CD38 signaling in T cells is initiated within a subset of membrane rafts containing Lck and the CD3-zeta subunit of the T cell antigen receptor

In this study we present data supporting that most CD38 is pre-assembled in a subset of Brij 98-resistant raft vesicles, which were stable at 37 degrees C, and have relatively high levels of Lck and the CD3-zeta subunit of T cell antigen receptor-CD3 complex in contrast with a Brij 98-soluble pool, where CD38 is associated with CD3-zeta, and Lck is not detected. Our data further indicate that following CD38 engagement, LAT and Lck are tyrosine phosphorylated exclusively in Brij 98-resistant rafts, and some key signaling components translocate into rafts (i.e. Sos and p85-phosphatidylinositol 3-kinase). Moreover, N-Ras results activated within rafts immediately upon CD38 ligation, whereas activated Erk was mainly found in soluble fractions with delayed kinetics respective to Ras activation. Furthermore, full phosphorylation of CD3-zeta and CD3-epsilon only occurs in rafts, whereas partial CD3-zeta tyrosine phosphorylation occurs exclusively in the soluble pool, which correlated with increased levels of c-Cbl tyrosine phosphorylation in the non-raft fractions. Taken together, these results suggest that, unlike the non-raft pool, CD38 in rafts is able to initiate and propagate several activating signaling pathways, possibly by facilitating critical associations within other raft subsets, for example, LAT rafts via its capacity to interact with Lck and CD3-zeta. Overall, these findings provide the first evidence that CD38 operates in two functionally distinct microdomains of the plasma membrane.     

Unravelling the activation mechanisms of protein kinase B/Akt

Over the past decade, protein kinase B (PKB, also termed Akt) has emerged as an important signaling mediator between extracellular cues and modulation of gene expression, metabolism, and cell survival. The enzyme is tightly controlled and consequences of its deregulation include loss of growth control and oncogenesis. Recent work has better characterized the mechanism of PKB activation, including upstream regulators and secondary binding partners. This minireview refreshes some old concepts with new twists and highlights current outstanding questions.     

Activation of the Akt/protein kinase B signaling pathway is associated with granulosa cell survival

Follicles from the hen ovary that have been selected into the preovulatory hierarchy are committed to ovulation and rarely become atretic under normal physiological conditions. In part, this is attributed to the resistance of the granulosa layer to apoptosis. The present studies were conducted to evaluate the role of the phosphatidylinositol (PI) 3-kinase/Akt signaling pathway in hen granulosa cell survival and, by implication, follicle viability. Cloning of the chicken akt2 homologue revealed a high degree of amino acid homology to its mammalian counterparts within the catalytic domain, plus complete conservation of the putative Thr(308) and Ser(474) phosphorylation sites. Treatment of granulosa cells from the three largest preovulatory follicles with insulin-like growth factor (IGF)-I and, to a lesser extent, transforming growth factor (TGF)-alpha induces rapid phosphorylation of Akt, and such phosphorylation is effectively blocked by the PI 3-kinase-inhibitor LY294006. Serum withdrawal from cultured cells for 33-44 h initiates oligonucleosome formation, an indicator of apoptotic cell death, whereas cotreatment with IGF-I prevents this effect. Moreover, treatment of cultured cells for 20 h with LY294006 induces apoptosis. The potential for nonspecific cell toxicity following LY294006 treatment is considered unlikely because of the ability of either LH or 8-bromo cAMP cotreatment to block LY294006-induced cell death. Finally, both IGF-I and TGF-alpha also activate mitogen-activated protein (MAP) kinase signaling, at least in part, through the phosphorylation of ERK: However, treatment with neither U0126 nor PD98059 (inhibitors of MAP kinase kinase) induced cell death in cultured granulosa cells, despite the ability of each inhibitor to effectively block Erk phosphorylation. Taken together, these results provide evidence for a role of the Akt signaling pathway in promoting cell survival within the preovulatory follicle granulosa layer. In addition, the data indicate the importance of an alternative survival pathway mediated via gonadotropins and protein kinase A independent of Akt signaling.     

