Stimulation of the creatine transporter SLC6A8 by the protein kinases SGK1 and SGK3

Creatine binds phosphate thus serving energy storage. Cellular creatine uptake is accomplished by the Na+,Cl-, creatine transporter CreaT (SLC6A8). The present study explored the regulation of SLC6A8 by the serum and glucocorticoid inducible kinase SGK1, a kinase upregulated during ischemia. In Xenopus oocytes expressing SLC6A8 but not in water injected oocytes creatine induced a current which was significantly enhanced by coexpression of wild type SGK1 and constitutively active (S422D)SGK1, but not inactive (K127N)SGK1. Kinetic analysis revealed that (S422D)SGK1 enhanced maximal current without significantly altering affinity. The effect of SGK1 was mimicked by the constitutively active isoform (S419D)SGK3 but not by inactive (K119N)SGK3, wild type isoform SGK2 or constitutively active related kinase (T308D,S473D)PKB. In conclusion, the kinases SGK1 and SGK3 increase SLC6A8 activity by increasing the maximal transport rate of the carrier. Deranged SGK1 and/or SGK3 dependent regulation of SLC6A8 may affect energy storage particularly in skeletal muscle, heart, and neurons.     

Stimulation of renal Na+ dicarboxylate cotransporter 1 by Na+/H+ exchanger regulating factor 2, serum and glucocorticoid inducible kinase isoforms, and protein kinase B

Renal tubular citrate transport is accomplished by electrogenic Na(+) coupled dicarboxylate transporter NaDC-1, a carrier subjected to regulation by acidosis. Trafficking of the Na(+)/H(+) exchanger NHE3 is controlled by NHE regulating factors NHERF-1 and NHERF-2 and the serum and glucocorticoid inducible kinase SGK1. To test for a possible involvement in NaDC-1 regulation, mRNA encoding NaDC-1 was injected into Xenopus oocytes with or without cRNA encoding NHERF-1, NHERF-2, SGK1, SGK2, SGK3, and/or the constitutively active form of the related protein kinase B ((T308,S473D)PKB). Succinate induced inward currents (I(succ)) were taken as a measure of transport rate. Coexpression of neither NHERF-1 nor NHERF-2 in NaDC-1 expressing oocytes significantly altered I(succ). On the other hand, coexpression of SGK1, SGK3, and (T308,S473D)PKB stimulated I(succ), an effect further stimulated by additional coexpression of NHERF-2 but not of NHERF-1. The action of the kinases and NHERF-2 may link urinary citrate excretion to proximal tubular H(+) secretion.     

Post-translational regulation of EAAT2 function by co-expressed ubiquitin ligase Nedd4-2 is impacted by SGK kinases

The human excitatory amino acid transporter (EAAT)2 is the major glutamate carrier in the mammalian CNS. Defective expression of the transporter results in neuroexcitotoxicity that may contribute to neuronal disorders such as amyotrophic lateral sclerosis (ALS). The serum and glucocorticoid inducible kinase (SGK) 1 is expressed in the brain and is known to interact with the ubiquitin ligase Nedd4-2 to modulate membrane transporters and ion channels. The present study aimed to investigate whether SGK isoforms and the related kinase, protein kinase B (PKB), regulate EAAT2. Expression studies in Xenopus oocytes demonstrated that glutamate-induced inward current (IGLU) was stimulated by co-expression of SGK1, SGK2, SGK3 or PKB. IGLU is virtually abolished by Nedd4-2, an effect abrogated by additional co-expression of either kinase. The kinases diminish the effect through Nedd4-2 phosphorylation without altering Nedd4-2 protein abundance. SGKs increase the transporter maximal velocity without significantly affecting substrate affinity. Similar to glutamate-induced currents, [3H] glutamate uptake and cell surface abundance of the transporter were increased by the SGK isoforms and down-regulated by the ubiquitin ligase Nedd4-2. In conclusion, all three SGK isoforms and PKB increase EAAT2 activity and plasma membrane expression and thus, may participate in the regulation of neuroexcitability.     

