GNAQ and GNA11 for over 70% of tumors and less frequently via V600E BRAF mutations. In this report, we describe the impact of dual pathway inhibition upon uveal melanoma cell lines with the MEK inhibitor selumetinib (AZD6244/ARRY-142886) and the ATP-competitive mTOR kinase inhibitor AZD8055. While synergistic reductions in cell viability were observed with AZD8055/selumetinib in both BRAF and GNAQ mutant cell lines, apoptosis was preferentially induced in BRAF mutant cells only. In vitro apoptosis assay results were predictive of in vivo drug efficacy as tumor regressions were observed only in a BRAF mutant xenograft model, but not GNAQ mutant model. We went on to discover that GNAQ promotes relative resistance to AZD8055/selumetinib-induced apoptosis in GNAQ mutant cells. For BRAF mutant cells, both AKT and 4E-BP1 phosphorylation were modulated by the combination; however, decreasing AKT phosphorylation alone was not sufficient and decreasing 4E-BP1 phosphorylation was not required for apoptosis. Instead, cooperative mTOR complex 2 (mTORC2) and MEK inhibition resulting in downregulation of the pro-survival protein MCL-1 was found to be critical for combination-induced apoptosis. These results suggest that the clinical efficacy of combined MEK and mTOR kinase inhibition will be determined by tumor genotype, and that BRAF mutant malignancies will be particularly susceptible to this strategy.Uveal melanomas possess activation of the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT/mammalian Target of Rapamycin (mTOR) pathways. MAPK activation occurs via somatic mutations in the heterotrimeric G protein subunits  Uveal melanoma is a primary intraocular malignancy that arises from melanocytes within the uveal tract, which includes the iris, ciliary body, and choroid. Over 50% of these patients develop incurable, metastatic disease for which there are no effective therapies BRAF (T1799A→V600E) mutations for MAPK activation in uveal melanoma is less clear. Though initial studies of uveal melanoma tumor samples reported that BRAF mutations are rare BRAF mutation and confirmed that the mutation can be limited to select areas within a tumor BRAF mutant tumors also can not be excluded.52% to 86% of uveal melanomas possess activation of the mitogen-activated protein kinase (MAPK) pathway GNAQras-like domain blocks intrinsic GTPase activity, keeping Gα in a GTP-bound, constitutively active state. Recently, a mutation at Q209 in the GNAQ paralogue gene GNA11 that also activates MAPK signaling was discovered in over 30% of uveal melanomas For over 40% of uveal melanomas, MAPK activation is driven by mutation of phosphatase and tensin homolog (PTEN) (a negative regulator of the PI3K/AKT/mTOR pathway) locus and mutations within the PTEN coding region translates to more than half of uveal melanomas having decreased or complete loss of PTEN expression It has been hypothesized that activation of the phosphoinositide 3-kinase (PI3K)/AKT/mammalian Target of Rapamycin (mTOR) pathway cooperates with MAPK activation to generate and maintain the malignant phenotype in uveal melanomas. High rates of loss of heterozygosity (LOH) at the For inhibition of the PI3K/AKT signaling, effort has been put into targeting mTOR , which is a serine/threonine kinase activated by the pathway. The drugs rapamycin and its analogues inhibit mTOR complex 1 (mTORC1), which includes mTOR and other regulatory proteins such as regulatory associated protein of mTOR (Raptor). However, clinical activity with rapamycin and its analogues has been modest to date, and several molecular mechanisms potentially limiting clinical efficacy have been proposed, including: 1) incomplete mTORC1 inhibition, 2) activation of AKT by release of negative feedback pathways regulated by mTORC1 substrates and 3) minimal inhibition of a second rapamycin-resistant complex called mTOR complex 2 (mTORC2), which contains a protein called “rapamycin-insensitive component of mTOR” (Rictor) instead of Raptor. mTORC2 is the protein dependent kinase 2 (PDK2) responsible for phosphorylating AKT at serine 473 in vitro (IC50 of 0.8+/−0.2 nM), while exhibiting >1000 fold selectivity against closely related kinases such as PI3K, ATM, and DNA-PK, and no activity against a panel of 260 other kinases at a concentration of 10 µM in vitro with an IC50 of 14.1+/−0.79 nM In this study, we explored combined MAPK and PI3K/AKT/mTOR pathway inhibition with an ATP-competitive mTOR inhibitor, AZD8055, and the allosteric MEK inhibitor, selumetinib, in uveal melanoma cell lines of various genotypic backgrounds. AZD8055 potently inhibits the mTOR kinase BRAF, GNAQ, GNA11, and RAS , two cell lines which possess the V600E BRAF mutation , and two cell lines which possess activating GNAQ mutations at Q209 (92.1 (Q209L) and Mel270 (Q209P), referred to as “GNAQ”); these cell lines were otherwise negative for common mutations in several receptor tyrosine kinases (RTKs), PIK3CA, and AKT1 (Table S1). PTEN protein expression was detectable by Western blot in all cell lines .We investigated the impact of dual pathway inhibition with the MEK1/2 inhibitor selumetinib and the mTOR kinase inhibitor AZD8055 upon uveal melanoma cell lines of distinct tumor genotypes. This panel included two cell lines which lack activating mutations in Figure S1). In both GNAQ and BRAF cells, the AZD8055/selumetinib combination at different dose combinations consistently reduced cell viability by more than 50%  >0.5) in a synergistic manner (combination index (CI) values <1) (The viability of cells treated with various concentrations of AZD8055 (0 to 1000 nM) and selumetinib (0 to 5000 nM) alone and in different combinations were evaluated  for each compound alone are depicted in Figure S2).We next investigated how differences in cellular viability correlated with apoptosis. 100 nM AZD8055 and 1000 nM selumetinib were utilized for this and all subsequent experiments after it was determined that these are the lowest concentrations that effectively inhibit mTOR and MEK, respectively, in cells  and GNAQ mutant (92.1) xenograft tumor model. In the BRAF mutant model, the combination resulted in tumor regressions at Day 19 with tumor volumes that were lower than those achieved with either drug alone  images). In the GNAQ mutant model, only a non-statistically significant trend towards enhanced tumor growth inhibition with the combination was achieved; neither tumor regression nor changes in Ki67 and TUNEL staining were observed . Hence, we concluded that the in vitro apoptosis assay results were more predictive of in vivo drug effectiveness than the in vitro viability data, and we subsequently focused our efforts upon investigating the mechanism(s) by which these drugs elicit distinct apoptotic outcomes in the BRAF and GNAQ mutant uveal tumor genotypes.We hypothesized that the preferential induction of apoptosis with the combination in GNAQ expression was suppressed with pooled small interfering RNA (siRNA) constructs in the GNAQ mutant cell line 92.1  and the mTORC2 substrate AKT ; selumetinib, however, failed to inhibit ERK phosphorylation in a sustained manner  due to rebound ERK phosphorylation . This lack of sustained target inhibition is one possible explanation for the lack of cooperative antitumor effect observed with the combination in these cells. Additionally, AKT phosphorylation was slightly increased with selumetinib in only these cells .To investigate how AZD8055/selumetinib preferentially elicits apoptosis in , lane 2 of Blots #1 and #2; Figure S2A). This was not due to ineffective mTORC2 inhibition, but instead rebound AKT phosphorylation: while 100 nM AZD8055 inhibited AKT phosphorylation after 2 hours in all three tumor genotypes, by 24 hours AKT phosphorylation rebounded only in BRAF and GNAQ cells . Interestingly, the addition of selumetinib to AZD8055 substantially decreased AKT phosphorylation in BRAF cells, but not significantly in GNAQ cells .In BRAF and GNAQ cells, AZD8055 potently suppressed S6K1 phosphorylation, but failed to completely inhibit AKT phosphorylation (BRAF mutant cell line OCM1A increased phosphorylation of the insulin-like growth factor type 1 receptor (IGF-1R) . In combination with AZD8055, NVP-AEW541 suppressed AKT re-phosphorylation without impacting ERK phosphorylation , consistent with the model that IGF-1R activation regulates AKT downstream of MEK/ERK activity in these cells. However, despite modulating AKT phosphorylation, the addition of NVP-AEW541 to AZD8055 was insufficient to induce apoptosis as assayed by PARP cleavage and quantifying sub-G1 cell populations . Hence, while selumetinib suppresses AZD8055-mediated activation of the IGF-1R/AKT axis, modulation of these targets alone is not sufficient for the apoptosis induced by the AZZD8055/selumetinib combination, implying that other target(s) downstream of selumetinib-mediated ERK inhibition is (are) critical for the induction of apoptosis.These results suggest that inhibition of IGF-1R activity may be one mechanism downstream of MEK/ERK inhibition by which selumetinib inhibits AZD8055-induced, rebound AKT phosphorylation. To test this hypothesis, we utilized the small molecule inhibitor NVP-AEW541 RAS and PIK3CA mutations BRAF mutant uveal melanomas.4E-BP1 (eIF4E binding protein-1) has recently been implicated as the key protein target cooperatively modulated by an AKT and MEK inhibitor combination to induce apoptosis in tumors harboring both 4E-BP1 is an mTORC1 substrate that negatively regulates translation by binding and blocking the function of eIF4E, an initiation factor that binds to the 5′ mRNA cap structure to promote mRNA translation. mTORC1 activity leads to 4E-BP1 phosphorylation at several sites  in the BRAF cells, suggesting no significant change upon 4E-BP1 function .Phosphorylation at all the 4E-BP1 sites assayed was inhibited with AZD8055 alone in WT cells. In the function  failed tBRAF cell apoptosis of mTORC1 versus mTORC2 inhibition in combination with selumetinib MEK inhibition. First, AZD8055 effects were compared to those of rapamycin, an mTORC1 inhibitor. As predicted, rapamycin suppressed mTORC1 substrate phosphorylation and increased AKT phosphorylation (presumably via release of negative feedback regulation), which was incompletely reduced by selumetinib ) was observed with the rapamycin/selumetinib combination compared to the AZD8055/selumetinib combination. Hence, mTORC1 inhibition with rapamycin alone is not sufficient to replicate the apoptosis produced with AZD8055.We next compared the impact upon mutant , lanes 2, 4, and 6), consistent with previously published data , lane 4); the rapamycin/selumetinib combination failed to further decrease MCL-1 levels over that observed with either drug alone .mTOR has been shown to stimulate cell survival by promoting expression of myeloid leukemia sequence-1 (MCL-1), a pro-survival member of the bcl-2 family of genes , lane 2 versus 1). Alternatively, partial suppression of Raptor expression failed to reduce MCL-1 levels relative to control siRNA-transfected cells . With the addition of selumetinib, cells with suppressed Rictor levels achieved modestly lower MCL-1 levels and BIM upregulation, resulting in an induction of apoptosis as indicated by the appearance of cleaved PARP that was not observed with raptor siRNA . These observations suggest that in BRAF mutant uveal melanoma the modulation of MCL-1 levels and apoptosis by AZD8055 in combination with selumetinib may be dependent upon mTORC2, not mTORC1, inhibition.Taking a previously published approach BRAF mutant OCM1A cell line was the level of MCL-1 decreased by AZD8055 and selumetinib cooperatively  to a level comparable to that observed with AZD8055 alone . This analysis revealed that apoptosis was induced with the combination only in the subset of cells expressing lower levels of MCL-1, while the drug combination failed to induce more apoptosis than that observed with selumetinib alone in the high MCL-1 expressers . In the xenograft model of the BRAF mutant cell line OCM1A, Western analysis of frozen tumor tissue revealed that cooperative MCL-1 downregulation and increased BIM levels were also achieved in vivo . Taken together, these studies demonstrate that MCL-1 downregulation is both necessary and sufficient to induce apoptosis with selumetinib, and strongly argues that MCL-1 is a singular target that is cooperatively modulated by the combination to induce apoptosis in BRAF mutant uveal melanoma.We next transfected into OCM1A cells a plasmid driving MCL-1 expression under the control of a constitutively active viral promoter in order to determine if MCL-1 downregulation is necessary for AZD8055/selumetinib-induced apoptosis  