Consensus Phosphorylation Sites Research Articles

Saccharomyces CDK1 Phosphorylates Rad53 Kinase in Metaphase, Influencing Cellular Morphogenesis

Rad53 is an essential protein kinase governing DNA damage and replication stress checkpoints in budding yeast. It also appears to be involved in cellular morphogenesis processes. Mass spectrometry analyses revealed that Rad53 is phosphorylated at multiple SQ/TQ and at SP/TP residues, which are typical consensus sites for phosphatidylinositol 3-kinase-related kinases and CDKs, respectively. Here we show that Clb-CDK1 phosphorylates Rad53 at Ser(774) in metaphase. This phosphorylation event does not influence the DNA damage and replication checkpoint roles of Rad53, and it is independent of the spindle assembly checkpoint network. Moreover, the Ser-to-Asp mutation, mimicking a constitutive phosphorylation state at site 774, causes sensitivity to calcofluor, supporting a functional linkage between Rad53 and cellular morphogenesis.

Water permeability through aquaporin-4 is regulated by protein kinase C and becomes rate-limiting for glioma invasion

Glial-derived tumors, gliomas, are highly invasive cancers that invade normal brain through the extracellular space. To navigate the tortuous extracellular spaces, cells undergo dynamic changes in cell volume, which entails water flux across the membrane through aquaporins (AQPs). Two members of this family, AQP1 and AQP4 are highly expressed in primary brain tumor biopsies and both have a consensus phosphorylation site for protein kinase C (PKC), which is a known regulator of glioma cell invasion. AQP4 colocalizes with PKC to the leading edge of invading processes and clustered with chloride channel (ClC2) and K +–Cl − cotransporter 1 (KCC1), believed to provide the pathways for Cl − and K + secretion to accomplish volume changes. Using D54MG glioma cells stably transfected with either AQP1 or AQP4, we show that PKC activity regulates water permeability through phosphorylation of AQP4. Activation of PKC with either phorbol 12-myristate 13-acetate or thrombin enhanced AQP4 phosphorylation, reduced water permeability and significantly decreased cell invasion. Conversely, inhibition of PKC activity with chelerythrine reduced AQP4 phosphorylation, enhanced water permeability and significantly enhanced tumor invasion. PKC regulation of AQP4 was lost after mutational inactivation of the consensus PKC phosphorylation site S180A. Interestingly, AQP1 expressing glioma cells, by contrast, were completely unaffected by changes in PKC activity. To demonstrate a role for AQPs in glioma invasion in vivo, cells selectively expressing AQP1, AQP4 or the mutated S180A-AQP4 were implanted intracranially into SCID mice. AQP4 expressing glioma cells showed significantly reduced invasion compared to AQP1 and S180 expressing tumors as determined by quantitative stereology, consistent with a differential role for AQP1 and AQP4 in this process.

CAMP-dependent kinase does not modulate the Slack sodium-activated potassium channel

The Slack gene encodes a Na +-activated K + channel and is expressed in many different types of neurons. Like the prokaryotic Ca 2+-gated K + channel MthK, Slack contains two ‘regulator of K + conductance’ (RCK) domains within its carboxy terminal, domains likely involved in Na + binding and channel gating. It also contains multiple consensus protein kinase C (PKC) and protein kinase A (PKA) phosphorylation sites and although regulated by protein kinase C (PKC) phosphorylation, modulation by PKA has not been determined. To test if PKA directly regulates Slack, nystatin-perforated patch whole-cell currents were recorded from a human embryonic kidney (HEK-293) cell line stably expressing Slack. Bath application of forskolin, an adenylate cyclase activator, caused a rapid and complete inhibition of Slack currents however, the inactive homolog of forskolin, 1,9-dideoxyforskolin caused a similar effect. In contrast, bath application of 8-bromo-cAMP did not affect the amplitude nor the activation kinetics of Slack currents. In excised inside-out patch recordings, direct application of the PKA catalytic subunit to patches did not affect the open probability of Slack channels nor was open probability affected by direct application of protein phosphatase 2B. Preincubation of cells with the protein kinase A inhibitor KT5720 also did not change current density. Finally, mutating the consensus phosphorylation site located between RCK domain 1 and domain 2 from serine to glutamate did not affect current activation kinetics. We conclude that unlike PKC, phosphorylation by PKA does not acutely modulate the function and gating activation kinetics of Slack channels.

