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Originally published In Press as doi:10.1074/jbc.M708208200 on March 5, 2008

J. Biol. Chem., Vol. 283, Issue 18, 12446-12455, May 2, 2008
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Protein Kinase C{zeta}-dependent LKB1 Serine 428 Phosphorylation Increases LKB1 Nucleus Export and Apoptosis in Endothelial Cells*

Ping Song, Zhonglin Xie, Yong Wu, Jian Xu, Yunzhou Dong, and Ming-Hui Zou1

From the Section of Endocrinology and Diabetes, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, 73104

Received for publication, October 3, 2007 , and in revised form, March 4, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
LKB1 is a serine-threonine protein kinase that, when inhibited, may result in unregulated cell growth and tumor formation. However, how LKB1 is regulated remains poorly understood. The aim of the present study was to define the upstream signaling events responsible for peroxynitrite (ONOO-)-induced LKB1 activation. Exposure of cultured human umbilical vein endothelial cells to a low concentration of ONOO- (5 µM) significantly increased the phosphorylation of LKB1 at Ser428 and protein kinase C{zeta} (PKC{zeta}) at Thr410. These effects were accompanied by increased activity of the lipid phosphatase PTEN, decreased activity and phosphorylation (Ser473) of Akt, and induction of apoptosis. ONOO- enhanced Akt-Ser473 phosphorylation in LKB1-deficient HeLa S3 cells or in HeLa S3 cells transfected with kinase-dead LKB1. Conversely, ONOO- inhibited Akt Ser473 phosphorylation when wild type LKB1 were reintroduced in HeLa S3 cells. Further analysis revealed that PKC{zeta} directly phosphorylated LKB1 at Ser428 in vitro and in intact cells, resulting in increased PTEN phosphorylation at Ser380/Thr382/383. Finally, ONOO- enhanced PKC{zeta} nuclear import and LKB1 nuclear export. We conclude that PKC{zeta} mediates LKB1-dependent Akt inhibition in response to ONOO-, resulting in endothelial apoptosis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
LKB1 is a serine-threonine protein kinase of the calcium calmodulin family, and LKB1 mRNA is ubiquitously expressed in all tissues including liver, skeletal muscle, and cardiac tissue (1, 2). In humans, loss-of-function mutations of LKB1 are associated with the development of a gastrointestinal disorder called Peutz-Jehgers syndrome (3). LKB1 has also been shown to play a role in the formation of sporadic tumors of the lung (4, 5). Therefore, LKB1 is thought to function as a tumor suppressor, with inhibition of LKB1 activity precipitating unregulated cell growth and tumor formation. In support of this tumor suppressor role, LKB1 has been shown to regulate growth and survival processes in several tumor cell lines. For example, reintroduction of LKB1 to LKB1-deficient tumor cells suppresses growth by inducing G1 arrest (5, 6). In addition, overexpression of LKB1 in a breast cancer cell line reduces cell migration and tumor genesis when injected into an animal cancer model (7). LKB1 is required for p53-dependent apoptosis (8). Importantly, LKB1 is thought to function as a tumor suppressor through its ability to negatively regulate the Akt signaling pathway. In this regard, aberrant Akt signaling is widely recognized as a contributor to the growth of many LKB1-deficient tumors (5), as well as to the induction of vascular endothelial growth factor and angiogenesis in these tumors (9). Moreover, LKB1 can interact with the tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome ten) (10), which is considered a key negative regulator of the phosphatidylinositol 3-kinase/Akt pathway (11).

LKB1 signaling is regulated through two main mechanisms: 1) phosphorylation and 2) subcellular localization. LKB1 can be phosphorylated on several residues, and its phosphorylation is thought to play a key role in cell cycle arrest (12), tumor suppression (4), and cell polarity (13, 14). In addition, LKB1 activity appears to be regulated by the formation of complexes with STRAD and MO25. In the absence of these proteins, overexpressed LKB1 is localized to the nucleus. On the other hand, formation of the LKB1·MO25·STRAD complex causes a relocalization of LKB1 to the cytosol (a major site of LKB1 action) and enhances LKB1 activity (15). The activation of LKB1 by these pseudokinases may regulate signaling pathways downstream of LKB1, including AMP-activated protein kinase and Akt pathways. However, the site(s) of LKB1 phosphorylation and the enzymes upstream of LKB1 remain enigmatic.

