were expressed by the RCAS vector in chicken embryo fibroblasts (CEF). Myr-Akt2 and Myr-Akt3 were strongly oncogenic, inducing multilayered foci of transformed cells. In contrast, wild-type Akt2 and Akt3 were only poorly transforming, their efficiencies of focus formation were more than 100-fold lower; foci appeared later and showed less multilayering. Addition of the myristylation signal not only enhanced oncogenic potential but also increased kinase activities. Myr-Akt2 and Myr-Akt3 also induced hemangiosarcomas in the animal, whereas wild type Akt2 and Akt3 were not oncogenic in vivo. Furthermore, Akt2, driven by the lck (lymphocyte specific kinase) promoter in transgenic mice, induced lymphomas. The oncogenic effects of Akt2 and Akt3 described here are indistinguishable from those of Akt1. The downstream targets relevant to oncogenic transformation are therefore probably shared by the three Akt kinases.

Tpl-2 induces apoptosis by promoting the assembly of protein complexes that contain caspase-9, the adapter protein Tvl-1, and procaspase-3.

The Tpl-2 proto-oncoprotein promotes cellular proliferation when overexpressed in a variety of tumor cell lines. Here, we present evidence that when overexpressed in immortalized non-transformed cells, Tpl-2 induces apoptosis by promoting the activation of caspase-3 via a caspase-9-dependent mechanism, and that apoptosis is enhanced when Tpl-2 is co-expressed with the newly identified ankyrin repeat protein Tvl-1. The activation of caspase-3 by caspase-9 is known to depend on the assembly of a multimolecular complex that includes Apaf-1 and caspase-9. Data presented here show that co-expression of Tpl-2 with Tvl-1 promotes the assembly of a complex that involves several proteins that bind Apaf-1 including Tvl-1, itself, Tpl-2 and phosphorylated procaspase-9. More important, procaspase-3, which under normal growth conditions is not associated with the complex, binds Tvl-1 conditionally in response to Tpl-2-generated apoptotic signals. The conditional association of procaspase-3 with Tvl-1 promotes the in vivo proteolytic maturation of procaspase-3 by caspase-9, a process casually linked to apoptosis.

Activation of the Ikappa B kinases by RIP via IKKgamma /NEMO-mediated oligomerization.

To understand the mechanism of activation of the IkappaB kinase (IKK) complex in the tumor necrosis factor (TNF) receptor 1 pathway, we examined the possibility that oligomerization of the IKK complex triggered by ligand-induced trimerization of the TNF receptor 1 complex is responsible for activation of the IKKs. Gel filtration analysis of the IKK complex revealed that TNFalpha stimulation induces a large increase in the size of this complex, suggesting oligomerization. Substitution of the C-terminal region of IKKgamma, which interacts with RIP, with a truncated DR4 lacking its cytoplasmic death domain, produced a molecule that could induce IKK and NF-kappaB activation in cells in response to TRAIL. Enforced oligomerization of the N terminus of IKKgamma or truncated IKKalpha or IKKbeta lacking their serine-cluster domains can also induce IKK and NF-kappaB activation. These data suggest that IKKgamma functions as a signaling adaptor between the upstream regulators such as RIP and the IKKs and that oligomerization of the IKK complex by upstream regulators is a critical step in activation of this complex.

Tyrosine 1062 of RET-MEN2A mediates activation of Akt (protein kinase B) and mitogen-activated protein kinase pathways leading to PC12 cell survival.

The RET tyrosine kinase is a functional receptor for neurotrophic ligands of the glial cell line-derived neurotrophic factor (GDNF) family. Loss of function of RET is associated with congenital megacolon or Hirschsprung's disease, whereas germ-line point mutations causing RET activation are responsible for multiple endocrine neoplasia type 2 (MEN2A, MEN2B, and familial medullary thyroid carcinoma) syndromes. Here we show that the expression of a constitutively active RET-MEN2A oncogene promotes survival of rat pheochromocytoma PC12 cells upon growth factor withdrawal. Moreover, we show that the RET-MEN2A-mediated survival depends on signals transduced by the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) cascades. Thus, in PC12 cells, RET-MEN2A associates with the PI3K regulatory subunit p85 and promotes activation of Akt (also referred to as protein kinase B) in a PI3K-dependent fashion; in addition, RET-MEN2A promotes MAPK activation. PI3K recruitment and Akt activation as well as MAPK activation depend on RET-MEN2A tyrosine residue 1062. As a result, tyrosine 1062 of RET-MEN2A is essential for RET-MEN2A-mediated survival of PC12 cells cultured in growth factor-depleted media.