Interaction of hookworm 14-3-3 with the forkhead transcription factor DAF-16 requires intact Akt phosphorylation sites

BackgroundThird-stage infective larvae (L3) of hookworms are in an obligatory state of developmental arrest that ends upon entering the definitive host, where they receive a signal that re-activates development. Recovery from the developmentally arrested dauer stage of Caenorhabditis elegans is analogous to the resumption of development during hookworm infection. Insulin-like signaling (ILS) mediates recovery from arrest in C. elegans and activation of hookworm dauer L3. In C. elegans, phosphorylation of the forkhead transcription factor DAF-16 in response to ILS creates binding cites for the 14-3-3 protein Ce-FTT-2, which translocates DAF-16 out of the nucleus, resulting in resumption of reproductive development.ResultsTo determine if hookworm 14-3-3 proteins play a similar role in L3 activation, hookworm FTT-2 was identified and tested for its ability to interact with A. caninum DAF-16 in vitro. The Ac-FTT-2 amino acid sequence was 91% identical to the Ce-FTT-2, and was most closely related to FTT-2 from other nematodes. Ac-FTT-2 was expressed in HEK 293T cells, and was recognized by an antibody against human 14-3-3β isoform. Reciprocal co-immunoprecipitations using anti-epitope tag antibodies indicated that Ac-FTT-2 interacts with Ac-DAF-16 when co-expressed in serum-stimulated HEK 293T cells. This interaction requires intact Akt consensus phosphorylation sites at serine107 and threonine312, but not serine381. Ac-FTT-2 was undetectable by Western blot in excretory/secretory products from serum-stimulated (activated) L3 or adult A. caninum.ConclusionThe results indicate that Ac-FTT-2 interacts with DAF-16 in a phosphorylation-site dependent manner, and suggests that Ac-FTT-2 mediates activation of L3 by binding Ac-DAF-16 during hookworm infection.

Phosphorylation of ribosomal protein S19 at Ser59 by CaM Kinase Iα

In order to examine the possible involvements of Ca(2+)/calmodulin-dependent protein kinases (CaM kinases) in the regulation of ribosomal functions, we tested the phosphorylation of rat ribosomal protein S19 (RPS19) by various CaM kinases in vitro. We found that CaM kinase Ialpha, but not CaM kinase Ibeta1, Ibeta2, II, or IV, robustly phosphorylated RPS19. From the consensus phosphorylation site sequence, Ser59, Ser90, and Thr124 were likely to be phosphorylated; therefore, we mutated each amino acid to alanine and found that the mutation of Ser59 to alanine strongly attenuated phosphorylation by CaM kinase Ialpha, suggesting that Ser59 was a major phosphorylation site. Furthermore, we produced a specific antibody against RPS19 phosphorylated at Ser59, and found that Ser59 was phosphorylated both in GT1-7 cells and rat brain. Phosphorylation of RPS19 in GT1-7 cells was inhibited by KN93, an inhibitor of CaM kinases. Immunoblot analysis after subcellular fractionation of rat brain demonstrated that phosphorylated RPS19 was present in 80S ribosomes. Phosphorylation of RPS19 by CaM kinase Ialpha augmented the interaction of RPS19 with the previously identified S19 binding protein. These results suggest that CaM kinase Ialpha regulates the functions of RPS19 through phosphorylation of Ser59.

MAP kinase phosphatase-1: a link between cell signaling and histone phosphorylation. Focus on “Histone H3 as a novel substrate for MAP kinase phosphatase-1”

the dna of eukaryotic cells is highly folded and compacted into chromatin that can be propagated during mitosis. Chromatin contains the entire genome and histones that are subjected to different posttranslational modifications, generally in the amino-terminal domain. Distinct modifications including

Destabilization of the Postsynaptic Density by PSD-95 Serine 73 Phosphorylation Inhibits Spine Growth and Synaptic Plasticity