Annexin V binds to viable B cells and colocalizes with a marker of lipid rafts upon B cell receptor activation

Recombinant annexin V (rAnV) has been used to identify apoptotic cells based on its ability to bind phosphatidylserine (PS), a lipid normally restricted to the cytoplasmic face of the plasma membrane, but externalized early during apoptosis. However, this association of rAnV binding and apoptosis is not an obligatory one. We demonstrate that rAnV binds to a large fraction of murine B cells bearing selectable Ag receptors despite the fact that these cells are not apoptotic. Phosphatidylserine, which is uniformly distributed on resting B cells, is mobilized to co-cap with IgM on anti-IgM-treated B cells and to colocalize with GM1, a marker of lipid rafts. Cross-linking PS before anti-IgM treatment sequesters this lipid and alters signaling through IgM. Thus, PS exposed on the majority of B cells in vivo does not reflect early apoptosis, but, instead, plays a role in receptor-mediated signaling events.     

Internalization of EGF receptor following lipid rafts disruption in keratinocytes is delayed and dependent on p38 MAPK activation

The receptor for epidermal growth factor (EGF) plays an important role in epidermal keratinocytes and is known to move out of lipid raft after cholesterol depletion, leading to ligand-independent activation. Accumulation of evidence indicates the ability of EGF receptor (EGFR) to undergo internalization without participation of the ligand under the control of p38 MAPK during stress conditions. Since cholesterol depletion using methyl-beta-cyclodextrin is known to induce ligand-independent activation of EGFR in keratinocytes, we investigated by confocal microscopy and ligand-binding tests the processing and localization of EGFR following lipid raft disruption. Here, we report the dimerization and the slow internalization of the receptor accompanied by the delayed phosphorylation of tyrosine 1068 and its degradation by the proteasome. We also demonstrate the involvement of p38 MAPK during the process of internalization, which can be considered as a protective response to stress. Moreover, cholesterol-depleted keratinocytes recover their ability to proliferate during the recovery period that follows lipid raft disruption.     

Acute activation of Erk1/Erk2 and protein kinase B/akt proceed by independent pathways in multiple cell types

We used two inhibitors of the signaling enzyme phosphatidylinositol 3-kinase (PtdIns3K), wortmannin and LY294002, to evaluate the potential involvement of PtdIns3K in the activation of the MAP kinases (MAPK), Erk1 and Erk2. In dose-response studies carried out on six different cell lines and a primary cell culture, we analyzed the ability of the inhibitors to block phosphorylation of protein kinase B/akt (PKB/akt) at Ser473 as a measure of PtdIns3K activity, or the phosphorylation of Erk1/2 at activating Thr/Tyr sites as a measure of the extent of activation of MAPK/Erk kinase (MEK/Erk). In three different hemopoietic cell lines stimulated with cytokines, and in HEK293 cells, stimulated with serum, either wortmannin or LY294002, but never both, could partially block phosphorylation of Erks. The same observations were made in a B-cell line and in primary fibroblasts. In only one cell type, the A20 B cells, was there a closer correlation between the PtdIns3K inhibition by both inhibitors, and their corresponding effects on Erk phosphorylation. However, this stands out as an exception that gives clues to the mechanism by which cross-talk might occur. In all other cells, acute activation of the pathway leading to Erk phosphorylation could proceed independently of PtdIns3K activation. In a biological assay comparing these two pathways, the ability of LY294002 and the MEK inhibitor, U0126, to induce apoptosis were tested. Whereas LY294002 caused death of cytokine-dependent hemopoietic cells, U0126 had little effect, but both inhibitors together had a synergistic effect. The data show that these two pathways are regulating very different downstream events involved in cell survival.     