Regulation of Glucose Transporter SGLT1 by Ubiquitin Ligase Nedd4‐2 and Kinases SGK1, SGK3, and PKB

Objectives : Serum‐ and glucocorticoid‐inducible kinase 1 (SGK1) inhibits the ubiquitin ligase neuronal cell expressed developmentally downregulated 4‐2 (Nedd4‐2), which retards the retrieval of the epithelial Na⁺ channel ENaC. Accordingly, SGK1 enhances ENaC abundance in the cell membrane. The significance of this effect is shown by an association of an E8CC/CT;I6CC polymorphism in the SGK1 gene with increased blood pressure. However, strong expression of SGK1 in enterocytes not expressing ENaC points to further functions of SGK1. This study was performed to test for regulation of Na⁺‐coupled glucose transporter 1 (SGLT1) by Nedd4‐2, SGK1, and/or the related kinases SGK3 and PKB. Additional studies searched for an association of the SGK1 gene with BMI. Research Methods and Procedures : mRNA encoding SGLT1, wild‐type Nedd4‐2, inactive ^C938SNedd4‐2, wild type SGK1, constitutively active ^S422DSGK1 or inactive ^K127NSGK1, wild‐type SGK3, and constitutively active ^T308DS473DPKB or inactive ^T308AS473APKB were injected into Xenopus oocytes, and glucose transport was quantified from glucose‐induced current (I_glc). BMI was determined in individuals with or without the E8CC/CT;I6CC polymorphism. Results: I_glc was significantly decreased by coexpression of Nedd4‐2 but not of ^C938SNedd4‐2. Coexpression of SGK1, ^S422DSGK1, SGK3, or ^T308DS473DPKB, but not of ^K127NSGK1 or ^T308AS473APKB, enhanced I_glc and reversed the effect of Nedd4‐2. SGK1 and SGK3 phosphorylated Nedd4‐2. Deletion of the SGK/PKB phosphorylation sites in Nedd4‐2 blunted the kinase effects. BMI was significantly (p < 0.008) greater in individuals with the E8CC/CT;I6CC polymorphism than in individuals without. Discussion : Overactivity of SGK1 may lead not only to excessive ENaC activity and hypertension but also to enhanced SGLT1 activity and obesity.     

Properties and regulation of glutamine transporter SN1 by protein kinases SGK and PKB

The amino acid transporter SN1 with substrate specificity identical to the amino acid transport system N is expressed mainly in astrocytes and hepatocytes where it accomplishes Na(+)-coupled glutamine uptake and efflux. To characterize properties and regulation of SN1, substrate-induced currents and/or radioactive glutamine uptake were determined in Xenopus oocytes injected with cRNA encoding SN1, the ubiquitin ligase Nedd4-2, and/or the constitutively active serum and glucocorticoid inducible kinase S422DSGK1, its isoform SGK3, and the constitutively active protein kinase B T308D,S473DPKB. The substrate-induced currents were enhanced by increasing glutamine and/or Na(+) concentrations, hyperpolarization, and alkalinization (pH 8.0). They were inhibited by acidification (pH 6.0). Coexpression of Nedd4-2 downregulated SN1-mediated transport, an effect reversed by coexpression of S422DSGK1, SGK3, and T308D,S473DPKB. It is concluded that SN1 is a target for the ubiquitin ligase Nedd4-2, which is inactivated by the serum and glucocorticoid inducible kinase SGK1, its isoform SGK3, and protein kinase B.     

Role of SGK1 kinase in regulating glucose transport via glucose transporter GLUT4

Insulin stimulates glucose transport into muscle and fat cells by enhancing GLUT4 abundance in the plasma membrane through activation of phosphatidylinositol 3-kinase (PI3K). Protein kinase B (PKB) and PKCzeta are known PI3K downstream targets in the regulation of GLUT4. The serum- and glucocorticoid-inducible kinase SGK1 is similarly activated by insulin and capable to regulate cell surface expression of several metabolite transporters. In this study, we evaluated the putative role of SGK1 in the modulation of GLUT4. Coexpression of the kinase along with GLUT4 in Xenopus oocytes stimulated glucose transport. The enhanced GLUT4 activity was paralleled by increased transporter abundance in the plasma membrane. Disruption of the SGK1 phosphorylation site on GLUT4 ((S274A)GLUT4) abrogated the stimulating effect of SGK1. In summary, SGK1 promotes glucose transporter membrane abundance via GLUT4 phosphorylation at Ser274. Thus, SGK1 may contribute to the insulin and GLUT4-dependent regulation of cellular glucose uptake.     