Long-term potentiation (LTP) is accompanied by dendritic spine growth and changes in the composition of the postsynaptic density (PSD). We find that activity-dependent growth of apical spines of CA1 pyramidal neurons is accompanied by destabilization of the PSD that results in transient loss and rapid replacement of PSD-95 and SHANK2. Signaling through PSD-95 is required for activity-dependent spine growth and trafficking of SHANK2. N-terminal PDZ and C-terminal guanylate kinase domains of PSD-95 are required for both processes, indicating that PSD-95 coordinates multiple signals to regulate morphological plasticity. Activity-dependent trafficking of PSD-95 is triggered by phosphorylation at serine 73, a conserved calcium/calmodulin-dependent protein kinase II (CaMKII) consensus phosphorylation site, which negatively regulates spine growth and potentiation of synaptic currents. We propose that PSD-95 and CaMKII act at multiple steps during plasticity induction to initially trigger and later terminate spine growth by trafficking growth-promoting PSD proteins out of the active spine.

CAMP-dependent and cholinergic regulation of the electrogenic intestinal/pancreatic Na+/HCO3- cotransporter pNBC1 in human embryonic kidney (HEK293) cells

BackgroundThe renal (kNBC1) and intestinal (pNBC1) electrogenic Na+/HCO3- cotransporter variants differ in their primary structure, transport direction, and response to secretagogues. Previous studies have suggested that regulatory differences between the two subtypes can be partially explained by unique consensus phosphorylation sites included in the pNBC1, but not the kNBC1 sequence. After having shown activation of NBC by carbachol and forskolin in murine colon, we now investigated these pathways in HEK293 cells transiently expressing a GFP-tagged pNBC1 construct. ResultsNa+- and HCO3--dependent pHi recovery from an acid load (measured with BCECF) was enhanced by 5-fold in GFP-positive cells compared to the control cells in the presence of CO2/HCO3-. Forskolin (10-5 M) had no effect in untransfected cells, but inhibited the pHi recovery in cells expressing pNBC1 by 62%. After preincubation with carbachol (10-4 M), the pHi recovery was enhanced to the same degree both in transfected and untransfected cells, indicating activation of endogenous alkalizing ion transporters. Acid-activated Na+/HCO3- cotransport via pNBC1 expressed in renal cells is thus inhibited by cAMP and not affected by cholinergic stimulation, as opposed to the findings in native intestinal tissue. ConclusionRegulation of pNBC1 by secretagogues appears to be not solely dependent on its primary structure, but also on properties of the cell type in which it is expressed.

Participation of HERG channel cytoplasmic structures on regulation by the G protein-coupled TRH receptor

Human ether-a-go-go-related gene (HERG) channels heterologously expressed in Xenopus oocytes are regulated by the activation of G protein-coupled hormone receptors that, like the thyrotropin-releasing hormone (TRH) receptor, activate phospholipase C. Previous work with serially deleted HERG mutants suggested that residues 326-345 located in the proximal domain of the channels amino terminus might be required for the hormonal modulation of HERG activation. Generation of new channel mutants deleted in this region further point to the amino acid sequence between residues 326 and 332 as a possible determinant of the TRH effects, but individual or combined single-point mutations in this sequence demonstrate that maintenance of its consensus sites for phosphorylation and/or interaction with regulatory components is not important for the modulatory response(s). The TRH-induced effects also remained unaltered when a basic amino acid cluster located between residues 362 and 366 is eliminated. Additionally, no effect of TRH was observed in channels carrying single-point mutations at the beginning of the intracellular loop linking transmembrane domains S4 and S5. Our results indicate that a correct structural arrangement of the amino terminal domains is essential for the hormone-induced modifications of HERG activation. They also suggest that the hormonal regulatory action is transmitted to the transmembrane channel core through interactions between the cytoplasmic domains and the initial portion of the S4-S5 linker.

Reflections

Brain type II Ca2+/calmodulin-dependent protein kinase is a holoenzyme composed of several copies each of three subunits, alpha (50 kd), beta (60 kd), and beta' (58 kd), in varying proportions. The deduced amino acid sequences of alpha (reported here) and beta are highly similar but not identical. The major difference between them is the deletion from alpha of two short segments (residues 316-339 and 354-392 in beta). cDNAs that appear to encode beta' are identical to beta except for the deletion of a segment encoding residues 378-392. Thus, the structural differences among alpha, beta, and beta' arise primarily from deletions (or insertions) in a variable region lying immediately carboxyl to the protein kinase and calmodulin-binding domains. The alpha and beta subunits are encoded by distinct genes expressed primarily, if not exclusively, in brain. Rather than being encoded by a third gene, beta' may arise by alternative splicing of the beta gene transcript.