Akt/protein kinase B promotes survival and hormone-independent proliferation of thyroid cells in the absence of dedifferentiating and transforming effects.

The Akt/protein kinase B serine/threonine kinase is a downstream effector of phosphoinositide 3-kinase (PI3K). Akt is an important component of mitogenic and antiapoptotic signaling pathways and is implicated in neoplastic transformation. Thyroid cells in culture retain a differentiated phenotype consisting of epithelial cell morphology and the expression of several tissue-specific genes. The survival and proliferation of these cells depend on thyrotropin and a mixture of five additional hormones that includes insulin. The regulation of proliferation and the expression of the thyroid differentiation program are intimately connected processes. As a result, oncogenes that induce hormone-independent proliferation invariably impair the expression of the thyroid-specific differentiation markers. Given that thyrotropin and insulin stimulate Akt activation in thyroid cells, we set out to determine the effects of Akt on thyroid cell proliferation, survival, and differentiation. To this end, we expressed constitutively active myristylated Akt (myrAkt) in PC Cl 3 thyroid cells. The myrAkt-expressing cells continued to proliferate, even in the absence of hormones, and they were resistant to programmed cell death induced by starvation. These effects were paralleled by the induction of the G1 cyclins D3 and E and by the inhibition of induction of the proapoptotic Fas, Fas ligand, and BAD genes in starved cells. However, in marked contrast with several other oncogenes, myrAkt did not interfere with the expression of thyroid differentiation functions. These results unveil the existence of an Akt-triggered thyroid cell pathway that modulates proliferation and survival without affecting the expression of the thyroid cell differentiated phenotype.

A novel plant glutathione S-transferase/peroxidase suppresses Bax lethality in yeast.

The mammalian inducer of apoptosis Bax is lethal when expressed in yeast and plant cells. To identify potential inhibitors of Bax in plants we transformed yeast cells expressing Bax with a tomato cDNA library and we selected for cells surviving after the induction of Bax. This genetic screen allows for the identification of plant genes, which inhibit either directly or indirectly the lethal phenotype of Bax. Using this method a number of cDNA clones were isolated, the more potent of which encodes a protein homologous to the class theta glutathione S-transferases. This Bax-inhibiting (BI) protein was expressed in Escherichia coli and found to possess glutathione S-transferase (GST) and weak glutathione peroxidase (GPX) activity. Expression of Bax in yeast decreases the intracellular levels of total glutathione, causes a substantial reduction of total cellular phospholipids, diminishes the mitochondrial membrane potential, and alters the intracellular redox potential. Co-expression of the BI-GST/GPX protein brought the total glutathione levels back to normal and re-established the mitochondrial membrane potential but had no effect on the phospholipid alterations. Moreover, expression of BI-GST/GPX in yeast was found to significantly enhance resistance to H(2)O(2)-induced stress. These results underline the relationship between oxidative stress and Bax-induced death in yeast cells and demonstrate that the yeast-based genetic strategy described here is a powerful tool for the isolation of novel antioxidant and antiapoptotic genes.

TNF-alpha induction by LPS is regulated posttranscriptionally via a Tpl2/ERK-dependent pathway.