Serum and glucocorticoid inducible kinases functionally regulate ClC-2 channels

ClC-2 participates in the regulation of neuronal excitability, chloride secretion, and cell volume. The ClC-2 sequence contains a consensus site (Ser82) for phosphorylation by the serum and glucocorticoid inducible kinase isoforms SGK1-3. Thus, the present study explored whether ClC-2 is regulated by those kinases. ClC-2 expression in Xenopus oocytes induced inwardly rectifying currents that increased upon coexpression of SGK1-3 and the related kinase PKB. The stimulatory effect was still present upon disruption of the SGK phosphorylation site. SGKs can phosphorylate the ubiquitin ligase Nedd4-2 and prevent Nedd4-2 from binding to its target. Therefore, the role of Nedd4-2 in ClC-2 modulation was investigated. ClC-2 activity decreased upon Nedd4-2 coexpression, an effect reversed by the kinases. According to chemiluminescence ClC-2 membrane abundance was enhanced by SGKs and diminished by Nedd4-2. These observations suggest that SGK1-3 and Nedd4-2 regulate ClC-2 at least in part by modulating ClC-2 abundance at the plasma membrane.     

Regulation of the Ca2+ Channel TRPV6 by the Kinases SGK1, PKB/Akt, and PIKfyve

The serum- and glucocorticoid-inducible kinase SGK1 and the protein kinase PKB/Akt presumably phosphorylate and, by this means, activate the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3), which has in turn been shown to regulate transporters and channels. SGK1-regulated channels include the Ca²⁺ channel TRPV6, which is expressed in a variety of epithelial and nonepithelial cells including tumor cells. SGK1 and protein kinase B PKB/Akt foster tumor growth. The present study thus explored whether TRPV6 is regulated by PIKfyve. TRPV6 was expressed in Xenopus laevis oocytes with or without additional coexpression of constitutively active ^S422DSGK1, constitutively active ^T308D,S473DPKB, wild-type PIKfyve, and ^S318APIKfyve lacking the SGK1 phosphorylation site. TRPV6 activity was determined from the current (I_Ca) resulting from TRPV6-induced Ca²⁺ entry and subsequent activation of Ca²⁺-sensitive endogenous Cl^? channels. TRPV6 protein abundance in the cell membrane was determined utilizing immunohistochemistry and Western blotting. In TRPV6-expressing oocytes I_H was increased by coexpression of ^S422DSGK1 and by ^T308D,S473DPKB. Coexpression of wild-type PIKfyve further increased I_H in TRPV6 + ^S422DSGK1-expressing oocytes but did not significantly modify I_Ca in oocytes expressing TRPV6 alone. ^S318APIKfyve failed to significantly modify I_Ca in the presence and absence of ^S422DSGK1. ^S422DSGK1 increased the TRPV6 protein abundance in the cell membrane, an effect augmented by additional expression of wild-type PIKfyve. We conclude that PIKfyve participates in the regulation of TRPV6.     

Multiple phosphoinositide 3-kinase-dependent steps in activation of protein kinase B

The protein kinase B (PKB)/Akt family of serine kinases is rapidly activated following agonist-induced stimulation of phosphoinositide 3-kinase (PI3K). To probe the molecular events important for the activation process, we employed two distinct models of posttranslational inducible activation and membrane recruitment. PKB induction requires phosphorylation of two critical residues, threonine 308 in the activation loop and serine 473 near the carboxyl terminus. Membrane localization of PKB was found to be a primary determinant of serine 473 phosphorylation. PI3K activity was equally important for promoting phosphorylation of serine 473, but this was separable from membrane localization. PDK1 phosphorylation of threonine 308 was primarily dependent upon prior serine 473 phosphorylation and, to a lesser extent, localization to the plasma membrane. Mutation of serine 473 to alanine or aspartic acid modulated the degree of threonine 308 phosphorylation in both models, while a point mutation in the substrate-binding region of PDK1 (L155E) rendered PDK1 incapable of phosphorylating PKB. Together, these results suggest a mechanism in which 3' phosphoinositide lipid-dependent translocation of PKB to the plasma membrane promotes serine 473 phosphorylation, which is, in turn, necessary for PDK1-mediated phosphorylation of threonine 308 and, consequentially, full PKB activation.     