Concomitant activation of the MAPK and PI3K/AKT pathways in tumor cells confers relative resistance to drugs targeting either pathway in isolation. AKT activation in BRAF and GNAQ mutant cells; however, apoptosis was only induced in BRAF mutant cells. Ultimately, in vitro measurement of apoptosis correlated better than cell viability to in vivo effectiveness as tumor regressions were observed only in the BRAF mutant xenograft model. These distinct drug induced fates may in fact reflect biologic differences in how BRAF and GNAQ mutations activate MAPK signaling: while the former directly activates MEK→ERK activity, the latter does so via protein kinase C (PKC) activation which can mediate cell survival signals via several pathways parallel to MAPK GNAQ mutant cells predisposes to AZD8055/selumetinib-induced apoptosis argues that GNAQ activity in these cells activates MEK- and mTOR- independent pro-survival signals.In this study, we found that uveal cancer cell fate in response to combined MEK and mTOR inhibition was closely correlated to tumor genotype. AZD8055/selumetinib did not confer cooperative antitumor effects in WT cells, possibly related to incomplete suppression of MAPK activity with selumetinib. Alternatively, the combination did synergistically suppress the viability of both GNAQ and BRAF mutant uveal melanomas revealed that AKT, 4E-BP1, and MCL-1 were cooperatively regulated by the AZD8055/selumetinib combination only in BRAF mutant cells and not GNAQ cells . Hence, different targeted approaches may achieve the dual pathway inhibition that can impact BIM/MCL-1 levels and induce apoptosis in BRAF mutant cells.In GNAQ and BRAF mutation status. We did observe that BRAF mutant cell lines had overall less PTEN protein expression compared to WT or GNAQ cells, though this did not result in baseline differences of AKT or S6K1 phosphorylation . Since PTEN null uveal melanoma cell lines are not currently available PTEN status to drug susceptibility can not be fully addressed. We did investigate how siRNA-mediated depletion of PTEN expression in the GNAQ cell line 92.1 impacted drug-induced apoptosis and found this did not change the drug effects upon the relevant signaling pathways or MCL-1 levels . Though there was also no statistically significant increase in apoptosis with PTEN siRNA compared to control siRNA transfected cells, we did observe a very small trend towards higher apoptosis with the selumetinib/AZD8055 combination in cells with suppressed PTEN expression . We concluded that while PTEN status may modestly influence the induction of apoptosis, it does so in a manner distinct from the MCL-1 dependent mechanism we have delineated for BRAF mutant cells, though further study regarding the contribution of PTEN status apart from BRAF or GNAQ mutations is warranted.What remains unknown is how differences in uveal melanoma PTEN status may influence drug susceptibility independent of BRAF mutant malignancies. We have evaluated the AZD8055/selumetinib combination in cutaneous melanomas and have found that the combination preferentially induced apoptosis in most BRAF mutant cell lines (3 out of 4 lines tested), but not N-RAS mutant cells or cells lacking these mutations , supporting observations from Gopal et. al. GNAQ mutant cells, further study is warranted to develop more effective GNAQ targeting strategies in order to achieve better therapeutic efficacy.One question stemming from this study is how applicable the efficacy of combined mTOR and MEK inhibition may be for other Selumetinib and AZD8055 were supplied by AstraZeneca  and dissolved in DMS0 at 10 mM and stored at −20°C. NVP-AEW541 was purchased from Caymen Chemical  and dissolved in PBS at 5 mM and stored at −20°C. Rapamycin was purchased from Calbiochem  and dissolved at 5 mM in DMSO and stored at −20°C.OCM1A Cell viability assays were performed with Dojindo Molecular Technologies  per manufacturer’s instructions. For the combination index (CI) analysis, dose-effect curve parameters for both AZD8055 and selumetinib were used for the automated calculation of CI values conducted by the CompuSyn software  Bi-parameter flow cytometry analysis for DNA content (detected with propidium iodide) and another maker (including MPM-2 or MCL-1) was performed as previously described Cell lysates and Western blots were performed as previously described Experiments with small interfering RNAs (siRNA) were performed as previously described 7 GTP sepharose beads  were performed as previously described Assays with mCells were transiently transfected with an MCL-1 expression plasmid as previously described 3 diameter were treated orally (p.o.) with vehicle, AZD8055 (20 mg/kg/d), and/or selumetinib (25 mg/kg/d) as single agents and in combination, 5 days/week for 3 weeks. After the fifth treatment, two animals from each cohort were sacrificed and the tumors were assessed by hematoxylin & eosin staining (H&E), Ki67 staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and Western blot as previously described 8-week-old nu/nu SCID male mice bearing subcutaneously injected OCM1A or 92.1 tumors (7 mice/cohort) of ∼100 mmin vitro experiments were carried out at least 2–3 times. Standard error was calculated as the standard deviation divided by the square root of the number of samples. For in vivo studies, p-values were calculated using an exact version of the Wilcoxon Rank Sum test. We selected p values ≤0.05 being statistically significant. Tumor volumes were compared between groups of mice at various time points.All Figure S1GI50 values for uveal melanoma cell lines treated with various concentrations of selumetinib and AZD8055.A, Cells were treated with selumetinib at various concentrations  for 96 hours and treated wells then analyzed by viability assay. GI50 values were determined utilizing CompuSyn. B, Cells were treated with AZD8055 at various concentrations  for 96 hours and analysis/calculations for viability and GI50s were performed as described in A.(TIF)Click here for additional data file.Figure S2Impact of AZD8055 and selumetinib upon MAPK and AKT/mTOR pathway signaling.A, Western blot of S6K1, AKT, and ERK phosphorylation following AZD8055 for 24 hours. B, Western blot of ERK and AKT phosphorylation following exposure to selumetinib for 24 hours. C, AKT phosphorylation over time with 100 nM AZD8055. D, ERK phosphorylation over time with 1000 nM selumetinib. Interpretation: For AZD8055, 100 nM was the lowest concentration to suppress phosphorylation of the mTORC1 substrate S6 Kinase 1 (S6K1) at 24 hours in all the cell lines . 100 nM AZD8055 impacted the phosphorylation of mTORC2 substrate AKT (at S473) more variably as BRAF and GNAQ, but not WT, cells retained phosphorylated AKT at levels approximating those in vehicle-treated cells . This was due to rebound AKT phosphorylation: 100 nM AZD8055 inhibited AKT phosphorylation at 2 hours in all three genotypes and by 24 hours AKT phosphorylation rebounded only in BRAF and GNAQ cells . This implies that a PDK2 other than mTORC2 may be re-activated after prolonged AZD8055 exposure. Interestingly, higher concentrations of AZD8055 inhibited AKT phosphorylation , which we concluded represented off-target activity given 1) effective mTORC2 inhibition in all the cell lines at 2 hours with 100 nM AZD8055, and 2) discordance between inhibition of AKT and S6K1 phosphorylation in BRAF cells (a discrepancy not observed in WT cells), confirming that mTOR inhibition occurs at concentrations as low as 20 nM in BRAF cells. While rebound AKT phosphorylation was accompanied by rebound S6K1 phosphorylation in the GNAQ mutant cell line, this was not observed in the BRAF mutant cell line, suggesting different mechanism of rebound phosphorylation may exist. For selumetinib, 1000 nM was the lowest concentration that inhibited phosphorylation of the MEK substrates ERK1/2 at 2 hours, but rebound ERK phosphorylation occurred preferentially in WT cells after 24 hours .(TIF)Click here for additional data file.Figure S3Immunohistochemistry staining for TUNEL and Ki67 in BRAF and GNAQ mutant xenograft models.A and B, After the fifth drug(s) or vehicle dose, two animals from each cohort were sacrificed and the tumors were assessed for TUNEL and Ki67 staining. See Methods for the details of this analysis.(TIF)Click here for additional data file.Figure S4NVP-AEW541 inhibits IGF-1R phosphorylation.A, Cells were serum-starved for 24 hours and then exposed to vehicle or 1000 nM NVP-AEW541 either in serum-free conditions or in the presence of 5 µg/ml of IGF-1 ligand. Treatment was for 10 minutes. Cell lysates were created and Western blot was then performed. B, Cells were treated with the indicated vehicles or drugs for 24 hours and then cell lysates were created for RTK antibody array blots. Drug concentrations used: 100 nM AZD8055, 1000 nM selumetinib, and 1000 nM NVP-AEW541.(TIF)Click here for additional data file.Figure S5Bi-parametric flow cytometry for MCL-1 transfected OCM1A cells. Cells were transiently transfected with an MCL-1 cDNA expression plasmid under the control of a constitutively active viral promoter or an empty vector for 48 hours. Cells were then treated with vehicle, 100 nM AZD8055, 1000 nM selumetinib, or the combination for 24 hours and then analyzed by bi-parametric flow cytometry for DNA content and MCL-1 expression levels. Red circle, sub-G1 fraction in both the low- and high- MCL-1 expressing populations.(TIF)Click here for additional data file.Figure S6AZD8055/selumetinib cooperatively downregulates MCL-1 and upregulates BIM in an in vivo BRAF mutant xenograft model. With the xenograft experiment performed upon the BRAF mutant cell line OCM1A described in (TIF)Click here for additional data file.Figure S7Dual pathway inhibition with selumetinib and the IGF-1R inhibitor NVP-AEW541 induces apoptosis in OCM1A cells. Cells were treated with vehicle, 1000 nM selumetinib, 100 nM AZD8055, 1000 nM NVP-AEW541, the selumetinib/AZD8055 combination, or the selumetinib/NVP-AEW541 combination. After 24 hours, cell lysates were created and Western blot was performed. After 48 hours, flow cytometry for DNA content was performed and the percentage of sub-G1 cells was quantified. Results reflect triplicate samples for each condition. The selumetinib/NVP-AEW541 combination achieved the same dual pathway inhibition , cooperative MCL-1 downregulation, BIM upregulation, and induction of apoptosis that was observed with the selumetinib/AZD8055 combination. See the RTK array blots in Figure S4B confirming that NVP-AEW541 inhibits IGF-1R phosphorylation alone and in combination with selumetinib in these conditions. Error bars, SE.(TIF)Click here for additional data file.Figure S8PTEN levels and the susceptibility to the selumetinib/AZD8055 combination.A, Protein expression of PTEN and several other phospho-proteins in the six uveal and two cutaneous melanoma (PTEN-negative Mel 133 and PTEN-positive Mel 32) cell lines was examined by Western blot. B, siRNA mediated suppression of PTEN in the GNAQ cell line 92.1 does not significantly change the impact of the selumetinib/AZD8055 combination upon the targeted pathways or apoptosis. Cells were transfected with pooled siRNA constructs targeting PTEN or unrelated control constructs for 48 hours and then treated with the indicated drugs for 24 hours. Western blots were then performed. After 48 hours of drug treatment, flow cytometry for DNA content was performed and the percentage of sub-G1 cells was quantified. Results reflect triplicate samples for each condition. Error bars, SE.(TIF)Click here for additional data file.Figure S9AZD8055/selumetinib induces apoptosis preferentially in BRAF mutant cutaneous melanomas. A panel of cutaneous melanoma cell lines of the indicated genotypes were treated with the indicated drugs for 72 hours (with the exception with MEL 267 which was treated for 48 hours) and then assayed by flow cytometry for DNA content in order to determine the percentage of cells with sub-G1 DNA, indicative of apoptosis. WT, defined as cells lacking BRAF, RAS, or any other known pathway mutations; N-RAS mutation, MEL 30 has a Q61K mutation and MEL 103 has a Q61R mutation in N-RAS; BRAF mutation, V600E.(TIF)Click here for additional data file.Table S1List of the uveal melanoma cell lines utilized in this study. The gene mutations for each cell line listed were previously described/discovered by other groups. We also analyzed the cell lines on a mass spectrometry platform to assess for other hotspot gene mutations. Some of the relevant mutations tested include: AKT1 , PIK3CA (E542Q), ARAF, BRAF, EGFR, ERBB2, FGFR1-4, KIT, PDGFR A/B, KRAS, NRAS, HRAS, and MEK1.(TIF)Click here for additional data file.