Tpl2 knockout mice produce low levels of TNF-alpha when exposed to lipopolysaccharide (LPS) and they are resistant to LPS/D-Galactosamine-induced pathology. LPS stimulation of peritoneal macrophages from these mice did not activate MEK1, ERK1, and ERK2 but did activate JNK, p38 MAPK, and NF-kappaB. The block in ERK1 and ERK2 activation was causally linked to the defect in TNF-alpha induction by experiments showing that normal murine macrophages treated with the MEK inhibitor PD98059 exhibit a similar defect. Deletion of the AU-rich motif in the TNF-alpha mRNA minimized the effect of Tpl2 inactivation on the induction of TNF-alpha. Subcellular fractionation of LPS-stimulated macrophages revealed that LPS signals transduced by Tpl2 specifically promote the transport of TNF-alpha mRNA from the nucleus to the cytoplasm.

Differential roles of Akt, Rac, and Ral in R-Ras-mediated cellular transformation, adhesion, and survival.

Multiple biological functions have been ascribed to the Ras-related G protein R-Ras. These include the ability to transform NIH 3T3 fibroblasts, the promotion of cell adhesion, and the regulation of apoptotic responses in hematopoietic cells. To investigate the signaling mechanisms responsible for these biological phenotypes, we compared three R-Ras effector loop mutants (S61, G63, and C66) for their relative biological and biochemical properties. While the S61 mutant retained the ability to cause transformation, both the G63 and the C66 mutants were defective in this biological activity. On the other hand, while both the S61 and the C66 mutants failed to promote cell adhesion and survival in 32D cells, the G63 mutant retained the ability to induce these biological activities. Thus, the ability of R-Ras to transform cells could be dissociated from its propensity to promote cell adhesion and survival. Although the transformation-competent S61 mutant bound preferentially to c-Raf, it only weakly stimulated the mitogen-activated protein kinase (MAPK) activity, and a dominant negative mutant of MEK did not significantly perturb R-Ras oncogenicity. Instead, a dominant negative mutant of phosphatidylinositol 3-kinase (PI3-K) drastically inhibited the oncogenic potential of R-Ras. Interestingly, the ability of the G63 mutant to induce cell adhesion and survival was closely associated with the PI3-K-dependent signaling cascades. To further delineate R-Ras downstream signaling events, we observed that while a dominant negative mutant of Akt/protein kinase inhibited the ability of R-Ras to promote cell survival, both dominant negative mutants of Rac and Ral suppressed cell adhesion stimulated by R-Ras. Thus, the biological actions of R-Ras are mediated by multiple effectors, with PI3-K-dependent signaling cascades being critical to its functions.

CLAP, a novel caspase recruitment domain-containing protein in the tumor necrosis factor receptor pathway, regulates NF-kappaB activation and apoptosis.

Molecules that regulate NF-kappaB activation play critical roles in apoptosis and inflammation. We describe the cloning of the cellular homolog of the equine herpesvirus-2 protein E10 and show that both proteins regulate apoptosis and NF-kappaB activation. These proteins were found to contain N-terminal caspase-recruitment domains (CARDs) and novel C-terminal domains (CTDs) and were therefore named CLAPs (CARD-like apoptotic proteins). The cellular and viral CLAPs induce apoptosis downstream of caspase-8 by activating the Apaf-1-caspase-9 pathway and activate NF-kappaB by acting upstream of the NF-kappaB-inducing kinase, NIK, and the IkB kinase, IKKalpha. Deletion of either the CARD or the CTD domain inhibits both activities. The CARD domain was found to be important for homo- and heterodimerization of CLAPs. Substitution of the CARD domain with an inducible FKBP12 oligomerization domain produced a molecule that can induce NF-kappaB activation, suggesting that the CARD domain functions as an oligomerization domain, whereas the CTD domain functions as the effector domain in the NF-kappaB activation pathway. Expression of the CARD domain of human CLAP abrogates tumor necrosis factor-alpha-induced NF-kappaB activation, suggesting that cellular CLAP plays an essential role in this pathway of NF-kappaB activation.

The ankyrin repeat-containing adaptor protein Tvl-1 is a novel substrate and regulator of Raf-1.