Conditional knock-out of integrin-linked kinase demonstrates an essential role in protein kinase B/Akt activation

Protein kinase B (PKB/Akt) plays a pivotal role in signaling pathways downstream of phosphatidylinositol 3-kinase, regulating fundamental processes such as cell survival, cell proliferation, differentiation, and metabolism. PKB/Akt activation is regulated by phosphoinositide phospholipid-mediated plasma membrane anchoring and by phosphorylation on Thr-308 and Ser-473. Whereas the Thr-308 site is phosphorylated by PDK-1, the identity of the Ser-473 kinase has remained unclear and controversial. The integrin-linked kinase (ILK) is a potential regulator of phosphorylation of PKB/Akt on Ser-473. Utilizing double-stranded RNA interference (siRNA) as well as conditional knock-out of ILK using the Cre-Lox system, we now demonstrate that ILK is essential for the regulation of PKB/Akt activity. ILK knock-out had no effect on phosphorylation of PKB/Akt on Thr-308 but resulted in almost complete inhibition of phosphorylation on Ser-473 and significant inhibition of PKB/Akt activity, accompanied by significant stimulation of apoptosis. The inhibition of PKB/Akt Ser-473 phosphorylation was rescued by kinase-active ILK but not by a kinase-deficient mutant of ILK, suggesting a role for the kinase activity of ILK in the stimulation of PKB/Akt phosphorylation. ILK knock-out also resulted in the suppression of phosphorylation of GSK-3beta on Ser-9 and cyclin D1 expression. These data establish ILK as an essential upstream regulator of PKB/Akt activation.     

Stimulation of the EAAT4 glutamate transporter by SGK protein kinase isoforms and PKB

The serum and glucocorticoid inducible kinase (SGK) 1 is expressed in brain tissue and upregulated by ischemia, neuronal excitation, and dehydration. The present study has been performed to elucidate the expression of SGK1 in cerebellar Purkinje cells and to explore whether it influences the colocalized glutamate transporter EAAT4. Intense SGK1 staining was observed in Purkinje cells following 48h of water deprivation. The kinase activates glutamate induced current (I(GLU)) in Xenopus oocytes heterologously expressing EAAT4, an effect mimicked by its isoforms SGK2, 3 and PKB. I(GLU) was decreased by the ubiquitin ligase Nedd4-2, an effect partially but not completely reversed by additional coexpression of the SGK kinase isoforms or PKB. According to immunohistochemistry EAAT4 protein abundance in the cell membrane was enhanced by SGK1 and decreased by Nedd4-2. In conclusion, SGK1 expression is upregulated by ischemia, excitation, and dehydration in cerebellar Purkinje cells. The upregulation of SGK1 may serve to stimulate EAAT4 and thus to reduce neuroexcitotoxicity.     

EAAT4 phosphorylation at the SGK1 consensus site is required for transport modulation by the kinase

EAAT4 (SLC1A6) is a Purkinje-Cell-specific post-synaptic excitatory amino acid transporter that plays a major role in clearing synaptic glutamate. EAAT4 abundance and function is known to be modulated by the serum and glucocorticoid inducible kinase (SGK) 1 but the precise mechanism of kinase action has not been defined yet. The present work aims to identify the molecular mechanism of EAAT4 modulation by the kinase. The EAAT4 sequence bears two putative SGK1 consensus sites (at Thr40 and Thr504) at the amino and carboxy terminus that are conserved among species. Expression studies in Xenopus oocytes demonstrated that EAAT4-mediated [(3)H] glutamate uptake and cell surface abundance are enhanced by co-expression of SGK1. Disruption of the SGK1 phosphorylation site at threonine 40 ((T40A)EAAT4) or of both phosphorylation sites ((T40AT504A)EAAT4) abrogated the effect of SGK1 on transporter function and expression. SGK1 modulates several transport proteins via inhibition of the ubiquitin ligase Nedd4-2. Co-expression of Nedd4-2 inhibited wild-type EAAT4 but not the (T40AT504A)EAAT4 mutant. Besides, RNA interference-mediated reduction of endogenous Nedd4-2 (xNedd4-2) expression increased the activity of the transporter. In conclusion, maximal glutamate transport modulation by SGK1 is accomplished by direct EAAT4 stimulation and to a lesser extent by inhibition of intrinsic Nedd4-2.     