Despite the widespread clinical use of agents that target β-ARs, the signaling pathways that operate downstream of β-AR stimulation have not yet been completely elucidated. Here, we utilized a lysate microarray approach to obtain a broad-scale perspective of phosphoprotein signaling downstream of β-AR. We monitored the time course of phosphorylation states of 54 proteins after β-AR activation mouse embryonic fibroblast (MEF) cells. In response to stimulation with the non-selective β-AR agonist isoproterenol, we observed previously described phosphorylation events such as ERK1/2(T202/Y204) and CREB(S133), but also novel phosphorylation events such as Cdc2(Y15) and Pyk2(Y402). All of these events were mediated through cAMP and PKA as they were reproduced by stimulation with the adenylyl cyclase activator forskolin and were blocked by treatment with H89, a PKA inhibitor. In addition, we also observed a number of novel isoproterenol-induced protein dephosphorylation events in target substrates of the PI3K/AKT pathway: GSK3β(S9), 4E-BP1(S65), and p70s6k(T389). These dephosphorylations were dependent on cAMP, but were independent of PKA and correlated with reduced PI3K/AKT activity. Isoproterenol stimulation also led to a cAMP-dependent dephosphorylation of PP1α(T320), a modification known to correlate with enhanced activity of this phosphatase. Dephosphorylation of PP1α coincided with the secondary decline in phosphorylation of some PKA-phosphorylated substrates, suggesting that PP1α may act in a feedback loop to return these phosphorylations to baseline. In summary, lysate microarrays are a powerful tool to profile phosphoprotein signaling and have provided a broad-scale perspective of how β-AR signaling can regulate key pathways involved in cell growth and metabolism.  Beta-adrenergic receptors (β-AR) are G-protein coupled receptors that mediate the effects of the catecholamines, epinephrine and norepinephrine, in the sympathetic nervous system. Three β-AR subtypes have been identified  ,5. More It is known that upon binding agonist ligands, all three types of β-ARs couple to the stimulatory G-protein (Gs), resulting in the activation of adenylyl cyclase and generation of the second messenger cAMP . cAMP thWhereas β1-ARs couple only to the stimulatory G-protein (Gs), β2-ARs have the ability to couple to both Gs and the inhibitory G-protein (Gi) . CouplinIn order to gain a more broad and unbiased perspective of β-AR signaling and to understand how the different mechanisms contribute to signaling, we utilized a lysate microarray approach to profile protein phosphorylation events downstream of β-AR in an immortalized mouse embryonic fibroblast (MEF) cell line. After stimulating MEFs with various β-AR agonists, lysates were harvested at different time-points and were used to construct lysate microarrays, which were probed with a panel of 54 well-characterized phospho-specific antibodies. Because a large number cell lysates were spotted onto the slides, many different stimulatory conditions could be assessed simultaneously, thus providing a broad-scale view of the kinetics of the β-AR signaling pathway. Using this approach, we identified a number of novel tyrosine phosphorylation events downstream of β-AR. We also identified novel dephosphorylation events downstream of β-AR and cAMP that were independent of PKA and Epac, but correlated with reduced AKT activity. Together, these studies shed light on the diverse and important cellular functions mediated by β-AR activation. Furthermore, they highlight the utility of lysate microarray technique to provide broad overviews of signaling and to uncover novel signaling events.2. The following inhibitors and stimuli were purchased from Sigma-Aldrich  unless otherwise specified: isoproterenol, forskolin, 8-pCPT-2'-O-Me-cAMP , H89, LY294002 , wortmannin, okadaic acid, CGP 20712A, ICI 118,551, pertussis toxin, prazosin, epinephrine, and norepinephrine. All antibodies used in this study were purchased from Cell Signaling Technologies  unless otherwise specified. A complete list of antibodies used in this study is available online (http://utzlab.stanford.edu/protocols/). A MEF cell line  was mainFor stimulation experiments, cells were seeded into 6-well plates. After the cells were confluent, they were serum starved overnight and were stimulated for various times. Following stimulations, cells were washed with ice-cold PBS and then lysed in buffer containing 50 mM Tris pH 6.8, 2% SDS, 5% glycerol, 1% 2-mercaptoethanol, 2.5 mM EDTA, 1.5x Halt phosphatase inhibitor cocktail , and 1x complete protease inhibitor cocktail . Lysate samples were immediately snap-frozen in a dry ice/ethanol bath and stored at -80°C. Prior to printing, lysates were boiled at 100°C for 10 minutes and were briefly centrifuged. Protein levels of all lysates were quantified using the Quant-It Protein Assay Kit (Life Technologies) and diluted with additional lysis buffer to equalize protein concentrations. Samples were loaded into wells of a 384-well plate in preparation for printing.Lysate microarrays were printed and processed as previously described . BrieflyMFI-B) was determined and averaged to obtain a fluorescence value for each lysate representing a single time point from a time course experiment. The values for each lysate were normalized to the MFI-B values of the respective vehicle-treated control spots providing a fold change in phosphorylation state. These data were imported into TIGR Multiple Experiment Viewer software (TMEV) dihydroalprenolol hydrochloride  and different concentrations of ICI 118,551 (Sigma-Aldrich). After a two hour incubation at room-temperature, the binding reactions were terminated by rapid filtration over glass fiber filters (Millipore). Radioactivity in the filters was then quantified using a liquid scintillation counter. Non-specific binding was determined in the presence of 1 μM alprenolol (Sigma-Aldrich). Binding data were analyzed with GraphPad Prism software .MEFs were grown to confluence, collected in lysis buffer  with a cell scraper, and then subjected to dounce homogenization. Following a low speed centrifugation (500 x g) to remove cellular debris, membranes were pelleted by a high speed centrifugation . Membranes were then resuspended in binding buffer . MEFs were used as a model system because they express a number of β-ARs and because β-AR signaling has been previously studied in these cells . MEFs alWe detected many protein phosphorylation and dephosphorylation events after isoproterenol stimulation . The patBased on prior studies that showed that the β2-AR is able to couple to the inhibitory G-protein Gi and phosphorylate ERK1/2 ,16, we aNext, we used specific antagonists of β1-AR (CGP 20712A) and β2-AR  to distinguish the role of these β-AR subtypes in mediating phospho-protein signaling downstream of isoproterenol stimulation in MEFs. For these experiments we focused on the signaling pathways that showed the greatest changes in phosphorylation status in our primary screen. We found that pre-treatment with β2-AR antagonist, but not the β1-AR antagonist significantly blunted the isoproterenol-induced phosphorylation of Cdc2(Y15), Pyk2(Y402), CREB(S133), Raf(S259), and PKA substrate , confirming that these phosphorylation events are coupled more to the β2-AR signaling pathway . InteresTo determine whether the isoproterenol-induced phosphorylation and dephosphorylation events are dependent on protein kinase A (PKA), we next pretreated MEFs with the PKA inhibitor H89 prior to conducting these stimulations. As seen in To investigate the cAMP-dependence of these phosphoprotein changes, we also stimulated MEFs with forskolin, a direct activator of adenylyl cyclase. As can be seen in Our studies using H89 and forskolin suggested that the dephosphorylation events induced by isoproterenol were dependent on cAMP, but were independent of PKA. We then reasoned that the cAMP sensor Epac may be a potential effector of these changes. To investigate Epac involvement, we treated MEFs with the Epac agonist 8-pCPT-2'-O-Me-cAMP (100 μM to 1 mM) to see if we could recapitulate the isoproterenol-induced dephosphorylation events. Surprisingly, stimulation of MEFs with this agent had no effect on either phosphorylation or dephosphorylation of signaling molecules (data not shown). In order to verify that 8-pCPT-2'-O-Me-cAMP was active in MEF cells, Rap GAP assays were also performed. These experiments confirmed that 8-pCPT-2'-O-Me-cAMP activated Rap1 similarly to forskolin in MEFs , PP1α(T320), p70S6K(T389), and AKT(T308) . In a siIn reviewing the list of signaling molecules that exhibited isoproterenol- and forskolin- induced patterns of dephosphorylation, we consistently observed a significant dephosphorylation of AKT at threonine 308. Furthermore, we recognized that many of the other proteins that were subject to dephosphorylation are known targets of the PI3K/AKT signaling pathway  . This leOf note, we observed that one of these forskolin-induced dephosphorylation events PP1α(T320), could not be reproduced with treatment with LY294002 and wortmannin, suggesting that PP1α(T320) is regulated in an AKT-independent fashion. Interestingly, this dephosphorylation event, which is known to result in increased PP1 activity , coincidUsing the lysate microarray technique, we conducted broad-scale phosphoprotein profiling of signaling molecules downstream of β-AR activation. This technique facilitated the simultaneous screening of a large number of samples with many different antibodies and provided us with a comprehensive view of the signaling network downstream of β-AR activation that would have otherwise not been possible using traditional western blot analysis. Using this approach we identified a number of novel phosphorylation and dephosphorylation events induced by β-AR stimulation. These studies, thus, shed light on the complexity of β-AR signaling.In addition to detecting phosphorylation events such as ERK1/2(T202/Y204) and CREB(S133), which are known to be downstream of PKA in β-AR signaling, our approach detected a number of novel tyrosine phosphorylation events in response to isoproterenol stimulation: Cdc2(Y15) and Pyk2(Y402). These phosphorylations were dependent on PKA as they were blocked by treatment with H89, however they occurred on a much slower timescale than ERK1/2(T202/Y204) and CREB(S133) suggesting that they were more distal in the pathway. The phosphorylation of Cdc2(Y15) downstream of PKA could have occurred either through activation of Wee1 kinase or deactivation of the Cdc2 effector phosphatase Cdc25 , as prevAnother insight from these lysate microarray studies is that GSK3β(S9) can be phosphorylated by both PKA and AKT as shown in the model . GSK3β iWe observed that β-AR activation also led to a number of key dephosphorylation events in protein targets of PI3K/AKT: GSK3β(S9), 4E-BP1(S65), and p70S6K(T389). PI3K/AKT is a master regulator of cell growth, and activation of this signaling pathway is critical for cell survival and is upregulated in many cancers . We demoIt is still unclear how cAMP inhibited PI3kinase/AKT in our MEF cells. One possibility is that it occurs through another unknown cAMP sensor. It is also possible that this effect on PI3kinase/AKT occurred through the regulatory units of PKA. Although H89 inhibits the catalytic activity of PKA, it does not prevent the dissociation of PKA into its catalytic and regulatory units, and a prior study showed that the regulatory units of PKA may have important signaling functions . Future The other dephosphorylation event that was cAMP-dependent, but independent of PI3K was of PP1α(T320). PP1α is a central regulator of protein phosphorylation that also has been shown to control cell cycle progression . DephospOur data also suggest that activation of PP1α may be part of a feedback loop by which cAMP returns PKA-phosphorylated substrates