We have previously shown that high glucose increases endothelial apoptosis by LKB1-dependent PTEN-mediated Akt inhibition (16). In endothelial cells, LKB1 activates AMP-activated protein kinase in response to reactive nitrogen species or metformin through a protein kinase C (PKC)-dependent2 mechanism (17). These findings have prompted us to investigate whether PKC acts upstream of LKB1-dependent Akt inhibition and apoptosis induced by ONOO-. Here, we provide evidence that PKC{zeta}-dependent LKB1 phosphorylation at Ser428 is essential for ONOO--induced Akt inhibition and apoptosis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Human umbilical vein endothelial cells (HUVECs) and cell culture medium were purchased from Cascade Biologics (Portland, OR). HUVECs were maintained in Medium 200 supplemented with low serum growth supplement. HeLa S3 and A549 cells obtained from ATCC (Manassas, VA) were grown in F-12K media (ATCC) containing 10% heat-inactivated fetal bovine serum. Human breast cancer MDA-MB-468 cells were a generous gift from Dr. Bolin Liu (University of Colorado Health Science Center, Denver, CO) and were grown in Dulbecco's modified Eagle's medium with 10% heat-inactivated fetal bovine serum and 2 mM L-glutamine. All of the culture media were supplemented with both penicillin (100 units/ml) and streptomycin (100 µg/ml). Plasmid for wild type and mutant PTEN (PTEN-C124S lacks both lipid and protein phosphatase activity) were kindly provided by Dr. Kaz Matsumoto (NIDCR, National Institutes of Health). Antibodies against PTEN, phospho-PTEN-Ser380/Thr382/383, Akt, phospho-Akt-Ser473, phospho-LKB1-Ser428, phospho-PKC{zeta}-Thr410, phospho-GSK-3{alpha}/β-Ser21/9, and phospho-GSK-3β-Ser9, as well as GSK-3 fusion protein and Akt kinase assay kit were obtained from Cell Signaling Technology (Beverly, MA). Anti-β-actin was from Abcam Inc. (Cambridge, MA). Anti-histone H2AX was from Bethyl Laboratories, Inc. (Montgomery, TX). Recombinant human Akt1 and myristoylated pseudosubstrate peptides for PKC{zeta} (Myr-Ser-Ile-Tyr-Arg-Arg-Gly-Ala-Arg-Arg-Trp-Arg-Lys-Leu-OH) were from BIOSOURCE International, Inc. (Camarillo, CA). Recombinant LKB1, and the PTEN Malachite Green Assay kit were acquired from Upstate Group LLC. (Lake Placid, NY). Antibodies against LKB1, and PKC{zeta} (C-20) were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Protein A-Sepharose CL-4B beads were from GE Healthcare Bio-Sciences Corp. (Piscataway, NJ). Recombinant human PTEN was purchased from R & D Systems, Inc. (Minneapolis, MN). ONOO- and bisindolylmaleimide I (a pan-PKC inhibitor (18)) were obtained from Calbiochem. Sin-1 was from Dojindo (Tokyo, Japan). Plasmid and siRNA delivery agent LipofectamineTM 2000 was from Invitrogen. Recombinant LKB1 mutants (S428A) were kindly provided by Dr. Dietbert Neumann (Institute of Cell Biology, ETH Zurich, Switzerland). Other chemicals and organic solvents of highest grade were obtained from Sigma-Aldrich.