Regulation of the glutamate transporter EAAT1 by the ubiquitin ligase Nedd4-2 and the serum and glucocorticoid-inducible kinase isoforms SGK1/3 and protein kinase B

Surface expression of the glial glutamate transporter EAAT1 is stimulated by insulin-like growth factor 1 through activation of phosphatidylinositol-3-kinase. Downstream targets include serum and glucocorticoid-sensitive kinase isoforms SGK1, SGK2 and SGK3, and protein kinase B. SGK1 regulates Nedd4-2, a ubiquitin ligase that prepares cell membrane proteins for degradation. To test whether Nedd4-2, SGK1, SGK3 and protein kinase B regulate EAAT1, cRNA encoding EAAT1 was injected into Xenopus oocytes with or without additional injection of wild-type Nedd4-2, constitutively active S422DSGK1, inactive K127NSGK1, wild-type SGK3 and/or constitutively active T308D,S473DPKB. Glutamate induces a current in Xenopus oocytes expressing EAAT1, but not in water-injected oocytes, which is decreased by co-expression of Nedd4-2, an effect reversed by additional co-expression of S422DSGK1, SGK3 and T308D,S473DPKB, but not K127NSGK1. Site-directed mutagenesis of the SGK1 phosphorylation sites in the Nedd4-2 protein (S382A,S468ANedd4-2) and in the EAAT1 protein (T482AEAAT1, T482DEAAT1) significantly blunts the effect of S422DSGK1. Moreover, the current is significantly larger in T482DEAAT1- than in T482AEAAT1-expressing oocytes, indicating that a negative charge mimicking phosphorylation at T482 increases transport. The experiments reveal a powerful novel mechanism that regulates the activity of EAAT1. This mechanism might participate in the regulation of neuronal excitability and glutamate transport in other tissues.     

Ceramide inhibits protein kinase B/Akt by promoting dephosphorylation of serine 473

The second messenger ceramide (N-alkylsphingosine) has been implicated in a host of cellular processes including growth arrest and apoptosis. Ceramide has been reported to have effects on both protein kinases and phosphatases and may constitute an important component of stress response in various tissues. We have examined in detail the relationship between ceramide signaling and the activation of an important signaling pathway, phosphatidylinositol (PI) 3-kinase and its downstream target, protein kinase B (PKB). PKB activation was observed following stimulation of cells with the cytokine granulocyte-macrophage colony-stimulating factor. Addition of cell-permeable ceramide analogs, C(2)- or C(6)-ceramide, caused a partial loss (50-60%) of PKB activation. This reduction was not a result of decreased PI(3,4,5)P(3) or PI(3,4)P(2) generation by PI 3-kinase. Two residues of PKB (threonine 308 and serine 473) require phosphorylation for maximal PKB activation. Serine 473 phosphorylation was consistently reduced by treatment with ceramide, whereas threonine 308 phosphorylation remained unaffected. In further experiments, ceramide appeared to accelerate serine 473 dephosphorylation, suggesting the activation of a phosphatase. Consistent with this, the reduction in serine 473 phosphorylation was inhibited by the phosphatase inhibitors okadaic acid and calyculin A. Surprisingly, threonine 308 phosphorylation was abolished in cells treated with these inhibitors, revealing a novel mechanism of regulation of threonine 308 phosphorylation. These results demonstrate that PI 3-kinase-dependent kinase 2-catalyzed phosphorylation of serine 473 is the principal target of a ceramide-activated phosphatase.     