mTOR.RICTOR is the Ser473 kinase for Akt/protein kinase B in 3T3-L1 adipocytes

The insulin-signaling pathway leading to the activation of Akt/protein kinase B has been well characterized except for a single step, the phosphorylation of Akt at Ser-473. Double-stranded DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM) gene product, integrin-linked kinase (ILK), protein kinase Calpha (PKCalpha), and mammalian target of rapamycin (mTOR), when complexed to rapamycin-insensitive companion of mTOR (RICTOR), have all been identified as playing a critical role in Akt Ser-473 phosphorylation. However, the apparently disparate results reported in these studies are difficult to evaluate, given that different stimuli and cell types were examined and that all of the candidate proteins have never been systematically studied in a single system. Additionally, none of these studies were performed in a classical insulin-responsive cell type or tissue such as muscle or fat. We therefore examined each of these candidates in 3T3-L1 adipocytes. In vitro kinase assays, using different subcellular fractions of 3T3-L1 adipocytes, revealed that phosphatidylinositol 3,4,5-trisphosphate-stimulated Ser-473 phosphorylation correlated well with the amount of DNA-PK, mTOR, and RICTOR but did not correlate with levels of ATM, ILK, and PKCalpha. PKCalpha was completely absent from compartments with Ser-473 phosphorylation activity. Although purified DNA-PK could phosphorylate a peptide derived from Akt that contains amino acid Ser-473, it could not phosphorylate full-length Akt2. Vesicles immunoprecipitated from low density microsomes using antibodies directed against mTOR or RICTOR had phosphatidylinositol 3,4,5-trisphosphate-stimulated Ser-473 activity that was sensitive to wortmannin but not staurosporine. In contrast, immunopurified low density microsome vesicles containing ILK could not phosphorylate Akt on Ser-473 in vitro. Small interference RNA knockdown of RICTOR, but not DNA-PK, ATM, or ILK, suppressed insulin-activated Ser-473 phosphorylation and, to a lesser extent, Thr-308 phosphorylation in 3T3-L1 adipocytes. Based on our cell-free kinase and small interference RNA results, we conclude that mTOR complexed to RICTOR is the Ser-473 kinase in 3T3-L1 adipocytes.     

Trypanosoma cruzi: phosphatidylinositol 3-kinase and protein kinase B activation is associated with parasite invasion

Multiple signal transduction events are triggered in the host cell during invasion by the protozoan parasite Trypanosoma cruzi. Here, we report the regulation of host cell phosphatydilinositol 3-kinase (PI3K) and protein kinase B (PKB/Akt) activities by T. cruzi during parasite-host cell interaction. Treatment of nonphagocytic cells (Vero, L(6)E(9), and NIH 3T3) and phagocytic cells (human and J774 murine macrophages) with the selective PI3K inhibitors Wortmannin and LY294002 significantly impaired parasite invasion in a dose-dependent fashion. A strong activation of PI3K and PKB/Akt activities in Vero cells was detected when these cells were incubated with trypomastigotes or their isolated membranes. Consistently, we were unable to detect activation of PI3K or PKB/Akt activities in host cells during epimastigote (noninfective) membrane-host cell interaction. Infection of transiently transfected cells containing an inactive mutant PKB showed a significant inhibition of invasion compared with the active mutant-transfected cells. T. cruzi PI3K-like activity was also required in host cell invasion since treatment of trypomastigotes with PI3K inhibitors prior to infection reduced parasite entry. Taken together, these results indicate that PI3K and PKB/Akt activation in parasites, as in host cells induced by T. cruzi, is an early invasion signal required for successful trypomastigote internalization.     

Reelin-mediated signaling locally regulates protein kinase B/Akt and glycogen synthase kinase 3beta

Reelin is a large secreted protein that controls cortical layering by signaling through the very low density lipoprotein receptor and apolipoprotein E receptor 2, thereby inducing tyrosine phosphorylation of the adaptor protein Disabled-1 (Dab1) and suppressing tau phosphorylation in vivo. Here we show that binding of Reelin to these receptors stimulates phosphatidylinositol 3-kinase, resulting in activation of protein kinase B and inhibition of glycogen synthase kinase 3beta. We present genetic evidence that this cascade is dependent on apolipoprotein E receptor 2, very low density lipoprotein receptor, and Dab1. Reelin-signaling components are enriched in axonal growth cones, where tyrosine phosphorylation of Dab1 is increased in response to Reelin. These findings suggest that Reelin-mediated phosphatidylinositol 3-kinase signaling in neuronal growth cones contributes to final neuron positioning in the mammalian brain by local modulation of protein kinase B and glycogen synthase kinase 3beta kinase activities.     