Tvl-1 is a 269-amino acid ankyrin repeat protein expressed primarily in thymus, lung, and testes that was identified by screening a murine T-cell two-hybrid cDNA library for proteins that associate with the serine-threonine kinase Raf-1. The interaction of Tvl-1 with Raf-1 was confirmed by co-immunoprecipitation of the two proteins from COS-1 cells transiently transfected with Tvl-1 and Raf-1 expression constructs as well as by co-immunoprecipitation of the endogenous proteins from CV-1 and NB2 cells. Tvl-1 interacts with Raf-1 via its carboxyl-terminal ankyrin repeat domain. The same domain also mediates Tvl-1 homodimerization. Tvl-1 was detected by immunofluorescence in both the cytoplasm and the nucleus suggesting that in addition to Raf-1 it may also interact with nuclear proteins. Activated Raf-1 phosphorylates Tvl-1 both in vitro and in vivo. In baculovirus-infected Sf9 insect cells, Tvl-1 potentiates the activation of Raf-1 by Src and Ras while in COS-1 cells it potentiates the activation of Raf-1 by EGF. These data suggest that Tvl-1 is both a target as well as a regulator of Raf-1. The human homologue of Tvl-1 maps to chromosome 19p12, upstream of MEF2B with the two genes in a head to head arrangement.

Hyperglycemia inhibits insulin activation of Akt/protein kinase B but not phosphatidylinositol 3-kinase in rat skeletal muscle.

Sustained hyperglycemia impairs insulin-stimulated glucose utilization in the skeletal muscle of both humans and experimental animals--a phenomenon referred to clinically as glucose toxicity. To study how this occurs, a model was developed in which hyperglycemia produces insulin resistance in vitro. Rat extensor digitorum longus muscles were preincubated for 4 h in Krebs-Henseleit solution containing glucose or glucose + insulin at various concentrations, after which insulin action was studied. Preincubation with 25 mmol/l glucose + insulin (10 mU/ml) led to a 70% decrease in the ability of insulin (10 mU/ml) to stimulate glucose incorporation into glycogen and a 30% decrease in 2-deoxyglucose (2-DG) uptake, compared with muscles incubated with 0 mmol/l glucose. Glucose incorporation into lipid and its oxidation to CO2 were marginally diminished, if at all. The alterations of glycogen synthesis and 2-DG uptake were first evident after 1 h and were maximal after 2 h of preincubation; they were not observed in muscles preincubated with 25 mmol/l glucose + insulin for 5 min. Preincubation for 4 h with 25 mmol/l glucose in the absence of insulin produced a similar although somewhat smaller decrease in insulin-stimulated glycogen synthesis; however, it did not alter 2-DG uptake, glucose oxidation to CO2, or incorporation into lipids. Studies of insulin signaling in the latter muscles revealed that activation of Akt/protein kinase B (PKB) was diminished by 60%, compared with that of muscles preincubated in a glucose-free medium; whereas activation of phosphatidylinositol (PI) 3-kinase, an upstream regulator of Akt/PKB in the insulin-signaling cascade, and of mitogen-activated protein (MAP) kinase, a parallel signal, was unaffected. Immunoblots demonstrated that this was not due to a change in Akt/PKB abundance. The results indicate that hyperglycemia-induced insulin resistance can be studied in rat skeletal muscle in vitro. They suggest that impairment of insulin action in these muscles is related to inhibition of Akt/PKB by events that do not affect PI 3-kinase.

p50(cdc37) acting in concert with Hsp90 is required for Raf-1 function.