Mechanism of Activation of PKB/Akt by the Protein Phosphatase Inhibitor Calyculin A

The protein phosphatase inhibitor calyculin A activates PKB/Akt to ~50% of the activity induced by insulin-like growth factor 1 (IGF1) in HeLa cells promoting an evident increased phosphorylation of Ser473 despite the apparent lack of Thr308 phosphorylation of PKB. Nevertheless, calyculin A-induced activation of PKB seems to be dependent on basal levels of Thr308 phosphorylation, since a PDK1-dependent mechanism is required for calyculin A-dependent PKB activation by using embryonic stem cells derived from PDK1 wild-type and knockout mice. Data shown suggest that calyculin A-induced phosphorylation of Ser473 was largely blocked by LY294002 and SB-203580 inhibitors, indicating that both PI3-kinase/TORC2-dependent and SAPK2/p38-dependent protein kinases contributed to phosphorylation of Ser473 in calyculin A-treated cells. Additionally, our results suggest that calyculin A blocks the IGF1-dependent Thr308 phosphorylation and activation of PKB, likely due to an enhanced Ser612 phosphorylation of insulin receptor substrate 1 (IRS1), which can be inhibitory to its activation of PI3-kinase, a requirement for PDK1-induced Thr308 phosphorylation and IGF1-dependent activation of PKB. Our data suggest that PKB activity is most dependent on the level of Ser473 phosphorylation rather than Thr308, but basal levels of Thr308 phosphorylation are a requirement. Additionally, we suggest here that calyculin A regulates the IGF1-dependent PKB activation by controlling the PI3-kinase-associated IRS1 Ser/Thr phosphorylation levels.     

Regulation of intestinal phosphate cotransporter NaPi IIb by ubiquitin ligase Nedd4-2 and by serum- and glucocorticoid-dependent kinase 1

Serum and glucocorticoid-inducible kinase 1 (SGK1) is highly expressed in enterocytes. The significance of the kinase in regulation of intestinal function has, however, remained elusive. In Xenopus laevis oocytes, SGK1 stimulates the epithelial Na(+) channel by phosphorylating the ubiquitin ligase Nedd4-2, which regulates channels by ubiquitination leading to subsequent degradation of the channel protein. Thus the present study has been performed to explore whether SGK1 regulates transport systems expressed in intestinal epithelial cells, specifically type IIb sodium-phosphate (Na(+)-P(i)) cotransporter (NaPi IIb). Immunohistochemistry in human small intestine revealed SGK1 colocalization with Nedd4-2 in villus enterocytes. For functional analysis cRNA encoding NaPi IIb, the SGK isoforms and/or the Nedd4-2 were injected into X. laevis oocytes, and transport activity was quantified as the substrate-induced current (I(P)). Exposure to 3 mM phosphate induces an I(P) in NaPi IIb-expressing oocytes. Coinjection of Nedd4-2, but not the catalytically inactive mutant (C938S)Nedd4-2, significantly downregulates I(P), whereas the coinjection of (S422D)SGK1 markedly stimulates I(P) and even fully reverses the effect of Nedd4-2 on I(P). The effect of (S422D)SGK1 on NaPi IIb is mimicked by wild-type SGK3 but not by wild-type SGK2, constitutively active (T308D,S473D)PKB, or inactive (K127N)SGK1. Moreover, (S422D)SGK1 and SGK3 phosphorylate Nedd4-2. In conclusion, SGK1 stimulates the NaPi IIb, at least in part, by phosphorylating and thereby inhibiting Nedd4-2 binding to its target. Thus the present study reveals a novel signaling pathway in the regulation of intestinal phosphate transport, which may be important for regulation of phosphate balance.     

The envelope glycoprotein of human endogenous retrovirus type W uses a divergent family of amino acid transporters/cell surface receptors