Aurora A Is Essential for Early Embryonic Development and Tumor Suppression

Aurora A is a serine/threonine kinase that functions in various stages of mitosis. Accumulating evidence has demonstrated that gene amplification and overexpression of Aurora A are linked to tumorigenesis, suggesting that Aurora A is an oncogene. In addition, Aurora A overexpression has been used as a negative prognostic marker, because it is associated with resistance to anti-mitotic agents commonly used for cancer therapy. To understand the physiological functions of Aurora A, we generated Aurora A knock-out mice. Aurora A null mice die early during embryonic development before the 16-cell stage. These Aurora A null embryos have defects in mitosis, particularly in spindle assembly, supporting critical functions of Aurora A during mitotic transitions. Interestingly, Aurora A heterozygosity results in a significantly increased tumor incidence in mice, suggesting that Aurora A may also act as a haploinsufficient tumor suppressor. Consistently, Aurora A heterozygous mouse embryonic fibroblasts have higher rates of aneuploidy. We further discovered that VX-680, an Aurora kinase inhibitor currently in phase II clinical trials for cancer treatment, could induce aneuploidy in wild type mouse embryonic fibroblasts. We conclude that a balanced Aurora A level is critical for maintaining genomic stability and one needs to be fully aware of the potential side effects of anti-cancer therapy based on the use of Aurora A-specific inhibitors.

High-resolution x-ray structure of human aquaporin 5

Human aquaporin 5 (HsAQP5) facilitates the transport of water across plasma membranes and has been identified within cells of the stomach, duodenum, pancreas, airways, lungs, salivary glands, sweat glands, eyes, lacrimal glands, and the inner ear. AQP5, like AQP2, is subject to posttranslational regulation by phosphorylation, at which point it is trafficked between intracellular storage compartments and the plasma membrane. Details concerning the molecular mechanism of membrane trafficking are unknown. Here we report the x-ray structure of HsAQP5 to 2.0-A resolution and highlight structural similarities and differences relative to other eukaryotic aquaporins. A lipid occludes the putative central pore, preventing the passage of gas or ions through the center of the tetramer. Multiple consensus phosphorylation sites are observed in the structure and their potential regulatory role is discussed. We postulate that a change in the conformation of the C terminus may arise from the phosphorylation of AQP5 and thereby signal trafficking.

S6K1 Phosphorylates and Regulates Fragile X Mental Retardation Protein (FMRP) with the Neuronal Protein Synthesis-dependent Mammalian Target of Rapamycin (mTOR) Signaling Cascade

Fragile X syndrome is a common form of cognitive deficit caused by the functional absence of fragile X mental retardation protein (FMRP), a dendritic RNA-binding protein that represses translation of specific messages. Although FMRP is phosphorylated in a group I metabotropic glutamate receptor (mGluR) activity-dependent manner following brief protein phosphatase 2A (PP2A)-mediated dephosphorylation, the kinase regulating FMRP function in neuronal protein synthesis is unclear. Here we identify ribosomal protein S6 kinase (S6K1) as a major FMRP kinase in the mouse hippocampus, finding that activity-dependent phosphorylation of FMRP by S6K1 requires signaling inputs from mammalian target of rapamycin (mTOR), ERK1/2, and PP2A. Further, the loss of hippocampal S6K1 and the subsequent absence of phospho-FMRP mimic FMRP loss in the increased expression of SAPAP3, a synapse-associated FMRP target mRNA. Together these data reveal a S6K1-PP2A signaling module regulating FMRP function and place FMRP phosphorylation in the mGluR-triggered signaling cascade required for protein-synthesis-dependent synaptic plasticity.