Treatment of Cells with ONOO-—The concentrations of ONOO- were determined spectrophotometrically in 0.1 M NaOH ({epsilon}302 = 1670 M-1 cm-1). To avoid a sharp shift in pH, ONOO- was diluted in 0.1 M NaOH before use. Treatment with ONOO- was performed as described previously (19). In addition, decomposed ONOO- (ONOO- was added in 1 M Tris buffer, pH 7.4, and kept in room temperature for 5 min or overnight) was used as a control. The same results were observed for either method of preparation of decomposed ONOO-. HUVECs serum-starved for 6 h were preincubated with protein kinase inhibitors at the indicated concentration for 30 min and subsequently treated with ONOO- for 15 min. HeLa S3 and breast cancer MDA-MB-468 cells were starved in serum-free medium overnight before being exposed to 5–25 µM ONOO- for 15 min.

siRNA Gene Silencing of PKC{zeta}—siRNA duplex oligonucleotides used in this study are based on the human cDNAs encoding PKC{zeta}. PKC{zeta} siRNA as well as a nonsilencing control siRNA were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The working concentration of siRNA duplexes applied was 100 nM. HUVECs were transfected with PKC{zeta} siRNA or nonspecific control siRNA for 48 h using LipofectamineTM 2000 (Invitrogen) according to the manufacturer's instructions. Transfected cells were starved in serum-free medium for 6 h and then exposed to the indicated concentrations of ONOO- for 15 min.

Western Blot Analysis—HUVECs were washed once with phosphate-buffered saline and lysed with ice-cold buffer from Cell Signaling Technology (Beverly, MA) containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptine, 10 µg/ml aprotinin, 1 mM Na3VO4, and 10 mM NaF. Lysates were clarified by centrifugation at 4 °C for 18 min. Protein concentration was measured using the BCA protein assay (Pierce). Samples containing 20–50 µg of proteins were separated on polyacrylamide gel with Tris-glycine-SDS running buffer (Bio-Rad) and transferred onto a nitrocellulose membrane (Bio-Rad) for 2 h. The membranes were blocked in 5% milk in Tris-buffered saline-Tween 20 for 1 h and incubated overnight with the primary antibody. The membranes were washed and incubated with a peroxidase-linked secondary antibody, and the reactive bands were detected by ECLTM Western blotting detection reagents (Amersham Biosciences).

Akt Activity Assay—Akt kinase activity was measured using an in vitro Akt kinase assay kit. Briefly, HUVECs were collected in lysis buffer according to the manufacturer's instructions. Lysate (200 µg protein) was incubated overnight with the anti-Akt antibody provided in the kit. Captured Akt was incubated with 1 µg of recombinant glycogen synthase kinase-3β in the reaction buffer for 30 min at 37 °C. The reaction was terminated by the addition of SDS sample buffer, and reaction mixtures were electrophoresed on SDS-polyacrylamide gels. Phosphorylation of GSK-3β was used as an index of Akt activity and measured by Western blot using a phospho-GSK-3{alpha}/β (Ser21/9) antibody.

PTEN Activity Assay—Anti-PTEN antibody (10 µl) was incubated with 450 µg of cell lysates for 2 h, and the mixture was then incubated with protein A-Sepharose CL-4B beads overnight at 4 °C. Immunoprecipitates were washed with lysis buffer, and PTEN phosphatase activity was measured with a Malachite Green-based assay (Upstate) using phophatidylinositol 3,4,5-triphosphate as a substrate. An affinity-purified, constitutively active form of PTEN supplied with the kit was used as a positive control.

LKB1 Immunocytochemical Staining—HUVECs or A549 cells transfected with LKB1 wild type and mutated plasmids were cultured on cover glasses and then fixed with 4% paraformaldehyde. After blocking, HUVECs were incubated with a goat anti-LKB1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight. Several commercially available anti-LKB1 antibodies failed to recognize recombinant LKB1 in A549 cells. Because LKB1 plasmids encoded a His tag in the N terminus of the protein, mouse anti-His tag antibody (Upstate Cell Signaling Solutions, Temecula, CA) was used as an alternative. After three washes, the slides were incubated with a fluorescein isothiocyanate-conjugated donkey anti-goat or a fluorescein isothiocyanate-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), at a dilution of 1:150 for 1 h. The slides were then rinsed, counter-stained with 4',6-diamidino-2-phynylindole, mounted in Vectashield medium (Vector Laboratories, Burlingame, CA), and viewed on a SLM 510 laser scanning confocal microscope (CARL Zeiss Meditec, Inc., Jena, Germany).