SKAP55 recruits to lipid rafts and positively mediates the MAPK pathway upon T cell receptor activation

T cell receptor (TCR) engagement triggers a series of events including protein tyrosine kinase activation, tyrosine phosphorylation of adapter proteins, and multiple protein-protein interactions. We observed that adapter protein SKAP55, the Src kinase-associated phosphoprotein, formed homodimers through its SH3 domain and SK region. SKAP55 as a substrate interacted with Fyn kinase in vivo. In Jurkat cells, interaction between SKAP55 and Fyn kinase depended on TCR activation. Stable overexpression of SKAP55 in Jurkat cells caused mitogen-activated protein kinase activation following TCR engagement. Anti-CD3 stimulation also promoted the interaction of SKAP55 with Grb-2 in T cells. Mutational analysis revealed that tyrosine 271 in SKAP55 played a pivotal role for interaction with both Fyn kinase and adapter protein Grb-2, indicating that the Fyn-phosphorylated SKAP55 transiently associates with adapter Grb-2 to mediate mitogen-activated protein kinase activation. Intriguingly, T cell receptor engagement dramatically induced the translocation of endogenous SKAP55 to lipid rafts where SKAP55 was found to interact with Fyn kinase, suggesting that the positive function of SKAP55 via its association with Fyn and other signaling components may have been involved in raft-mediated T cell activation.     

The v-Crk oncogene enhances cell survival and induces activation of protein kinase B/Akt

The v-Crk oncogene encodes an adaptor protein containing an SH2 domain and an SH3 domain. v-Crk-transformed fibroblast cells display enhanced tyrosine phosphorylation levels, and the v-Crk protein localizes in focal adhesions, suggesting that transformation may be due to enhanced focal complex signaling. Here we investigated the mechanism of transformation and found that v-Crk-transformed NIH 3T3 cells display growth rates and serum requirements similar to control cells. However, v-Crk enhanced survival in conditions of serum starvation. Both an intact SH2 and SH3 domain are required; moreover, SH2 mutants displayed dominant interfering properties, enhancing cell death. Using other cell death-inducing stimuli, it appeared that v-Crk in general inhibits apoptosis and enhances cell survival. In search of the signaling pathways involved, we found that v-Crk-transformed cells show constitutively higher levels of phospho-protein kinase B (PKB)/Akt and PKB/Akt activity, especially in conditions of serum starvation. These data strongly suggest involvement of the phosphatidylinositol 3-kinase/PKB survival pathway in the v-Crk-induced protection against apoptosis. In accordance, inhibition of this pathway by wortmannin or LY924002 reduced protection against starvation-induced apoptosis. In addition to the phosphatidylinositol 3-kinase/PKB pathway, a MEK-dependent pathway and an unknown additional pathway are also implicated in resistance against apoptosis. Activation of survival pathways may be the most important function of v-Crk in its oncogenic properties.     

Expression and activation of Akt/protein kinase B in sexually immature and mature rat uterus

This study investigated the expression and activation of Akt/PKB in developing and adult rat uterus. Expression of Akt was observed in uteri from adult ovariectomized and 7–35-day-old rats and no changes were observed in response to in vivo estradiol treatment (1–100 μg/100 g b.w.). To examine the mechanisms of PKB/Akt activation, phosphorylation at Thr³⁰⁸ and Ser⁴⁷³ regulatory sites were studied in uteri. Akt was constitutively phosphorylated on Ser⁴⁷³ residue in the untreated, control uteri, while phosphorylation of Thr³⁰⁸ was observed only after estradiol 17β (E2) treatment. The effects of E2 treatment were age dependent, no response was induced in 11-day-old uteri, while in 28 days and older rats the activation of Akt at both regulatory sites, Ser⁴⁷³ and Thr³⁰⁸, increased, the first response was detected 2 h after treatment, reaching the highest rate at 6 h. The rate of phosphorylation was stronger at Ser⁴⁷³ residue. The results suggest that the regulation of Akt activation at two regulatory sites in rat uteri are different, phosphorylation of Thr³⁰⁸ seems to be entirely estrogen dependent, while the phosphorylation of Ser⁴⁷³ is regulated by other factors as well as estrogen.     