Regulation of Intra-S Phase Checkpoint by Ionizing Radiation (IR)-dependent and IR-independent Phosphorylation of SMC3

Structure maintenance of chromosome 1 (SMC1) is phosphorylated by ataxia telangiectasia-mutated (ATM) in response to ionizing radiation (IR) to activate intra-S phase checkpoint. A role of CK2 in DNA damage response has been implicated in many previous works, but the molecular mechanism for its activation is not clear. In the present work, we report that SMC3 is phosphorylated at Ser-1067 and Ser-1083 in vivo. Ser-1083 phosphorylation is IR-inducible, depends on ATM and Nijmegen breakage syndrome 1 (NBS1), and is required for intra-S phase checkpoint. Interestingly, Ser-1067 phosphorylation is constitutive and is not induced by IR but also affects intra-S phase checkpoint. Phosphorylation of Ser-1083 is weakened in cells expressing S1067A mutant, suggesting interplay between Ser-1067 and Ser-1083 phosphorylation in DNA damage response. Consistently, small interfering RNA knockdown of CK2 leads to attenuated phosphorylation of Ser-1067 as well as intra-S phase checkpoint defect. Our data provide evidence that phosphorylation of a core cohesin subunit SMC3 by ATM plays an important role in DNA damage response and suggest that a constitutive phosphorylation by CK2 may affect intra-S phase checkpoint by modulating SMC3 phosphorylation by ATM.

What Are the Consequences of Phosphorylation and Hyperphosphorylation of Ryanodine Receptors in Normal and Failing Heart?

See related article, pages e65–e72 Marks and colleagues published some influential results in 2000, suggesting that protein kinase A phosphorylation regulates ryanodine receptor (RyR2) open probability ( P oRyR). The authors attributed this to the dissociation of the RyR-associated regulatory protein FKBP12.6.1 They went on to propose that, in failing hearts, RyR2 is hyperphosphorylated, which leads to defective channel function by increasing sensitivity to Ca-induced activation of the molecule. Moreover, these effects were attributable to phosphorylation of a single amino acid, serine 2809 (S2809). There are 4 of these serine molecules at location 2808 in the human RyR2 molecule, 1 on each subunit of the tetramer. Much subsequent work has referred to the 2809 residue, which is found in rabbits. Marks and colleagues further suggested that phosphorylation of S2809 was also responsible for the normal effects of adrenergic stimulation on excitation–contraction coupling (ECC) and that hyperphosphorylation was responsible for abnormal ECC during failure. The RyR is a tetramer that forms the largest membrane ion channel. In muscle, it is found in the sarcoplasmic reticulum (SR), where it functions as the Ca release channel. The heart isoform is designated RyR2. The pore sequences are largely found in membrane spanning domains.2 There is a large cytosolic domain. Although hyperphosphorylation refers, by definition, to the occupation of all the putative phosphorylation sites in a molecule in the context of the work by Marks and colleagues,1 hyperphosphorylation refers to 3 or perhaps 4 of the serine residues. In RyRs, there are a large number of consensus phosphorylation sites.3 RyRs are associated with a number of proteins, and these include anchoring protein phosphatases and kinases, as well as the regulatory protein FKBP12.6. From the standpoint of this editorial, this last protein is perhaps the most interesting, and it is considered …

Modulation of the Mitotic Regulatory Network by APC-Dependent Destruction of the Cdh1 Inhibitor Acm1

The completion of mitosis depends on protein ubiquitination by the anaphase-promoting complex (APC). The APC is activated by association with Cdc20 in midmitosis and Cdh1 in late mitosis and G1. Here, we show that in budding yeast the activation of APC(Cdh1) is controlled in part by destruction of the Cdh1 inhibitor Acm1. We find that Acm1 uses pseudosubstrate and other sequence motifs to bind and inhibit Cdh1, but not Cdc20. Acm1 also contains a destruction sequence that promotes its ubiquitination by APC(Cdc20), resulting in the disappearance of Acm1 in early anaphase. Later in mitosis, Acm1 destruction is also promoted by APC(Cdh1). Finally, Cdk1-dependent phosphorylation of Acm1 modulates its localization and destruction. We conclude that ubiquitination of a Cdh1 inhibitor by APC(Cdc20) helps establish the order of activation of the two APC isoforms. We also speculate that the ability of APC(Cdh1) to target its own inhibitor enhances the bistability of the late mitotic regulatory system.