Preparation of Subcellular Fractions—Cellular cytosolic and nuclear fractions were prepared as described previously (20). Briefly, HUVECs were harvested in homogenization buffer (10 mM MOPS, pH 7.0, 10 mM KCl) containing protease inhibitors (Complete; Roche Applied Science) and processed in a Dounce homogenizer. The nuclei were pelleted by centrifugation at 800 x g at 4 °C for 10 min, the supernatant was then centrifuged (40,000 x g, 4 °C) for 40 min, and the resulting supernatant (cytosolic fraction) was collected. Pelleted nuclei were resuspended in lysis buffer (50 mM Tris-HCl, pH 6.8, 6.5 M urea, 2% SDS, 2 mM dithiothreitol, 1% Triton X-100, protease inhibitor mixture), and centrifuged at 14,000 x g at 4 °C for 30 min, and the resulting supernatants (nuclear fraction) were collected.

Cell Viability Assay—Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide assay (ATCC). After being pretreated with 10 µM PKC{zeta}-PS or 1 mM uric acid for 30 min, HUVECs were incubated in 1 mM Sin-1 in 0.4% serum for 18 h. Cell viability was measured according to the manufacturer's protocol. Absorbance was measured with a Bio-Rad Benchmark microplate reader at 570 nm.

Quantitative Detection of DNA Fragmentation by Enzyme-linked Immunosorbent Assay—To assay endothelial apoptosis, DNA fragmentation was quantified using the apoptosis detection enzyme-linked immunosorbent assay kit (Roche Applied Science). Briefly, the cells were lysed, and nuclei-free supernatant was incubated with anti-histone antibodies. Fragmented nucleosomal DNA was detected with anti-DNA peroxidase-conjugated antibody and the peroxidase substrate 2,2'-azinodi(3-ethylbenzthiazolin-sulfonate). The absorbance was measured at 405 and 490 nm. DNA fragmentation was expressed as fold increase over the control values.

Site-directed Mutagenesis of Ser428 or Asp194 of Human LKB1 and Plasmid Transfection—LKB1 mutants were generated as described elsewhere (17) and 2 µg of plasmid DNA were transfected into HeLa S3 cells cultured in 35-mm-diameter dishes using the LipofectamineTM 2000 kit from Invitrogen, according to the manufacturer's instructions. Forty hours after transfection, the cells were treated as indicated. A LacZ expression vector served as a control.

Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling Staining—After being transfected with LKB1 or mutated plasmid DNA for 24 h, A549 cells were incubated with 1 mM Sin-1 for 18 h. After treatment, the cells were fixed with 4% paraformaldehyde in phosphate buffer. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining (TMR red) using a kit from Roche Applied Science and following the provided instruction manual. The percentage of apoptosis was calculated from the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells divided by the total number of cells counted.

In Vitro Kinase Assays—For determination of the effects of PKC{zeta} on Akt activity, GSK-3β fusion protein was incubated with recombinant Akt1 with or without recombinant PKC{zeta} for 30 min at 37 °C in kinase buffer (25 mM Tris, pH 7.5, 5 mM β-glycerophosphate, 2 mM dithiothreitol, 0.1 mM sodium orthovanadate, 10 mM MgCl2) with 0.2 mM ATP. To determine the effect of LKB1 Ser428 phosphorylation on PTEN phosphorylation, PTEN was incubated with mutant (LKB1-S428A) or wild type LKB1 under the same reaction conditions. To define the influence of PKC{zeta} on LKB1-mediated PTEN phosphorylation, recombinant LKB1 was incubated with PTEN ± recombinant PKC{zeta} under the same conditions. The reactions were terminated by adding SDS sample buffer and were heated for 5 min at 95 °C. The samples were then subjected to SDS-polyacrylamide gel electrophoresis and Western blotting using the indicated phospho-specific antibodies.