Akt/protein kinase B is regulated by autophosphorylation at the hypothetical PDK-2 site

The function of Akt (protein kinase B) is regulated by phosphorylation on two sites conserved within the AGC kinase family: the activation loop (Thr-308) in the kinase core and a hydrophobic phosphorylation site on the carboxyl terminus (Ser-473). Thr-308 is phosphorylated by the phosphoinositide-dependent kinase-1, (PDK-1), whereas the mechanism of phosphorylation of the hydrophobic site, tentatively referred to as the PDK-2 site, is unknown. Here we report that phosphorylation of the hydrophobic motif requires catalytically competent Akt. First we show that a kinase-inactive construct of Akt fails to incorporate phosphate at Ser-473 following IGF-1 stimulation in vivo but does incorporate phosphate at Thr-308 and a second carboxyl-terminal site, Thr-450; this ligand triggers the phosphorylation of both sites in wild-type enzyme. Neither does a catalytically inactive construct in which phosphorylation at the activation loop is blocked, T308A, become phosphorylated on the hydrophobic site in response to stimulation. Second, we show that Akt autophosphorylates on the hydrophobic site in vitro: phosphorylation of the activation loop by PDK-1 triggers the phosphorylation of the hydrophobic site in kinase-active, but not thermally inactivated, Akt alpha. Thus, Akt is regulated by autophosphorylation at the Ser-473 hydrophobic site.     

The protein kinase PKR is required for p38 MAPK activation and the innate immune response to bacterial endotoxin

Protein kinase RNA-regulated (PKR) is an established component of innate antiviral immunity. Recently, PKR has been shown to be essential for signal transduction in other situations of cellular stress. The relationship between PKR and the stress-activated protein kinases (SAPKs), such as p38 mitogen-activated protein kinase (MAPK), is not clear. Using embryonic fibroblasts from PKR wild-type and null mice, we established a requirement for PKR in the activation of SAPKs by double-stranded RNA, lipopolysaccharide (LPS) and proinflammatory cytokines. This does not reflect a global failure to activate SAPKs in the PKR-null background as these kinases are activated normally by anisomycin and other physicochemical stress. Activation of p38 MAPK was restored in immortalized PKR-null cells by reconstitution with human PKR. We also show that LPS induction of interleukin-6 and interleukin-12 mRNA is defective in PKR-null cells, and that production of these cytokines is impaired in PKR-null mice challenged with LPS. Our findings indicate, for the first time, that PKR is required for p38 MAPK signaling and plays a potentially important role in the innate response against bacterial endotoxin.     

Impact of estrogen replacement on ventricular myocyte contractile function and protein kinase B/Akt activation

Women with functional ovaries have a lower cardiovascular risk than men and postmenopausal women. However, estrogen replacement therapy remains controversial. This study examined the effect of ovarian hormone deficiency and estrogen replacement on ventricular myocyte contractile function and PKB/Akt activation. Nulliparous female rats were subjected to bilateral ovariectomy (Ovx) or sham operation (sham). A subgroup of Ovx rats received estrogen (E(2)) replacement (40 microg. kg(-1). day(-1)) for 8 weeks. Mechanical and intracellular Ca(2+) properties were evaluated including peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR(90)), maximal velocity of shortening/relengthening (+/-dL/dt), fura 2 fluorescence intensity (FFI), and decay rate. Levels of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a), phospholamban (PLB), and Akt were assessed by Western blot. Ovx promoted body weight gain associated with reduced serum E(2) and uterine weight, all of which were abolished by E(2). Ovx depressed PS and +/-dL/dt, prolonged TPS, TR(90), and decay rate, and enhanced resting FFI, all of which, with the exception of TPS, were restored by E(2). Ovx did not alter the levels of SERCA2a, PLB, and total Akt, but significantly reduced Akt activation [phosphorylated Akt (pAkt)], pAkt/Akt, and the SERCA2a-to-PLB ratio. These alterations in protein expression were restored by E(2). E(2) enhanced PS and +dL/dt in vitro, which was abolished by the E(2) receptor antagonist ICI-182780. Ovx reduced myocyte Ca(2+) responsiveness and lessened stimulating frequency-induced decline in PS, both ablated by E(2). These data suggest that mechanical and protein functions of ventricular myocytes are directly regulated by E(2).     