to basal state. In support of this concept is the finding that dephosphorylation of PP1α(T320) was a delayed β-AR signaling event that coincided with the secondary decline in phosphorylation levels of PKA substrate and CREB post stimulation. Although not specific for PP1, okadaic acid also increased the basal phosphorylation levels of PKA substrate, resulting in no induction and no secondary decline in phospho-substrate after β-AR stimulation. In the heart, PP1 has been shown to dephosphorylate protein targets that are phosphorylated by PKA, such phospholambam . PP1 actIn summary, we propose the following model of β-AR signaling to explain the observed phosphorylation and dephosphorylation events . The phoIn conclusion, we have shown how application of lysate microarrays can be used to provide a greater understanding of phosphoprotein signaling for a specific pathway. By applying lysate microarrays to β-AR signaling in MEFs, we identified novel β-AR signaling events, including protein phosphorylations and dephosphorylations. Further delineation of these pathways in MEFs and other cell types will be important for defining a role for β-AR agonists/antagonists in cancer treatment and will be useful to understanding how states of chronic catecholamine excess  can lead to proliferation or apoptosis of different cell types. File S1Figures S1-S6. Figure S1. Competition binding study in MEFs with ICI 118,551. Membranes were prepared from MEFs and were used in binding reactions. For each reaction, 60 μg of membranes was incubated with 10 nM [3H]dihydroalprenolol ([3H]DHA) and varying concentrations of the β2-AR selective antagonist ICI 188,551. Non-specific binding was determined in the presence of 1 μM alprenolol. Results show mean ± SEM values from three experiments. Competition data were best fit with a two-site model that included a high-affinity binding site (74.7%) and low-affinity binding site (25.3%) for ICI 118,551. The high-affinity site corresponds to β2-AR, whereas low-affinity sites are β1-AR or β3-AR. Figure S2. Phosphoprotein signaling in MEFs after stimulation with epinephrine. A heat map representation of phosphoprotein changes over time. MEFs were grown to confluence and then were stimulated with different doses of epinephrine (Epi)  for various times . Stimulations were performed in the presence or absence of the alpha 1 adrenergic receptor antagonist prazosin (Praz) (1 μM). Using data from the lysate microarrays, a heat map was constructed which revealed distinct clusters of phosphorylation (yellow) and dephosphorylation (blue) events after epinephrine stimulation. The color scale shows fold change as compared with unstimulated MEFs. Data are representative of two independent experiments. Figure S3. Phosphoprotein signaling in MEFs after stimulation with norepinephrine. A heat map representation of phosphoprotein changes over time. MEFs were grown to confluence and then were stimulated with different doses of norepinephrine (Norepi)  for various times . Stimulations were performed in the presence or absence of the alpha 1 adrenergic receptor antagonist prazosin (Praz) (1 μM). Using data from the lysate microarrays, a heat map was constructed which revealed distinct clusters of phosphorylation (yellow) and dephosphorylation (blue) events after norepinephrine stimulation. The color scale shows fold change as compared with unstimulated MEFs. Data are representative of two independent experiments. Figure S4. Western blotting confirmatory studies on MEF lysates used for lysate microarrays. Figures show phospho (denoted by “p-“) and total levels of proteins as detected by western blotting. Each blot represents an isoproterenol time course study with 0, 5, 10, 20, 30 and 60 minute stimulations. Dephosphorylated proteins are shown at the top of the figure and phosphorylated proteins are shown at the bottom of the figure. Figure S5. Isoproterenol induced phosphoprotein signaling in the presence of pertussis toxin. MEFs were pretreated with pertussis toxin (PTX) (1 μg/ml) or vehicle control for 30 minutes and then stimulated with isoproterenol (Iso) (1 μM) for various times . A heat map shows phosphorylation (yellow) and dephosphorylation (blue) events after isoproterenol stimulation. The isoproterenol time course study on the left was performed in the presence of PTX and the time course study on the right was performed in the presence of vehicle. The color scale shows fold change as compared with unstimulated MEFs. Data are means of three independent stimulation experiments. Figure S6. Epac activation as assessed by the Rap GAP assay. MEFs were stimulated with vehicle control (C), forskolin (FSK) (50 μM), or the Epac-specific analog 8-pCPT-2'-O-Me-cAMP (Epac) (250 μM) for 5 or 20 minutes. Following stimulation, cells were lysed and incubated with Ral GDS-Rap Binding Domain bound to glutathione-agarose beads to specifically pull down Rap1-GTP . Western blotting was then performed to detect Rap1. Positive (+) and negative (-) controls are included in the figure. Data depicted are representative of two independent stimulation experiments.(PDF)Click here for additional data file.


The process by which G protein-coupled receptors (GPCRs) are internalized through the clathrin-coated vesicles involves interactions of multifunctional adaptor proteins. These interactions are tightly controlled by phosphorylation and dephosphorylation mechanisms resulting in the regulation of receptor endocytosis. However, the identities of the kinases involved in this process remained largely unknown until recently. This paper discusses advances in our knowledge of the important role played by protein phosphorylation in the regulation of the endocytic machinery and how phosphorylation controls the coated vesicle cycle. Reversible protein phosphorylation by the interplay between kinases and phosphatases is a major regulatory mechanism for G protein-coupled receptors (GPCRs) internalization –3. Thereβ2-adrenergic receptor (β2-AR) studies. Briefly, following agonist stimulation, GPCRs undergo conformational change that allow binding of G proteins, leading to the activation of different effectors and signaling pathways  appeared to contribute to agonist-dependent phosphorylation of the receptor [By the mid-1980s, it had become clear that both rhodopsin  and the β2-AR  were phoβ2-AR . In the receptor . Now, 7Treceptor , 25. α-subunit by acting as GTPase-activating proteins (GAPs) [in vitro has not yet been defined, but tissue distribution, as well as levels of expression, probably contributes to their specificity in vivo [GRKs phosphorylate GPCRs at both serine and threonine residues localized within the third intracellular loop (i3) or C-terminal tail domain . Althougs (GAPs) . The spe in vivo –32. Howe in vivo , 34. Sev in vivo .βγ-mediated membrane association [β-arrestin to GPCRs, phosphorylates GRK-2 on tyrosine residues and targets GRK-2 for degradation [A regulatory complexity is added by the fact that GRK activity can be influenced by phosphorylation. Mitogen-activated protein kinase (MAPK) decreases its efficacy towards GPCRs substrates , whereasociation , 37 and ociation , 38. In ociation . Moreoveradation , 41. β-arrestin interaction is the phosphorylation status of the activated GPCRs. Thus, impairment of receptor phosphorylation by mutagenesis of key serine/threonine residues generally leads to diminished β-arrestin binding after stimulation of the M2 muscarinic cholinergic receptor, rhodopsin, the AT1R, or the V2R [GRK-phosphorylation of receptors is not sufficient for desensitization, but rather serves to create high affinity sites to promote the recruitment of the cytoplasmic accessory proteins, arrestins, and target the receptors for internalization via clathrin-coated pits. The arrestin protein was first identified by Wilden et al. in 1986 as, “a 48K protein”, bound to the phosphorylated rhodopsin, thereby interfering with its coupling to transducin . The pro the V2R –48. The  the V2R , 50. How the V2R . In addi the V2R  have sho the V2R  or with  the V2R  do not s the V2R .αo-coupled) contains also a putative CK2 site, which, when mutated, reduced GRK-6-mediated desensitization [s-coupled receptors [Casein kinase II has been reported to phosphorylate the M3–muscarinic receptor in cerebella granule neurons and to affect coupling to the Jun-kinase pathway . It was tization . Rebholzeceptors . Howeverβ-Arrestin are component of a multiprotein complex that assembles on the receptor after ligand stimulation. Acting as a protein scaffold, it recruits Src kinase and also binds to clathrin, and adaptor-protein 2 (AP-2), which serves to target the activated GPCRs to clathrin-coated pits for internalization. β-Arrestin also induce rapid homologous desensitization of the receptor by binding to the GPCRs and sterically inhibiting further G-protein coupling. However, several reports on arrestin phosphorylation provide some insight into potential regulation of arrestin function. Despite numerous studies suggesting an essential role of arrestins in GPCRs regulation, the detailed mechanism underlying the function of arrestins and receptor-mediated modulation of arrestin activity is unclear. β-arrestin 1 demonstrate that β-arrestin 1 was constitutively phosphorylated at Ser-412 by extracellular signal-regulated kinases 1 and 2 and became dephosphorylated following β2AR stimulation [β-arrestin 1 phosphorylation was proposed to regulate the interaction of arrestin with clathrin and Src, since a S412D mutant mimicking phosphorylated β-arrestin 1 had reduced binding to these proteins [β-arrestin 1 at Ser-412, as well as transfection of the S412A β-arrestin 1 mutant, which mimics dephosphorylated β-arrestin 1 and enhances clathrin and Src binding to β-arrestin 1, resulting in enhanced GPCRs internalization. Notably, β-arrestin 2 shares ~80% amino acid identity with β-arrestin 1 but does not contain the corresponding serine residue. Thus, β-arrestin 2 might be phosphorylated at other sites or perhaps be regulated by different mechanisms. Studies on mammalian mulation , 61. β-aproteins , 62. Thuβ-arrestin 1 has also been shown to be phosphorylated at Ser-412 by GRK-5 [Recently, by GRK-5 . This phβ-arrestin 2 is constitutively phosphorylated by casein kinase II at Thr-383 and suggested that β-arrestin 2 phosphorylation may regulate its interaction with novel binding partners unidentified [β-arrestins at the plasma membrane seems to be necessary for engaging endocytic partners.Kim et al. have reported that entified . Threonientified , 65. DepDrosophila, two visual system arrestins have been identified: Arr1 and Arr2. Both Arr1 and Arr2 are phosphorylated in a light-dependent manner, and it has been established that Arr2 is phosphorylated by a Ca2+/calmodulin-dependent protein kinase (CaMKII) [Drosophila visual arrestin-2 demonstrate that it undergoes light-dependent phosphorylation by calcium/calmodulin-dependent kinase II and that phosphorylation is necessary for dissociation of arrestin from rhodopsin [In (CaMKII) . Althoughodopsin , 68. It hodopsin , 69–71. β-arrestin. There are four tyrosines present in the N-terminall part of β-arrestin-1 that are not conserved in β-arrestin-2 [β-arrestin-1 tyrosine 54 by phenylalanine increases the β-arrestin-1 interaction with the μ-subunit of the AP-2 and enhances β-arrestin-1-mediated β2-AR internalization.Src kinases phosphorylates tyrosine residues on the clathrin coated pit structural proteins including clathrin itself and dynamin but also restin-2 . One of β-arrestin has been shown recently to mediate phosphorylation events by activating 38 protein kinases [β-arrestin-phosphorylation status.Furthemore,  kinases . Howeverin vitro and in vivo and are thought to serve as regulatory subunits [Clathrin is also subject to phosphorylation both subunits , 75. Thesubunits , 77. Tyrosine phosphorylation of clathrin was shown