Western Blot Quantification—The integrated intensity (area x density) of individual bands was quantified by densitometry (AlphaEaseFC, Alpha Innotech). The background was subtracted from the calculated area.

Statistical Analysis—All of the quantitative variables are presented as the means ± S.D. Differences between individual groups were analyzed by one-way, repeated measures analysis of variance with Student's t test. p < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Protein Kinase C{zeta} Mediates Akt Inhibition by ONOO-—Protein kinase B/Akt plays a significant role in cell survival and insulin actions such as glycolysis, gluconeogenesis, protein synthesis, and adipogenesis. We first investigated the effects of ONOO- on both Akt-Ser473 phosphorylation and Akt activity in cultured HUVECs. As shown in Fig. 1 (A and B), exposure of HUVECs to a pathologically relevant concentration of ONOO- (5 µM) (21, 22) for 15 min attenuated the Akt-Ser473 phosphorylation (70%, p < 0.01) and Akt activity (75% reduction, p < 0.01). This concentration of ONOO- did not alter Ser307 phosphorylation of insulin receptor substrate 1 (data not shown).

ONOO- is known to yield numerous reactive free radicals and oxidants including Formula, Formula, Formula, and HO· (23, 24). However, exposure of HUVECs to decomposed ONOO- did not alter Akt phosphorylation, excluding the possibility that the effects of ONOO- were from Formula or Formula, two major end products of ONOO-. Further, exposure of HUVECs to H2O2 (100 µM) or spermine N-[4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]-butyl]-1,3-propanediamine (100 µM) increased Akt phosphorylation at Ser473 (data not shown). Taken together, these results implied that ONOO- plays a key role in the inhibition of Akt, although we could not rule out the possible role of the trace and relatively unstable products from initial ONOO- decomposition.


Figure 1
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  		FIGURE 1.
Inhibition of protein kinase C{zeta} attenuates ONOO- inhibition of Akt-Ser473 phosphorylation and Akt activity. A and B, HUVECs were treated with protein kinase inhibitors (protein kinase G inhibitor, KT5832; protein kinase A inhibitor, H-89; bisindolylmaleimide I (BIS I), pan-PKC inhibitor) for 30 min before ONOO- exposure (5 µM, 15 min). A, phosphorylated Akt-Ser473 was detected by Western blotting. B, in vitro Akt activity was assessed by phosphorylation of GSK-3 fusion protein. The blots are representative of three individual experiments (n = 3; Figure 1, p < 0.01 compared with control; {dagger}, p < 0.01 compared with ONOO-). C and D, HUVECs were treated with PKC{zeta} pseudosubstrate peptides (PKC{zeta}-PS) for 30 min prior to ONOO- exposure. C, phosphorylated PKC{zeta}-Thr410 was detected by Western blotting. D, phosphorylation of Akt at Ser473 and Akt activity. The blots are representative of three independent experiments (n = 3; {dagger}, p < 0.01 compared with control; Figure 1, p < 0.01 compared with 0 µM PKC{zeta}-PS). E, PKC{zeta} cleavage was detected by Western blotting. Representative blots are shown from three separate experiments. Decomposed ONOO- served as control. IP, immunoprecipitation.

 
We next determined whether other protein kinases were involved in ONOO--induced Akt inhibition. Exposure of HUVECs to KT5823, a potent protein kinase G inhibitor, or H-89 (1 µM), a selective protein kinase A inhibitor, did not alter the basal levels of Akt Ser473 phosphorylation or Akt activity in HUVECs (data not shown). Neither KT5823 nor H-89 altered ONOO--induced Akt inhibition (Fig. 1, A and B). In contrast, bisindolylmaleimide I (5 µM), a nonselective PKC inhibitor (18), which had no effect on Akt phosphorylation or Akt activity in unstimulated cells, significantly attenuated ONOO--induced suppression of both Akt Ser473 phosphorylation and Akt activity (Fig. 1, A and B). These results suggest that Akt inhibition caused by ONOO- might be PKC-dependent.