Genetic screens in Drosophila have identified p50(cdc37) to be an essential component of the sevenless receptor/mitogen-activated kinase protein (MAPK) signaling pathway, but neither the function nor the target of p50(cdc37) in this pathway has been defined. In this study, we examined the role of p50(cdc37) and its Hsp90 chaperone partner in Raf/Mek/MAPK signaling biochemically. We found that coexpression of wild-type p50(cdc37) with Raf-1 resulted in robust and dose-dependent activation of Raf-1 in Sf9 cells. In addition, p50(cdc37) greatly potentiated v-Src-mediated Raf-1 activation. Moreover, we found that p50(cdc37) is the primary determinant of Hsp90 recruitment to Raf-1. Overexpression of a p50(cdc37) mutant which is unable to recruit Hsp90 into the Raf-1 complex inhibited Raf-1 and MAPK activation by growth factors. Similarly, pretreatment with geldanamycin (GA), an Hsp90-specific inhibitor, prevented both the association of Raf-1 with the p50(cdc37)-Hsp90 heterodimer and Raf-1 kinase activation by serum. Activation of Raf-1 via baculovirus coexpression with oncogenic Src or Ras in Sf9 cells was also strongly inhibited by dominant negative p50(cdc37) or by GA. Thus, formation of a ternary Raf-1-p50(cdc37)-Hsp90 complex is crucial for Raf-1 activity and MAPK pathway signaling. These results provide the first biochemical evidence for the requirement of the p50(cdc37)-Hsp90 complex in protein kinase regulation and for Raf-1 function in particular.

AKT/PKB and other D3 phosphoinositide-regulated kinases: kinase activation by phosphoinositide-dependent phosphorylation.

The protein kinase Akt/PKB is activated via a multistep process by a variety of signals. In the early steps of this process, PI-3 kinase-generated D3-phosphorylated phosphoinositides bind the Akt PH domain and induce the translocation of the kinase to the plasma membrane where it co-localizes with phosphoinositide-dependent kinase-1. By binding to the PH domains of both Akt and phosphoinositide-dependent kinase-1, D3-phosphorylated phosphoinositides appear to also induce conformational changes that permit phosphoinositide-dependent kinase-1 to phosphorylate the activation loop of Akt. The paradigm of Akt activation via phosphoinositide-dependent phosphorylation provided a framework for research into the mechanism of activation of other members of the AGC kinase group (p70S6K, PKC, and PKA) and members of the Tec tyrosine kinase family (TecI, TecII, Btk/Atk, Itk/Tsk/Emt, Txk/Rlk, and Bm/Etk). The result was the discovery that these kinases and Akt are activated by overlapping pathways. In this review, we present our current understanding of the regulation and function of the Akt kinase and we discuss the common and unique features of the activation processes of Akt and the AGC and Tec kinase families. In addition, we present an overview of the biosynthesis of phosphoinositides that contribute to the regulation of these kinases.

Tpl-2 induces IL-2 expression in T-cell lines by triggering multiple signaling pathways that activate NFAT and NF-kappaB.

The Tpl-2 kinase activates the nuclear factor of activated T cells (NFAT) and induces IL-2 expression in T-cell lines. Here we show that the activation of the IL-2 promoter by Tpl-2 is inhibited by mutant signaling molecules that inhibit the mitogen-activated protein kinase (MAPK) or the calcineurin/NFAT pathways and is promoted by combinations of signaling molecules that activate these pathways. We, therefore, conclude that signals generated by the convergence of the MAPK and the calcineurin/NFAT pathway are necessary and sufficient for the activation of the IL-2 promoter by Tpl-2. The activation of both the IL-2 promoter and an NFAT-driven minimal promoter were shown to depend on signals transduced by Raf1. However, it was only the IL-2 promoter whose activation by Tpl-2 was fully blocked by the dominant negative mutant MEK1S218/222A and the MEK1/MEK2 inhibitor PD098059. Since the activation of NFAT is MAPK-dependent these findings suggested that the activation of MAPK by Tpl-2 is either independent or only partially dependent on MEK1 and MEK2. In addition, they suggested that the activation of the IL-2 promoter is under the control of not only NFAT but also a second factor whose activation is MEK-dependent. Experiments in COS-1 and EL-4 cells confirmed both hypotheses and revealed that the second factor activated by Tpl-2 is NF-kappaB. While the activation of the IL-2 promoter and an NFAT-driven minimal promoter by Tpl-2 was fully blocked by the dominant negative mutant NFAT delta418, it was only partially blocked by the calcineurin inhibitor cyclosporin A suggesting that the Tpl-2-mediated NFAT activation is under the control of a combination of calcineurin-dependent and independent pathways. Both pathways were fully blocked by Bcl-2 or Bcl-X(L).