only for the epidermal growth factor receptor. Tyrosine phosphorylation of the clathrin heavy chain by Src-family kinases enhances the clathrin recruitment to the plasma membrane. It was shown that ligand binding to the epidermal growth factor receptor leads, via activation of protein kinase activity, to clathrin phosphorylation .in vitro [in vivo, but the functional role of this phosphorylation event in endocytosis is not entirely clear.Casein kinase 2 plays an essential role in endocytosis, the inhibition of CK2 leads to a significant decrease in transferrin uptake . CK2 is in vitro . As mentβ-arrestin and other endocytic regulatory proteins to stimulate the formation of the clathrin coat [α-adaptin and β2-adaptin), a medium subunit (μ2), and a small subunit (σ2). It has been known for some time that several proteins associated with clathrin-coated vesicles are substrates for protein kinases. These proteins include AP-2, dynamin, clathrin, synaptojanin 1, and amphiphysins. However, the identities of the kinases involved in this process remained largely unknown until recently. So far, only three of the four adaptor complex subunits of AP-2 have been shown to be phosphorylated. Clathrin-mediated endocytosis is the well-described mechanism for the entry of molecules into cells. This pathway is characterized by the recruitment of soluble clathrin from the cytoplasm to the plasma membrane. Clathrin-binding adaptors, such as the adaptor-protein 2 (AP-2), are the key components of this pathway. They bind directly to clathrin , as wellrin coat . In thisμ2 is phosphorylated both in vivo, and in vitro but the functional relevance of these posttranslational modifications was unclear [μ2 remained obscure for a long time. Two candidate kinases have been described in the literature [μ2 recombinant proteins in vitro [μ2 subunit in vitro [α-adaptin [trans-Golgi network (in HeLa cells at least). However, its overexpression completely prevents transferrin uptake [μ2 phosphorylation.It has been known for many years that  unclear . The ideterature –88. One in vitro . It was in vitro . GAK is in vitro . GAK was-adaptin . GAK/auxn uptake , 87, 89 in vitro, that μ2 phosphorylation is required for internalization of Transferrin receptor [ in vivo [μ2-subunit induced by α-adaptin-associated kinase-1 (AAK1), could regulate its binding to endocytic cargo [μ2 subunit of AP-2 in vitro [μ2 phosphorylation is not obligatory for receptor uptake, it is critical for maximizing internalization efficiency [It was shown, receptor . However in vivo , 84, it ic cargo , 84, 91.ficiency , 92. Addficiency . Thus, pficiency , their ificiency , and theficiency , 94.α− and β2-subunits of the AP-2 adaptor complex in vitro [in vivo. In vitro phosphorylation of AP-2 by CK2 prevents AP-2 from binding to clathrin cages, thus mimicking the effect observed in vivo [It was found that Casein kinase 2 (CK2) phosphorylates the in vitro . It is p in vivo .β-arrestins have been implicated includes not only components of the clathrin-endocytic machinery, but also signaling molecules such as the non-receptor tyrosine kinase Src family [β2AR was abolished [β2AR, c-Src rapidly associates with the receptor in a β-arrestin-dependent manner [β-arrestin (SH1-KD) function as dominant-negative inhibitors of clathrin-mediated internalization of receptors [The list of GPCRs-activated signaling pathways in which c family –101. Srcc family . Thus, tbolished . Upon stt manner . c-Src meceptors , 104. Alβ-arrestin/AP-2 complex is necessary for the agonist-dependent phosphorylation of the β2-subunit on Tyr-737, and regulates the disassembly of the endocytic complex and AT1R internalization [β2-adrenergic, vasopressin V2, bradykinin type 2, platelet-activating factor, and endothelin A receptors but also for other type of receptors such as epidermal growth factor receptor [It has been suggested that c-Src recruitment to the endocytic complexes may play a role in the activation of the endocytic machinery , 101. Thlization . This mereceptor .β-arrestin participated in the de novo formation of CCPs [β-arrestin targets receptors to existing pits at the cell surface and engage them in the internalization process [During receptor internalization, it was initially thought that  of CCPs , 108. Ho process , 110. Al process .β2-AR internalization. The proposed model is that β-arrestin will act as an adaptor/scaffold to bind c-Src, allowing the phosphorylation of dynamin [β2-AR. These data were also confirmed for the M1 muscarinic receptor [Dynamin is regulated by tyrosine phosphorylation during  dynamin . Ahn andreceptor , suggestreceptor . Src phoreceptor . in vitro and probably by PKC in intact nerve terminals [2+-dependent phosphatase calcineurin in vitro and in nerve terminals [Dynamin I is also phosphorylated by protein kinase C erminals –117. Dynerminals –121. Afterminals , 122. Reerminals . This PKerminals .in vitro studies, Cdk5 has recently been reported to phosphorylate dynamin both in vivo and in vitro, and the phosphorylation sites have been determined. Cdk5 is one of the proline-directed kinases and phosphorylates serine/threonine motifs [Although several kinases for dynamin have been reported based on e motifs . Cdk5 phe motifs . The phoin vivo [Synaptojanin 1 is a presynaptic inositol 5-phophatase enriched on endocytic intermediates . As mentin vivo , but theα-subunit of AP-2 [Amphiphysin dimer binds both dynamin and Synaptojanin 1 –130. Dis of AP-2 , 132. Th of AP-2 . At leasin vivo and in vitro, and the phosphorylation sites have been determined. Cdk5 phosphorylates Ser- residues 261, 272, 276, and 285 and Thr-310 in amphiphysin 1 [β-adaptin [Cdk5 has recently been reported also to phosphorylate amphiphysin 1 physin 1 . The phophysin 1 , 133. Cdphysin 1 . The sim-adaptin . TherefoAs a second kinase, mitogen-activated protein kinase/extra-cellular-signal-regulated kinase has been shown to phosphorylate Amphiphysin I at two sites, also within the proline/arginine rich domain (PRD) but distinct from those for cdk5. MAPK/ERK phosphorylates amphiphysin I at Ser-285 and Ser-293. This phosphorylation negatively regulates the amphiphysin binding to AP-2 . Phosphorylation of amphiphysin-1 can regulate its interaction with the heavy chain of clathrin. This interaction is mediated by two clathrin binding motifs of amphiphysin, the clathrin box and the W box. Two threonine residues, conserved in amphiphysin-1 and -2, are among several vertebrate species that can be phosphorylated by CK2. The phosphorylation of Thr-350 and Thr-387 in amphiphysin by CK2 regulates the interaction of amphiphysin and clathrin .Coated vesicle formation is a constitutive process that involves continuous cycling of the coat proteins from the cytosol onto the membrane. Reversible phosphorylation is, therefore, a plausible mechanism for how this might be regulated. Phosphorylation events can be classified into two functionally opposite classes: (i) those that are inhibitory for CCV formation and (ii) those that facilitate the assembly of CCVs.β2-AR internalization [β2-subunit of AP-2, to regulate its interaction with β-arrestin [α- and β2-subunits of the AP-2 complex. Phosphorylated AP-2 has a reduced ability to interact with clathrin and requires dephosphorylation for efficient interaction [in vitro dephosphorylation of rat brain extracts apparently promotes the assembly of dynamin I, synaptojanin, amphiphysin, clathrin, and AP-2 into complexes [Few examples of phosphorylation mechanisms occurring during GPCRs internalization were previously reported see . Indeed,lization . Interesarrestin , 105. Preraction , 77. Phoeraction . It was eraction . For exaomplexes . in vivo roles of dissociation. In others words, how and why these endocytic complexes dissociate? Close examination of AP-1 [β-arrestin [S. cerevisiae endocytic, eps15-like accessory factor governs the post budding inactivation of Arp2/3-dependent actin polymerization, preparing the transport vesicle for subsequent fusion [The other angle of the story that would be particularly interesting is the  of AP-1  and β-ararrestin  shows tht fusion . The rolβ2-adaptin [β2-adaptin as compared to phosphorylated β2-adaptin. A structural approach will be useful in demonstrating the ability of phosphorylated β2-adaptin to bind, or not, endocytic proteins. It is not unlikely that a protein with an SH2 domain would make an excellent binding partner for c-Src phosphorylated tyrosine residue within β2-adaptin. Other AP-2 subunits when phosphorylated increase their affinity for different proteins. The affinity of phosphorylated μ2 for peptides containing the tyrosine-containing internalization motif in the cytoplasmic tails of receptors has been shown to be much greater that of unphosphorylate μ2. Recent structural data on AP-2 suggest a possible molecular explanation for these observations. Apparently, the binding site in μ2 for the tyrosine-containing motif is buried in the AP-2 complex, thus a conformational change is required for its exposure. This could be triggered by phosphorylation of μ2 [Another explanation is that phosphorylation could also promote the recruitment of new interacting proteins with a SH2 domain. Nevertheless, proof of this model will require crystallization of the phosphorylated endocytic complexes. For example, since the crystal structure was obtained for -adaptin , it willon of μ2 . In paraHow endocytic phosphorylation cycles are regulated is an important question for the future. Further studies on regulatory inputs and control mechanisms will provide important insight into the events controlling receptor endocytosis.μ2 subunit of AP-2 [β2-subunit of AP-2 [β-arrestin. However, we can not exclude the fact that phosphorylation may also influence AP-2 localization. Previous studies have established that β2-adaptin is phosphorylated in vivo by a staurosporine-sensitive kinase whose function is balanced by the constitutive activity of protein phosphatase 2A (PP2A) [While the mechanisms involved in regulating the clathrin-mediated endocytic pathway are not fully understood, accumulating evidence suggests that phosphorylation cycles may be a key step , 141. In of AP-2 , Src kin of AP-2 , and the of AP-2 . Previou of AP-2 . Thus, iA (PP2A) . They alA (PP2A) , it is pversus dephosphorylation occurs. These issues are crucial to our understanding for the regulation of receptor internalization through clathrin-coated vesicles.In conclusion, phosphorylation appears to be an important mechanism by which GPCRs regulate their own internalization. We currently have only a few pieces of the puzzle available. The identity of many kinases and phosphatases, their location, and the timing of their action remain to be determined. In the future, it will be interesting to investigate the molecular consequences of protein phosphorylation, how kinases are regulated, and where and how phosphorylation


Results section.Following the publication of our article two sepaTiters of egg white-specific IgE, IgG, and IgE/IgG antibodies subsection, the last sentence stated: “In the PFC, the ratio of egg-specific IgE/IgG4 antibodies (IgE/IgG4) was 4.83 , whereas it was 46.71 (18.05-131.04) in the NFC group ”.Firstly, in the It should instead read “In the NFC, the ratio of egg-specific IgE/IgG4 antibodies (IgE/IgG4) was 4.83 , whereas it was 46.71(18.05-131.04) in the PFC group ”.Ratio of egg-specific IgE/IgG antibodies for each dosage of eggs in the challenge test subsection, the 4th sentence stated: “The IgE/IgG4 ratio in the NFC group was significantly lower than those of the other groups, except the A group ”.Secondly, in the It should instead read: “The IgE/IgG4 ratio in the NFC group was not significantly those of the other groups, except the A group than ”.Please note that the conclusions made in the original publication were based on the correct data, and as such they are unaffected by the errors made.