The human endogenous retrovirus type W (HERV-W) family includes proviruses with intact protein-coding regions that appear to be under selection pressure, suggesting that some HERV-W proviruses may remain active in higher primates. The envelope glycoprotein (Env) encoded by HERV-W is highly fusogenic, is naturally expressed in human placental syncytiatrophoblasts, and has been reported to function as a superantigen in lymphocyte cultures. Recent evidence suggested that HERV-W Env can mediate syncytium formation by interacting with the human sodium-dependent neutral amino acid transporter type 2 (hASCT2; gene name, SLC1A5) (J.-L. Blond, D. Lavillette, V. Cheynet, O. Bouton, G. Oriol, S. Chapel-Fernandez, B. Mandrand, F. Mallet, and F.-L. Cosset, J. Virol. 74:3321-3329, 2000) and that it can pseudotype human immunodeficiency virus cores (D. S. An, Y. Xie, and I. S. Y. Chen, J. Virol. 75:3488-3489, 2001). By using cell-cell fusion and pseudotype virion infection assays, we found that HERV-W Env efficiently uses both hASCT2 and the related transporter hASCT1 (gene name, SLC1A4) as receptors. In addition, although HERV-W Env mediates only slight syncytium formation or infection of mouse cells, it utilizes the mouse transporters mASCT1 and mASCT2 when their sites for N-linked glycosylation are eliminated by mutagenesis. Consistent with their role as a battlefield in host-virus coevolution, the viral recognition regions in ASCT1 and ASCT2 of humans and mice are highly divergent compared with other regions of these proteins, and their ratios of nonsynonymous to synonymous nucleotide sequence changes are extremely large. The recognition of ASCT1 and ASCT2 despite this divergence of their sequences strongly suggests that the use of both receptors has been highly advantageous for survival and evolution of the HERV-W family of retroviruses.     

Dual regulation of platelet protein kinase B

Protein kinase B (PKB) is a serine/threonine kinase that is activated by growth hormones and implicated in prevention of apoptosis, glycogen metabolism, and glucose uptake. A key enzyme in PKB activation is phosphatidylinositide 3-kinase (PI-3K), which triggers the dual phosphorylation of PKB by phosphatidylinositol-dependent kinases (PDKs). Here we report that the major PKB subtype in platelets is PKBalpha, which is activated by phosphorylation of Thr(308) and Ser(473) and has a constitutively phosphorylated Thr(450) that does not contribute to PKB activation. alpha-Thrombin and thrombopoietin activate PKBalpha via PI-3K and trigger the concurrent phosphorylation of Thr(308) (via PDK1) and Ser(473) (via a not yet identified PDK2). In addition, alpha-thrombin activates a PI-3K-independent pathway involving phospholipase Cbeta and calcium-dependent protein kinase C subtypes (PKCalpha/beta). This route is specific for phosphorylation of Ser(473) and can be initiated by direct PKC activation with phorbol ester or purified active PKC catalytic fragment in platelet lysate. Different degrees of Ser(473) and Thr(308) phosphorylation correlate with different degrees of enzyme activity. These data reveal a PI-3K-independent PKB activation in which PKCalpha/beta regulates the phosphorylation of Ser(473) in PKBalpha. The independent control of the two phosphorylation sites may contribute to fine regulation of PKBalpha activity.     

In vivo analysis of protein kinase B (PKB)/Akt regulation in DNA-PKcs-null mice reveals a role for PKB/Akt in DNA damage response and tumorigenesis

Full activation of protein kinase B (PKB/Akt) requires phosphorylation on Thr-308 and Ser-473. It is well established that Thr-308 is phosphorylated by 3-phosphoinositide-dependent kinase-1 (PDK1). Ser-473 phosphorylation is mediated by both mammalian target of rapamycin-rictor complex (mTORC2) and DNA-dependent protein kinase (DNA-PK) depending on type of stimulus. However, the physiological role of DNA-PK in the regulation of PKB phosphorylation remains to be established. To address this, we analyzed basal, insulin-induced, and DNA damage-induced PKB Ser-473 phosphorylation in DNA-PK catalytic subunit-null DNA-PKcs(-/-) mice. Our results revealed that DNA-PK is required for DNA damage-induced phosphorylation but dispensable for insulin- and growth factor-induced PKB Ser-473 phosphorylation. Moreover, DNA-PKcs(-/-) mice showed a tissue-specific increase in basal PKB phosphorylation. In particular, persistent PKB hyperactivity in the thymus apparently contributed to spontaneous lymphomagenesis in DNA-PKcs(-/-) mice. Significantly, these tumors could be prevented by deletion of PKBalpha. These findings reveal stimulus-specific regulation of PKB activation by specific upstream kinases and provide genetic evidence of PKB deregulation in DNA-PKcs(-/-) mice.