ONOO- caused a 2-fold increase of PKC{zeta} phosphorylation at Thr410 (p < 0.01). Earlier studies from us suggested that ONOO- activates PKC{zeta} in bovine aortic endothelial cells (17). To determine whether PKC{zeta} was responsible for ONOO--induced Akt inhibition, HUVECs were pretreated with PKC{zeta} pseudosubstrate peptide (PKC{zeta}-PS), a selective pharmacological inhibitor for PKC{zeta} (25). As expected, treatment with PKC{zeta}-PS prior to ONOO- addition suppressed ONOO--induced inhibition on both Akt-Ser473 phosphorylation and Akt activity in a dose-dependent manner (Fig. 1, C and D).

ONOO- is reported to activate PKC{zeta} by increasing caspase-dependent cleavage (26). We next determined whether ONOO- increased caspase-dependent PKC{zeta} cleavage. As shown in Fig. 1E, ONOO- increased the cleavage of both caspase 3 and PKC{zeta} in HUVECs.

LKB1 Is Required for ONOO--induced Akt Inhibition—We next determined whether PKC{zeta} directly inhibited Akt. To that end, recombinant Akt1 were incubated with recombinant PKC{zeta}. Interestingly, PKC{zeta} increased Akt activity, as measured by GSK-3β phosphorylation. Consistently, PKC{zeta} also increased Akt phosphorylation (Fig. 2A, lane 4 versus lane 2). Consistent with an earlier report (25), PKC{zeta} directly phosphorylated GSK-3β (Fig. 2A, lane 3 versus lane 1).

LKB1 is thought to function as a tumor suppressor through its ability to negatively regulate the Akt signaling pathway. In this regard, aberrant Akt signaling is widely recognized as a contributor to the growth of many LKB1-deficient tumors (5). In addition, our earlier work indicates that ONOO- significantly elevates Ser428 phosphorylation of LKB1 in bovine aortic endothelial cells (17). We next determined whether LKB1 was required for ONOO--induced Akt inhibition. In contrast to Akt inhibition in HUVECs, ONOO- dose-dependently increased the phosphorylation of both PKC{zeta} and Akt-Ser473 phosphorylation in LKB1-deficient HeLa S3 cells (Fig. 2B), suggesting that LKB1 might be required for ONOO--induced but PKC{zeta}-dependent Akt inhibition.

To further investigate the role of LKB1 in ONOO--induced Akt inhibition, LKB1-deficient HeLa S3 cells were transfected with WT LKB1, a kinase-dead LKB1 mutant (D194A) (13), or LacZ (as control). In HeLa S3 cells transfected with LacZ or D194A, ONOO- increased Akt-Ser473 phosphorylation (Fig. 2C). Conversely, overexpression of wild type LKB1 abrogated ONOO--induced Akt-Ser473 phosphorylation. These results confirm that ONOO--induced Akt inhibition is LKB1-dependent.

To examine how LKB1 was involved in PKC{zeta}-mediated Akt inhibition by ONOO-, we measured phosphorylation of LKB1 and PKC{zeta}, when present alone or together, using an in vitro kinase assay. In line with earlier reports (8, 17), recombinant LKB1 underwent autophosphorylation at Ser428 when incubated alone (Fig. 3A, lane 3). The addition of recombinant PKC{zeta} dramatically increased LKB1 Ser428 phosphorylation (lane 4), whereas the presence of LKB1 did not significantly alter PKC{zeta} autophosphorylation at Thr410 (Fig. 3A, lane 4 versus lane 2), suggesting that PKC{zeta} may activate as a LKB1 kinase.