A tonic form of synaptic inhibition occurs in discrete regions of the central nervous system and has an important role in controlling neuronal excitability. Recently, we reported that GABA present in astrocyte is the major source of tonic inhibition in cerebellum and that GABA is released through Bestrophin-1 channel by direct permeation. In this study, we screened for the presence of astrocytic GABA in various brain regions such as hippocampus, thalamus, hypothalamus and cerebellum using immunohistochemistry. We found that astrocytic GABA was present in the regions that were reported to show tonic inhibition. Because the existence of tonic inhibition in hippocampal CA1 is somewhat controversial, we compared the amount of astrocytic GABA and tonic inhibition between the hippocampal CA1 pyramidal cell layer and the cerebellar granule cell layer. Unlike cerebellar glial cells, hippocampal astrocytes did not contain GABA. The tonic inhibition was also much lower in the pyramidal neurons of hippocampal CA1 compared to the granule cells of cerebellum. Nevertheless, most of the hippocampal astrocytes expressed Bestrophin-1 channel. These data indicate that the absence of astrocytic GABA results in a low level of tonic inhibition in hippocampal CA1 region. A receptor blockers such as GABAzine, picrotoxin and bicuculline. Because of its persistent increase in input conductance, tonic inhibition dominates over the conventional (phasic) synaptic inhibition in controlling neuronal excitability [Tonic inhibition originates from the sustained activation of high affinity gamma-aminobutyric acid (GABA) receptors by ambient GABA . Tonic ctability . Thus, ttability , and it tability -6.Tonic inhibition was first identified in the cerebellum, where it is particularly prominent . RecentlUnlike those brain regions, Tonic inhibition in hippocampal CA1 region is somewhat controversial. It is reported to be absent in the pyramidal neurons of hippocampal CA1 and could be detected only in early development or in specific circumstances . Other i2+-activated anion channel, Bestrophin 1 (Best1), mediates tonic inhibition by releasing tonic GABA from glia. We demonstrated that GABA directly permeates through Best1 and that tonic inhibition is eliminated by silencing Best1. But the glia-specific expression of Best1 fully rescues the tonic inhibition [In cerebellum, we recently reported that Cahibition . Since thibition . TherefoTo test our hypothesis, we first screened for the presence of GABA and Best1 in several brain regions and focused on astrocytic GABA and its tonic release in the hippocampal CA1 region. We show that astrocytes in the hippocampal CA1 contain negligible amount of astrocytic GABA and this correlated well with a low level of tonic inhibition currents.To uncover the extent of astrocytic GABA, we used anti-GABA and anti-GFP antibody in GFAP-GFP transgenic mice and analyzed the percent of GABA containing cells out of all GFAP-GFP positive glial cells in several brain regions Table . As oppoAmong the several brain regions we screened, we selected two regions that extremely differed in the amount of astrocytic GABA for further investigation: hippocampal CA1 and cerebellum. As we reported previously , most ceA receptors mediating tonic inhibition show distinct pharmacological properties based on their subunit composition [Based on the immunohistochemical data, we expected that tonic inhibition in hippocampus should be marginal because the hippocampal glial cells contain negligible amount of GABA. To confirm this idea, we performed electrophysiological recordings from the cerebellar granule cells and the hippocampal CA1 pyramidal cells and compared the amplitude of tonic inhibition current sensitive to 10 μM SR95531 (GABAzine). From the cerebellar granule cells, we observed a considerable degree of tonic inhibition current  in hippocampal pyramidal cells in CA1. The hippocampal dentate gyrus region contained more astrocytic GABA than CA1 region, and accordingly, we could record more tonic inhibition current from the granule cell of dentate gyrus . Also, the previously reported amount of thalamic tonic inhibition currents  pA in hi matched A receptor on interneuron to induce tonic inhibition. We found that the interneurons of CA1 in hippocampus also express Best1 channels using immunohistochemistry (data not shown). This possibility needs to be tested in the future experiments.The tonic inhibition in CA1 pyramidal cells is clearly low level and correlated well with the absence of astrocytic GABA. However, the tonic inhibition was previously reported in the interneurons of the same CA1 region . An obviA receptor that is readily blocked by GABAzine. The major subunits of tonic GABAA receptor of cerebellar granule cells are α6, β2, and δ [We used 10 μM SR95531 (GABAzine) and 100 μM bicuculline to measure the tonic inhibition current of cerebellar granule cells and hippocampal pyramidal cells respectively. We used two different antagonists because the two cells - granule cells and pyramidal cells -have differential GABAzine sensitivity. The tonic current from the cerebellar granule cells can be measured using GABAzine because they express GABA2, and δ , but the2, and δ . Therefo2, and δ . TherefoWe investigated not only GABA, but also the Best1 expression pattern between hippocampus and cerebellum. There was a clear distinction in the distribution of Best1 within the cell. The cerebellar glial cells expressed Best1 mostly in their cell body and processes, but the hippocampal glial cells seem to express Best1 on near synapses rather than cell body, as evidenced by the immunostaining Figure . These rIn conclusion, this investigation verified the absence of tonic inhibition in hippocampal pyramidal cells in CA1 region. Also we report the positive correlation between the amount of astrocytic GABA and the degree of tonic inhibition currents in several brain regions. Furthermore, we can expect that Best1 has different function in gliotransmission and regulation of synaptic plasticity depending on brain regions. C57BL/6 mice of wild-type and GFAP-GFP genotypes, of either sex, were used. All experimental procedures described below were performed in accordance with the institutional guidelines of KIST .Adult mice were deeply anesthetized by 2% avertin (20 μl/g) and perfused with 0.1 M Phosphate buffered saline(PBS) followed by ice cold 4% paraformaldehyde (PFA). Excised brains were post-fixed overnight in 4% PFA at 4°C and immersed in 30% sucrose for 48 hrs for cryoprotection. Parasagittal cerebellar sections (30 μm) or coronal hippocampal sections were cut with a Cryostat, rinsed in PBS three times, and incubated for 1 hr with blocking solution . Sections were incubated overnight in a mixture of the following primary antibodies with blocking solution at 4°C on a shaker; rabbit anti-mouse bestrophin antibody (1:100), chicken anti-GFP antibody  and guinea pig anti GABA antibody  for GFAP-GFP mice. After washing three times in PBS, sections were incubated with corresponding secondary antibodies; conjugated Alexa 555 goat anti rabbit IgG (1:200), Alexa 488 conjugated goat anti chicken IgG (1:200), and conjugated Alexa 647 goat anti guinea pig (1:200), for one and a half hours, followed by three rinses in PBS, then mounted with fluorescent mounting medium. A series of fluorescence images were obtained with an Olympus confocal microscope (FV1000) and images were processed for later analysis using FLUOVIEW software.Animals were deeply anesthetized with halothane. After decapitation, the brain was quickly excised from the skull and submerged in ice-cold cutting solution that contained (in mM): 250 Sucrose, 26 NaHCO3, 10 D(+)-Glucose, 4 MgCl2, 3 myo-inositol, 2.5 KCl, 2 Sodium pyruvate, 1.25 NaH2PO4, 0.5 Ascorbic acid 0.1 CaCl2, and 1 Kynurenic acid, pH 7.4. All solution was gassed with 95% O2-5% CO2. After trimming either side of the vermis, 250 μm thick parasagittal slices were cut using a microtome and transferred to extracellular ACSF solution: 126 NaCl, 24 NaHCO3, 1 NaH2PO4, 2.5 KCl, 2.5 CaCl2, 2 MgCl2, and 10 D(+)-Glucose, pH 7.4. For hippocampus, 300 μm thick coronal slices were used. Slices were incubated at room temperature for at least one hour prior to recording. Slices were transferred to a recording chamber that was continuously perfused with ASCF solution (flow rate = 2 ml/min). Slice chamber was mounted on the stage of an upright Olympus microscope and viewed with a 60X water immersion objective (NA = 0.90) with infrared differential interference contrast optics. Cellular morphology was visualized by CCD camera and Axon Imaging Workbench software. Whole-cell recordings were made from granule cell somata located in lobules 2-5 or pyramidal cells located in hippocampal CA1 region. The holding potential was -70 mV. Pipette resistance was typically 10-12 MΩ for granule cells and 5-7 MΩ for pyramidal cells and pipette was filled with an internal solution: 135 CsCl, 4 NaCl, 0.5 CaCl2, 10 HEPES, 5 EGTA, 2 Mg-ATP, 0.5 Na2-GTP, 10 QX-314, pH adjusted to 7.2 with CsOH (278-285 mOsmol). Electrical signals were digitized and sampled at 50 μs intervals with Digidata 1440A and Multiclamp 700B amplifier (Molecular Devices) using pCLAMP 10.2 sofware. Data were filtered at 2 kHz.Off-line analysis was carried out using Clampfit, Minianalysis, SigmaPlot and Excel software. Numerial data are presented as means ± S.E.M. The significance of data for comparison was assessed by Student's two-tailed unpaired t-test and significance level was displayed as * (p < 0.05), ** (p < 0.01), ***(p <0.001).The authors declare that they have no competing interests.BEY carried out electrophysiology, immunohistochemistry and wrote the manuscript. SJ performed electrophysiology and immunohistochemistry. JW carried out electrophysiology. JHL and TK performed immunohistochemistry. CJL designed the most of experiments, edited the manuscript, and coordinated entire project. All authors read and approved the final manuscript.