G1/S Cyclin-dependent Kinase Regulates Small GTPase Rho1p through Phosphorylation of RhoGEF Tus1p inSaccharomyces cerevisiae

Rho1p is an essential small GTPase that plays a key role in the morphogenesis of Saccharomyces cerevisiae. We show here that the activation of Rho1p is regulated by a cyclin-dependent kinase (CDK). Rho1p is activated at the G1/S transition at the incipient-bud sites by the Cln2p (G1 cyclin) and Cdc28p (CDK) complex, in a process mediated by Tus1p, a guanine nucleotide exchange factor for Rho1p. Tus1p interacts physically with Cln2p/Cdc28p and is phosphorylated in a Cln2p/Cdc28p-dependent manner. CDK phosphorylation consensus sites in Tus1p are required for both Cln2p-dependent activation of Rho1p and polarized organization of the actin cytoskeleton. We propose that Cln2p/Cdc28p-dependent phosphorylation of Tus1p is required for appropriate temporal and spatial activation of Rho1p at the G1/S transition.

T1876 Lubiprostone Activation of Cl − Currents Does Not Involve CA 2+, cAMP, or PKA

Introduction Lubiprostone stimulates electrogenic Cltransport in human intestinal T84 cells and Clcurrents in HEK293 cells stably transfected with recombinant human ClC-2 (hClC-2) with an EC50 of approximately 20 nM [Cuppoletti et al AJP 287:C1173, 2004]. The purpose of this study was to determine whether lubiprostone (over the concentration range that activates electrogenic Cltransport) causes changes in [Ca]i, cAMP or PKA mediated phosphorylation. Methods T84 cells, HEK293 cells and HEK293 cells stably expressing hClC-2 or mutant hClC-2 were used. T84 cell [Ca]i, was measured using the calcium indicator dye indo-1/AM and intracellular [cAMP] was determined using a commercial ELISA kit. A commercial FLIPER assay was used to measure [Ca]i, and [cAMP] in HEK293 cells. Cl currents were measured by whole cell patch clamp. Results Lubiprostone at 1, 10 or 100 nM did not increase [Ca]i, in T84 cells and only slightly increased [cAMP] from 0.56 ± 0.02 to 4.0 ± 0.3 pmole/105 cells (n=4, P<0.001) at 100 nM (5 times the EC50 for lubiprostone activation of Cl currents). 1 μM PGE1 significantly (P<0.0005) increased [Ca]i from 80.8 ± 2.4 nM (control) to 1175.0 ± 18.7 nM (n=3) and greatly increased [cAMP] to 37.6 ± 0.6 pmole/105 cells (n=4) (P<0.0005). In contrast, 100 nM lubiprostone had no effect on [Ca]i or cAMP in HEK293 cells. Nevertheless hClC-2 in HEK293 cells is activated by lubiprostone, thereby demonstrating that neither [Ca]i or cAMP signaling are involved in lubiprostone activation of hClC-2. To rule out involvement of PKA phosphorylation of hClC-2 in lubiprostone activation of Clcurrents, a mutant hClC-2 Clchannel lacking the two unique PKA consensus phosphorylation sites (RRAT and RGET) essential for PKA activation of ClC-2, was used [Cuppoletti, et al JBC 279:21849, 2004]. In wild-type hClC-2 expressing HEK293 cells, control Clcurrents at -140 mV (n=6) of -27.2 ± 3.5 pA/pF, significantly increased (P<0.0005) to -100.9 ± 6.5 pA/pF with 20 nM lubiprostone and subsequently were reduced with 500 μM CdCl2 to control levels, -29.9 ± 5.6 pA/pF (P<0.0005). Cl currents in mutant hClC-2 expressing HEK293 cells was also significantly (P<0.0005) increased with 20 nM lubiprostone and inhibited by CdCl2 (n=6): control, -27.1 ± 6.9 pA/pF; with lubiprostone, -81.9 ± 5.1 (P<0.0005); after CdCl2, -29.4 ± 3.7 pA/pF (P<0.0005). Conclusions Activation of Clcurrents by lubiprostone is independent of [Ca]i and cAMP signaling and PKA mediated phosphorylation of hClC-2. More direct action of lubiprostone on hClC-2 Clchannels may be involved. Supported by Sucampo Pharmaceuticals, Inc. E001288 Conclusions Activation of Clcurrents by lubiprostone is independent of [Ca]i and cAMP signaling and PKA mediated phosphorylation of hClC-2. More direct action of lubiprostone on hClC-2 Clchannels may be involved.