Angiogenin-induced protein kinase B/Akt activation is necessary for angiogenesis but is independent of nuclear translocation of angiogenin in HUVE cells

Angiogenin, a potent angiogenic factor, binds to endothelial cells and is endocytosed and rapidly translocated to and concentrated in the nucleolus where it binds to DNA. In this study, we report that angiogenin induces transient phosphorylation of protein kinase B/Akt in cultured human umbilical vein endothelial (HUVE) cells. LY294002 inhibits the angiogenin-induced protein kinase B/Akt activation and also angiogenin-induced cell migration in vitro as well as angiogenesis in chick embryo chorioallantoic membrane in vivo without affecting nuclear translocation of angiogenin in HUVE cells. These results suggest that cross-talk between angiogenin and protein kinase B/Akt signaling pathways is essential for angiogenin-induced angiogenesis in vitro and in vivo, and that angiogenin-induced PKB/Akt activation is independent of nuclear translocation of angiogenin in HUVE cells.     

Colocalization of the B cell receptor and CD20 followed by activation-dependent dissociation in distinct lipid rafts

The B cell Ag receptor (BCR) and CD20, a putative calcium channel, inducibly associate with cholesterol-dependent membrane microdomains known as lipid rafts. A functional association between the BCR and CD20 is suggested by the effects of CD20-specific mAbs, which can modulate cell cycle transitions elicited by BCR signaling. Using immunofluorescence microscopy we show here that the BCR and CD20 colocalize after receptor ligation and then rapidly dissociate at the cell surface before endocytosis of the BCR. After separation, surface BCR and CD20 were detected in distinct lipid rafts isolated as low density, detergent-resistant membrane fragments. Pretreatment with methyl-beta-cyclodextrin, which we have previously shown to enhance receptor-mediated calcium mobilization, did not prevent colocalization of the BCR and CD20, but slowed their dissociation. The data demonstrate rapid dynamics of the BCR in relation to CD20 at the cell surface. Activation-dependent dissociation of the BCR from CD20 occurs before receptor endocytosis and appears to require in part the integrity of lipid rafts.     

Insulin-stimulated Interaction between insulin receptor substrate 1 and p85alpha and activation of protein kinase B/Akt require Rab5

Binding of insulin to the insulin receptor initiates a cascade of protein phosphorylation and effector recruitment events leading to the activation of multiple distinct signaling pathways. Previous studies suggested that the diversity and specificity of insulin signal transduction are accomplished by both subcellular localization of receptor and the selective activation of downstream signaling molecules. The small GTPase Rab5 is a key regulator of endocytosis. Three Rab5 isoforms (Rab5a, -5b, and -5c) have been identified. Here we exploited the RNA interference technique to specifically knock down individual Rab5 isoforms to determine the cellular function of Rab5 in distinct insulin signaling pathways. Small interference RNA against a single Rab5 isoform had no effect on protein kinase B (PKB)/Akt or MAPK activation by insulin in NIH3T3 cells overexpressing human insulin receptor. However, simultaneous knockdown of all three Rab5 isoforms dramatically attenuated PKB/Akt activation by insulin without affecting MAPK activation. This inhibition of PKB/Akt activation was because of the impaired interaction between insulin receptor substrate 1 and the p85alpha subunit of phosphatidylinositol 3-kinase. These results indicate a requirement of Rab5 in presenting p85 to insulin receptor substrate 1. Additional evidence supporting a role for Rab5 was suggested by studies with GAPex-5, a vps9 domain containing exchange factor. Down-regulation of GAPex-5 impaired insulin-stimulated PKB/Akt activation. Collectively, this study indicates the involvement of Rab5 in insulin signaling.