Enhancement of Akt activation by kb NB 142-70 was also evident in cells with other GPCR agonists, including vasopressin and lysophosphatidic acid. Cell treatment rovincial Hospital Affiliated to Shandong University, Jinan, China with the structurally unrelated PKD family inhibitor CRT0066101 increased Akt phosphorylation as potently as kb NB 142–70. Knockdown of PKD1 with two different siRNAs strikingly enhanced Akt phosphorylation in response to ANG II stimulation in IEC-18 cells. To determine whether treatment with kb NB 142–70 enhances accumulation of phosphatidylinositol -trisphosphate (PIP3) in the plasma membrane, we monitored the redistribution of Akt-pleckstrin homology domain-green fluorescent protein (Akt-PH-GFP) in single IEC-18 cells. Exposure to kb NB 142–70 strikingly increased membrane accumulation of Akt-PH-GFP in response to ANG II. The translocation of the PIP3 sensor to the plasma membrane and the phosphorylation of Akt was completed prevented by prior exposure to the class I p110α specific inhibitor A66. ANG II markedly increased the phosphorylation of p85α detected by a PKD motif-specific antibody and enhanced the association of p85α with PTEN. Transgenic mice overexpressing PKD1 showed a reduced phosphorylation of Akt at Ser473 in intestinal epithelial cells compared to wild type littermates. Collectively these results indicate that PKD1 activation mediates feedback inhibition of PI3K/Akt signaling in intestinal epithelial cells in vitro and in vivo.We examined whether protein kinase D1 (PKD1) mediates negative feeback of PI3K/Akt signaling in intestinal epithelial cells stimulated with G protein-coupled receptor (GPCR) agonists. Exposure of intestinal epithelial IEC-18 cells to increasing concentrations of the PKD family inhibitor kb NB 14270, at concentrations that inhibited PKD1 activation, strikingly potentiated Akt phosphorylation at Thr Specifically, Akt translocated to the plasma membrane in response to products of PI3K, is activated by phosphorylation at Thr308 by PDK1 and at Ser473 by mTORC2 The phosphoinositide 3-kinase (PI3K)/Akt pathway plays a critical role in regulating a plehora of biological processes, including metabolism, migration, survival, autophagy and growth 2, induce rapid but transient Akt activation, at least in part, via EGFR transactivation In contrast to the canonical pathway activated by tyrosine kinase receptors, the effect of G protein-coupled receptor (GPCR) activation on PI3K/Akt signaling is less well defined and appears to depend on cell context. In some epithelial cell systems, GPCR agonists, including angiotensin II, muscarinic agonists and PGEq, G12, Gi, and Rho 744 and Ser748 in the PKD1 activation loop 916q-coupled receptor agonists 3 has not been examined in any cell type.Protein kinase D1 (PKD1), the founding member of a new protein kinase family within the CAMK group 3. Our results demonstrate that PKD1 mediates potent feedback inhibition of PIP3/Akt activation in intestinal epithelial IEC-18 cells stimulated with multiple GPCR agonists.In the present study, we used non-transformed intestinal epithelial cells stimulated via GPCRs as a model system to examine whether PKD1 negatively regulates PI3K/Akt signaling and cellular levels of PIPThis study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Animal Research Committee of the University of California, Los Angeles (Protocol Number: 2001-142–23).in vivo, we used transgenic mice that express elevated PKD1 protein in the small intestine and colonic epithelium and control littermates. The generation of PKD1 transgenic mice was described elsewhere 2 chamber. Overexpression of PKD1 in the ileum was verified using epithelial cells isolated sequentially along the crypt-villus axis by timed incubations in EDTA-PBS solutions. To measure PKD1 expression and Akt phosphorylation, lysates of intestinal cells isolated from gender- and age-matched mice (4 PKD1 transgenic mice and 4 nontransgenic littermates) were subjected to immunoblotting, as described above.To assess the effect of PKD1 on Akt phosphorylation 2 incubator. Stock cultures were sub-cultured every 3–4 days.The non-transformed rat intestinal epithelial IEC-6 and IEC-18 cells 3OH. For detection of proteins, membranes were blocked using 5% nonfat dried milk in PBS (pH 7.2) and then incubated for at least 2 h with the desired antibodies diluted in PBS containing 3% nonfat dried milk. Primary antibodies bound to immunoreactive bands were visualized by enhanced chemiluminescence (ECL) detection with horseradish peroxidase-conjugated anti-mouse, anti-rabbit antibody and a FUJI LAS-4000 Mini Luminescent Image Analyzer. The phosphospecific polyclonal antibodies used  detect the phosphorylated state of residues Ser473 and Thr308 of Akt, Thr389 of S6K and Ser916 of PKD.Serum-starved, confluent intestinal epithelial IEC-18 cells treated with inhibitors and/or agonists were lysed in 2x SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer  and boiled for 10 min. After SDS-PAGE, proteins were transferred to Immobilon-P membranes. The transfer was carried out at 100 V, 0.4 A at 4°C for 4 h using a Bio-Rad transfer apparatus. The transfer buffer consisted of 200 mM glycine, 5 mM Tris, 0.01% SDS, and 20% CH5 cells/35 mm dish. Control transfections were carried out with Stealth siRNA neagative control . Four days after transfection, cells were used for experiments and subsequent Western blot analysis.The Stealth siRNA duplexes were purchased from Invitrogen  Two different PKD1 siRNAs were designed to target the mRNA of rat PKD1 (XM_234108): Oligo 1 GAACCUUCAUCACCCUGGUUGUA; Oligo2, GAGAAGAGGUCAAAUUCGCAGUCAU. For siRNA transfection the reverse transfection method was used, the siRNA pool was mixed with Lipofectamine RNAiMAX  according to the manufacturer's protocol and added to 35mm dishes. IEC-18 cells were then plated on top of the siRNA/Lipofectamine RNAiMAX complex at a density of 1×10IEC-18 cells were transfected with the plasmid containing a cDNA encoding a green fluorescent protein (GFP) tagged-Akt pleckstrin homology domain (Akt-PH-GFP) from Addgene (pcDNA3-Akt-PH-GFP cat #18836) by using Lipofectamine Plus (Invitrogen) as suggested by the manufacturer. Analysis of the cells transiently transfected were performed 24 h after transfection.Single live-cell imaging of the GFP tagged Akt-PH domain was achieved with a fluorescence microscope. The microscope used was a Zeiss  epifluorescent Axioskop with a Zeiss Achroplan 40×/0.75 water immersion objective (Zeiss). Images were captured as uncompressed 24-bit TIF files with a Pusuit cooled single CCD color digital camera driven by SPOT software . GFP fluorescence was observed with a HI Q filter set for fluorescein isothiocyanate .Quantitative analysis of the relative change in plasma membrane and cytosol fluorescence intensity of individual cells were performed by importing the TIF images into Zeiss LSM 510 software and performing profile scans with the largest line width. Five equally spaced line profiles were taken for each cell or cell pair. Intensities were background corrected, and the intensities at the membrane were divided by those in the immediately surrounding cytoplasm. We analyzed 20–30 cells in each experiment, and each experiment was performed in duplicate. The selected cells displayed in the figures were representative of 90% of the population of positive cells.Confluent IEC-18 cells were lysed in buffer A containing 50 mM Tris-HCl, pH 7.6, 2 mM EGTA, 2 mM EDTA, 1 mM dithiothreitol, 100 µg/ml leupeptin, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, hydrochloride (Pefabloc) and 1% Triton X-100. p85α was immunoprecipitated with p85α rabbit monoclonal antibody . The immune complexes were recovered using protein-A coupled to agarose and analyzed for PKD1-mediated phosphorylation (using a PKD1 motif antibody) or complex formation with tyrosine phosphorylated EGFR, p110α or PTEN, as described in in vivo, we used transgenic mice that express elevated PKD1 protein in the small intestine and colonic epithelium and control littermates. The generation of PKD1 transgenic mice was described elsewhere To assess the effect of PKD1 on Akt phosphorylation DMEM was obtained from Invitrogen . Angiotensin II, vasopressin and LPA were obtained from Sigma Chemical . kb NB 142-70 was obtained from R&D Systems  and CRT0066101 (41) was obtained from Cancer Research Technology Discovery Laboratories . Anti-phosphotyrosine 4G10 antibody was purchased from Millipore All other antibodies were purchased from Cell Signaling Technology . All other reagents were of the highest grade available.473, the residue phosphorylated by mTORC2 or Thr308, the residue in the activation loop phosphorylated by PDK1 (A). The level of Akt phosphorylation on these residues in response to ANG II was small, as compared with the effect induced by either EGF or insulin in parallel cultures . Because ANG II in contrast to EGF or insulin induces robust PKD1 activation in IEC-18 cells , we examined whether ANG II-induced Akt phosphorylation was restrained by feedback inhibition mediated by PKD1. Cultures of IEC-18 cells were treated with increasing concentrations of the selective PKD family inhibitor kb NB 142–70 for 1 h and then challenged with 50 nM ANG II A, prior exposure of IEC-18 cells to kb NB 142–70 strikingly potentiated Akt phosphorylation at Thr308 and Ser473 in a dose-dependent manner. Half-maximal potentiation was obtained at a concentration of 1.5 µM (B). The enhancements of Akt phosphorylation ocurred at concentrations of kb NB 142-70 that inhibited PKD1 activation in the same cells, as scored by PKD1 autophosphorylation at either Ser916 in the C-terminus or Ser748 in the activation loop .Stimulation of intestinal epithelial IEC-18 cells with angiotensin II (ANG II), a mitogenic agonist that activates Gq-coupled receptors endogenously expressed by these cells, induced a detectable increase in Akt activation, as judged by its phosphorylation at either Ser by PDK1 Fig1A.308 and Ser473 by treatment with kb NB 142–70 was also observed in IEC-18 cells stimulated with various concentrations of ANG II (0.1–50 nM); enhancement was detected in cells stimulated with ANG II at a concentration as low as 1 nM . ANG II induced PKD1 activation (scored by Ser916 autophosphorylation) at similar concentrations. Enhancement of Akt activation by addition of kb NB 142–70 reached a maximum as early as 10 min after ANG II stimulation and persisted at the maximal level for at least 2 h . We verified that treatment with kb NB 142–70 suppressed PKD1 autophosphorylation on Ser916 at all concentrations of ANG II  and times examined .The striking increase of Akt phosphorylation at ThrA) or LPA (B) as potently as in paralell cultures stimulated with ANG II. In contrast, prior exposure to kb NB 142–70 had only a small potentiating effect on Akt activation induced by EGF, an agonist that induces Akt activation but weak PKD1 activation in IEC-18 cells (C). These results, corroborated by quatification (D), demonstrate that the PKD family inhibitor kb NB 142–70 enhances Akt phosphorylation at Thr308 and Ser473 in response to multiple GPCR agonists in intestinal epithelial IEC-18 cells.We next determined whether inhibition of PKD1 enhances Akt phosphorylation in IEC-18 cells stimulated via GPCRs other than those that bind ANG II. The agonists vasopressin and LPA induced robust PKD activation in IEC-18 cells Fig3. EA, treatment with CRT0066101 (5 µM) increased Akt phosphorylation at Thr308 and Ser473 and inhibited PKD1 activation as potently as kb NB 142–70. These results substantiate the notion that PKD1 inhibition markedly enhances Akt activation in ANG II-stimulated IEC-18 cells.Having established that kb NB 142–70 potentiates Akt activation induced by GPCR agonists, we next determined whether similar enhancing effects can be elicited by a structurally unrelated PKD family inhibitor. We used CRT0066101 308 and Ser473 in these cells. Subconfluent cultures of IEC-18 cells were transiently transfected with PKD1 siRNA or nontargeted negative control duplex. To minimize the possibility that the siRNA oligonucleotide may be affecting the expression of a gene other than PKD1, IEC-18 cells were transfected with two different siRNAs targeting distinct regions of PKD1. The PKD1 protein level in IEC-18 cells transfected with either siRNA1 or siRNA2 was dramatically reduced (∼90%) compared with cells transfected with nontargeted negative control duplex ( B). In contrast, Akt protein levels, determined as a loading control, were not affected. The salient feature of the results shown in , B is that PKD1 knockdown by either siRNA1 or siRNA2 strikingly enhanced Akt phosphorylation at Thr308 and Ser473 in response to ANG II stimulation in IEC-18 cells. Collectively, the results with PKD family inhibitors and siRNAs indicate that PKD1 mediates feedback inhibition of Akt activation in response to GPCR agonists.To prove that PKD1 mediates feedback