Akt stabilizes estrogen receptor α with the concomitant reduction in its transcriptional activity

We have investigated the effect of Akt on estrogen receptor (ER) α protein level and its transcriptional activity. Transient transfection studies revealed that constitutively active Akt1 up-regulated ERα at the post-transcriptional level. Studies using Akt inhibitor and dominant-negative Akt1 showed that Akt1 kinase activity is required for the up-regulation of ERα. Cycloheximide decay assays and studies with proteasome inhibitor indicated that Akt1-mediated up-regulation of ERα was maintained by inhibiting proteasome-mediated degradation of ERα. When Akt consensus phosphorylation site mutant, ERαS167A was tested for Akt1-mediated up-regulation, increase of ERαS167A by Akt1 was significantly impaired as compared to wild type ERα. In addition, dominant-negative glycogen synthase kinase (GSK) 3β and LiCl could also partially up-regulate ERα protein level, suggesting that concerted action of Akt1-mediated phosphorylation on S167 and kinase activity of Akt-downstream GSK3β could affect ERα protein level. Paradoxically, co-expression of Akt1 could down-regulate transcriptional activity of ERα. The inhibitory effect of Akt1 on ERα transcriptional activity was not attributable to changes in subcellular distribution of ERα. Transfection studies using increasing amount of Akt1 and ERα indicated that the transcriptional activity of ERα was negatively regulated by ERα protein quantities at higher ERα concentrations. Chromatin immunoprecipitation assays revealed that at Akt1 concentration high enough to induce up-regulation of ERα, association of ERα to promoter region of ERα target pS2 gene was impaired. Taken together, these data suggest that Akt1 could increase ERα protein level with simultaneous reduction in its transcriptional activity, possibly by modulating association of ERα to the target gene promoters.

Endothelin‐1‐induced NOS3 activation is mediated by the PI3K/AKT pathway in the thick ascending limb

Endothelin‐1 (ET‐1) inhibits NaCl transport by the thick ascending limb (THAL) via increased NO. NOS3 is the primary source of NO in this segment. Phosphorylation of NOS3 at serine 1177 (pNOS3 Ser1177) positively regulates its activity. We hypothesized that ET‐1 stimulates pNOS3 Ser1177 via activation of the PI3K/AKT pathway. We measured: 1) pNOS3 Ser1177 by Western blot; 2) AKT activity using a FRET‐based AKT reporter; and 3) pAKT Ser473. Rat THALs were treated with ET‐1 (1nM) for 15 min. ET‐1 increased pNOS3 Ser1177 by 45 ± 9 % vs. control (p = 0.001; n = 6). Wortmannin (wort., 150nM), a PI3K inhibitor, blocked this effect (Δ + 3 ± 14 % vs. wort. alone, n = 5). The AKT reporter was expressed in the THAL by in vivo gene transfer. The probe is a fusion protein of the AKT consensus phosphorylation site, CFP and YFP (Kunkel et al, JBC. 280(7):5581–7). An increase in the CFP/YFP ratio indicates increased AKT activity. THALs were isolated and FRET measured by fluorescence microscopy. ET‐1 increased the CFP/YFP ratio by 0.16 ± 0.02 fluorescence units (f.u) (p &lt; 0.01; n = 5). Wort. blocked this effect (Δ + 0.01 ± 0.01 f.u; n = 5). ET‐1 increased pAKT Ser473 by 47 ± 14% vs. control (p &lt; 0.001; n = 5). Wort. blocked this effect (Δ + 2 ± 6 % vs. wort. alone). We concluded that ET‐1 acutely phosphorylates NOS3 at Ser1177 via PI3K/AKT activation in the THAL. This could be an important regulatory mechanism of THAL NaCl reabsorption by ET‐1.HL028982, HL070985