inhibition of Akt activation in response to GPCR agonits in IEC-18 cells, we determined whether knockdown of PKD1 expression using different siRNAs enhances ANG II-induced Akt phosphorylation at Thr308 and Ser473 is mediated by protein kinases, PDK1 and mTORC2, that respond to the generation of PIP3 by PI3K. The accumulation of PIP3 in the plasma membrane initiates Akt translocation to this location, a preriquisite for its subsequent phosphorylation and activation. We considered the possibility that PKD1 activation attenuates production of PIP3 and thereby restrains Akt phosphorylation. In order to examine this possibility, we monitored the redistribution of Akt-pleckstrin homology domain-green fluorescent protein (Akt-PH-GFP), an in vivo reporter of PIP33 sensor was located primarily in the cytosolic compartment without any detectable accumulation at the plasma membrane (A). Treatment with ANG II induced detectable translocation of Akt-PH-GFP to the plasma membrane. Prior exposure of the cells to kb NB 142–70 strikingly increased membrane accumulation of the PIP3 sensor in response to subsequent stimulation with ANG II . Translocation of Akt-PH-GFP to the plasma membrane was also detected at 5 min and 30 min after ANG II stimulation of IEC-18 cells treated with kb NB 142–70 .We next examined the mechanism by which PKD1 restrains Akt activation in response to GPCR agonists in intestinal epithelial cells. Akt phosphorylation at Thr A; corroborated by quatification in  B). These results indicate that exposure to kb NB 142–70 induces a striking increase in PIP3 at the plasma membrane via p110α in cells stimulated with ANG II.In order to verify that membrane accumulation of Akt-PH-GFP senses PI3K-generated lipid second messengers, we determined whether the recently developed class I p110α specific inhibitor A66  A) or the class IA p110α specific inhibitor A66 ( B) completely prevented the increase in Akt phosphorylation at Thr308 and Ser473 in IEC-18 cells exposed to kb NB 142–70 and subsequently challenged with ANG II. Similar results were obtained when the cells were stimulated with vasopressin instead of ANG II (data not shown).In view of the preceding results, we next determined whether the increase in Akt phosphorylation by ANGII in cells exposed to kb NB 142–70 is prevented by inhibition of PI3K activity within IEC-18 cells. Treatment with either the PI3K and mTOR inhibitor LY294002  induced by suppression of PKD1 activity in GPCR-stimulated intestinal epithelial cells requires EGFR transactivation. In line with this possibility, treatment of the cells with the specific inhibitor of EGFR tyrosine kinase activity AG1478 completely prevented the enhancement of Akt phosphorylation at Thr308 and Ser473 in IEC-18 cells exposed to kb NB 142–70 and stimulated with either ANG II or vasoppressin ( C). These results are consistent with the notion that endogenous GPCRs couple to class IA PI3K involving p110 α via EGFR transactivation in intestinal epithelial IEC-18 cells.The class IA PI3Ks are heterodimers consisting of a p110 catalytic subunit and a p85 regulatory subunit. Class I A heterodimers involving p110α are activated by tyrosine kinases. The results obtained with the specific p110α inhibitor A66 imply that the striking increase in PIPmulation Fig.5 a154, Ser361 and Ser652 As shown in  A, stimulation of cells with ANG II markedly increased the phosphorylation of p85α detected by the PKD motif-specific antibody. We verified that similar amounts of p85α were immunoprecipitated from lysates of cells exposed to different treatments. Crucially, the increase in the phosphorylation of p85α induced by stimulation with ANG II was completely prevented by prior cell exposure to the PKD family inhibitors kb NB 142–70 and CRT0066101 ( A). The results indicate that GPCR activation induces phosphorylation of the endogenous p85α regulatory subunit of PI3K through PKD1 in intestinal epithelial cells.Recent results demonstrated that treatment with the phorbol ester PMA inhibited PI3K activation via phosphorylation of the p85α regulatory subunit by PKD1 3 accumulation and Akt phosphorylation, we examined possible molecular events, including (1) inhibition of the binding of p85α to tyrosine phosphorylated residues; (2) complex formation between p85α and either p110α or PTEN. In order to determine whether PKD1-mediated phosphorylation of p85α inhibits its binding to the EGFR, cultures of IEC-18 cells treated without or with kb NB 142–70 or the specific EGFR tyrosine kinase inhibitor AG1478. Subsequently, the cultures were stimulated with ANG II for various times, lysed and p85α immunoprecipitates were analyzed by anti-phosphotyrosine immunoblotting. As shown in B, stimulation with ANGII induced rapid binding of p85α to a major tyrosine phosphorylated band migrating with the apparent molecular mass of the EGFR . The detection of this band was completely extinguished by treatment with AG1478, indicating that this band corresponds to the tyrosine phosphorylated EGFR. These results corroborated that GPCR stimulation of IEC-18 cells induces transactivation and tyrosine phosphorylation of the EGFR leading to complex formation with the p85α subunit of PI3K. Our results also show that binding of p85α to the EGFR was not altered by treatment with kb NB 142–70, implying that the increase in PI3K activity in response to PKD1 inhibition is not mediated by enhanced EGFR/p85α complex formation. Anti p110a immunoblotting of p85a immunoprecipitates showed that treatment with kb NB 142–70 did not alter the level of the p85α/p110α complex in quiescent and stimulated cells .To explore the mechanisms by which PKD1-mediated phosphorylation of the p85α subunit attenuates PIP154 to enhance its interaction with PTEN, thereby leading to PTEN activation in neutrophiles  C). A salient feature of these results is that prior exposure of the cells to kb NB 142–70 prevented the increase in p85α/PTEN complex formation induced by ANG II ( C). The results suggest that PKD1-mediated phosphorylation of p85α mediates negative feedback of PIP3 accumulation and Akt phosphorylation in GPCR-stimulated cells, at least in part, by enhancing the stimulatory association of p85α with PTEN.A recent study demonstrated that PKD1 phosphorylates p85α at Ser3 levels and Akt activity of intestinal epithelial cells in vivo. To test this hypothesis, we used transgenic mice that express elevated PKD1 protein in the small intestine epithelium  D). Overexpressed PKD1 was active, as revealed by Ser916 autophosphorylation. The salient feature of the results is that overexpression of PKD1 was associated with reduced phosphorylation of Akt at Ser473 in intestinal epithelial cells in vivo ( D). Collectively, our results indicate that PKD1 mediates negative feedback of PI3K/Akt activation within intestinal epithelial cells in vitro and in vivo.Collectively, our preceding results indicating that PKD1 mediates negative feedback of PI3K/Akt activation in IEC-18 cells prompted us to hypothesize that PKD1 regulates PIP1) Treatment with the preferential PKD family inhibitor kb NB 142–70, at concentrations that inhibited PKD1 activation, induced a striking enhancement of Akt phosphorylation at Thr308 and Ser473 in response to the Gq-coupled receptor agonist ANG II; 2) Enhancement of Akt activation was elicited in cells challenged with various concentrations of ANG II or with a fixed concentration of ANG II for various times. These studies revealed that inhibition of PKD family activity converted Akt signaling from transient to persistent; 3) Exposure to kb NB 142–70 enhanced Akt phosphorylation in IEC-18 cells stimulated with the GPCR agonists vasopressin or LPA as potently as in paralell cultures stimulated with ANG II. In contrast, kb NB 142–70 had only a small potentiating effect on Akt activation induced by EGF, an agonist that induces weak PKD1 activation in IEC-18 cells; 4) We verified that the potentiating effect of kb NB 142–70 on Akt phosphorylation can be mimicked by cell treatment with a structurally unrelated inhibitor of the PKD family, namely CRT0066101; 5) Extensive knockdown of PKD1 expression using siRNAs directed against different regions of PKD1 enhanced ANG II-induced Akt phosphorylation at Thr308 and Ser473. These results demonstrate, for the first time, that endogenous PKD1 mediates potent feedback inhibition of GPCR-induced Akt activation.GPCR agonists act as potent cellular growth factors and have been implicated in a variety of normal and abnormal processes, including development, inflammation, and malignant transformation in vivo. To test this hypothesis, we used transgenic mice that express elevated PKD1 protein in the small intestine epithelium 473 in intestinal epithelial cells. Collectively, the results indicate that PKD1 mediates negative feedback of Akt activation within intestinal epithelial cells in vitro and in vivo.Since PKD1 mediates negative feedback of PI3K/Akt activation in IEC-18 cells, we hypothesized that PKD1 regulates Akt activity of intestinal epithelial cells in vivo reporter of PIP33 sensor and the enhancement of Akt phosphorylation at Thr308 and Ser473 were completely blocked by exposure to the class I p110α specific inhibitor A66. Class IA PI3K heterodimers involving p110α are activated by tyrosine kinases, implying that PIP3 accumulation and Akt activation induced by suppression of PKD1 activity in GPCR-stimulated intestinal epithelial cells require GPCR-induced EGFR transactivation. Accordingly, treatment with the specific inhibitor of EGFR tyrosine kinase activity AG1478 completely prevented the enhancement of Akt phosphorylation in cells exposed to kb NB 142–70 and stimulated with either ANG II or vasoppressin. Consequently, we conclude that PKD1 mediates negative feedback of GPCR-induced Akt activation by preventing PIP3 accumulation in the plasma membrane, the second messenger that triggers the translocation and subsequent phosphorylation of Akt.In order to examine the mechanism by which PKD1 attenuates Akt activation, we evaluated the activity of PI3K in single cells by monitoring the redistribution of Akt-PH-GFP, an 608652361 and Ser652 interfered with PI3K activation. However, these studies used ectopically expressed proteins and phorbol esters, which are ultrapotent, non-physiological surrogates of rapidly turning over endogenous DAG. Furthermore, phorbol esters induce PKD1 activation via PKC at early and late times 154 to enhance its interaction with PTEN, thereby regulating neutrophil migration 3 has not been examined in any of the previous studies.As mentioned before, the class IA PI3Ks are heterodimers consisting of a p110 catalytic subunit and a p85 regulatory subunit 3 accumulation and Akt phosphorylation in response to GPCR activation in intestinal epithelial cells, we determined whether physiological PKD1 activation via GPCR-mediated pathways also induces phosphorylation of endogenous p85α. Here, we demonstrate that stimulation of IEC-18 cells with ANG II markedly increased PKD1-mediated phosphorylation of p85α in response to ANG II, as shown with a PKD family motif-specific antibody. The immureactive band detected by this antibody was completely extinguished by cell exposure to the PKD family inhibitors kb NB 142–70 or CRT0066101. Our results support a model in which PKD1 inhibits PIP3 accumulation within intestinal epithelial cells stimulated with GPCR agonists by phosphorylating the p85α regulatory subunit of class IA PI3Ks. We examined several potential mechanisms by which PKD1-mediated phosphorylation of p85α attenuates PIP3 accumulation. Co-immunoprecipitation experiments indicated that suppresion of PKD1 activity did not enhance EGFR/p85α complex formation or change the level of the p85α/p110α PI3K heterodimer. In contrast, we found that GPCR activation induced p85α binding to PTEN and inhibition of PKD1 prevented p85α/PTEN complex formation. Consequently, we conclude that PKD1-mediated phosphorylation of p85α mediates negative feedback of PIP3 accumulation and Akt phosphorylation, at least in part, by enhancing the stimulatory association of phosphorylated p85α with PTEN leading to PIP3 dephosphorylation.To asses the importance of p85α phosphorylation in the mechanism by which PKD1 attenuates PIP3 levels within cells. Based on the results presented here, we propose that PKD1 mediates negative feedback of PIP3 accumulation thereby contributing to dynamic PIP3/Akt signaling in the cell. Together with previous results demonstrating that PKD1 prolongs ERK activation but attenuates JNK The regulation of the intensity and duration of signaling pathways is of critical importance for determining cellular outcomes, including metabolism, proliferation, survival, growth arrest or senescence. While this notion is well supported by studies of the RAF/MEK/ERK pathway Figure S1(PDF)Click here for additional data file.Figure S2(PDF)Click here for additional data file.Figure S3(PDF)Click here for additional data file.