Figure 2
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  		FIGURE 2.
LKB1 is required for ONOO--induced Akt inhibition. A, activity and Ser473 phosphorylation of recombinant Akt1 in the presence or absence of recombinant PKC{zeta}. Akt1 activity was assessed by phosphorylation of GSK-3 fusion protein. Representative Western blots for phosphorylated Akt-Ser473 and GSK-3β are shown (n = 4; {dagger}, p < 0.001 compared with GSK-3β + Akt1; Figure 2, p < 0.01 compared with GSK-3β + Akt1). B, LKB1-null HeLa S3 cells were exposed to ONOO- (5–25 µM, 15 min). The lysates were analyzed for PKC{zeta} (total and Thr410-phosphorylated) and Akt (total and Ser473-phosphorylated) by Western blot. Representative blots are shown (n = 3; Figure 2, p < 0.01 compared with control). cPKC{zeta}, cleaved PKC{zeta}. C, HeLa S3 cells were transiently transfected with wild type LKB1 (WT), kinase-dead LKB1 (D194A), or LacZ prior to ONOO- exposure (25 µM, 15 min; n = 4; Figure 2, p < 0.01 compared with WT).

 
ONOO- Induces PKC{zeta} Nucleus Import and LKB1 Nucleus Export—LKB1 is predominantly localized within the nucleus, whereas PKC{zeta} is a cytoplasmic protein in unstimulated cells (10). We next determined whether ONOO- altered the subcellular distributions of both PKC{zeta} and LKB1. As depicted in Fig. 3B, ONOO- treatment induced translocation of PKC{zeta} from the cytoplasm into the nucleus. Intriguingly, ONOO- decreased the amount of LKB1 in the nucleus but increased LKB1 in the cytosol (Fig. 3, C and D). Consistent with previous reports (27), LKB1 was found to be localized primarily within the nucleus in cells overexpressing LKB1 mutants (LKB1-S428A). Further, ONOO- did not alter the subcellular distribution of LKB1-S428A mutants (Fig. 3D). These results suggest that LKB1 phosphorylation at Ser428 was required for LKB1 nucleus export, and ONOO- might increase LKB1 nucleus export by increasing PKC{zeta}-dependent LKB1 phosphorylation at Ser428.

Inhibition of Akt by ONOO- Is PTEN-dependent—PTEN is a phophatidylinositol 3,4,5-triphosphate D3-phosphatase that inhibits Akt signaling by dephosphorylating phophatidylinositol 3,4,5-triphosphate (28, 29), and LKB1 is known to phosphorylate PTEN (10, 16). Therefore, we tested the effect of ONOO- on PTEN lipid phosphatase activity. Low concentrations (5 µM) of ONOO- enhanced PTEN activity by 60% in HUVECs (Fig. 4A).

To address whether PTEN is responsible for ONOO--induced Akt inhibition, MDA-MB-468 cells, PTEN null breast cancer cells (30), were exposed to ONOO-. ONOO- increased the detection of LKB1 phosphorylation at Ser428 (Fig. 4B). In contrast to a strong inhibition seen in HUVECs, ONOO- markedly stimulated Akt Ser473 phosphorylation in a dose-dependent manner (Fig. 4B).

To further determine whether PTEN was required for LKB1-dependent Akt inhibition, MDA-MB-468 cells were transfected with wild type PTEN or phosphatasedead PTEN mutants, C124S, before the addition of ONOO-. Compared with the cells overexpressing either LacZ or C124S PTEN mutants, ONOO- significantly attenuated Akt Ser473 phosphorylation in cells overexpressing wild type PTEN (Fig. 4C), implying that active PTEN was required for ONOO--induced Akt inhibition.

LKB1 Ser428 Phosphorylation Is Required for LKB1-mediated PTEN Stabilization—We next determined the phosphorylation site of LKB1, which was important for LKB1-dependent PTEN stabilization and Akt inhibition. As shown in Fig. 5A, ONOO- (5 µM) significantly increased LKB1 Ser428 phosphorylation in both the nucleus and cytosol. Interestingly, more LKB1 phosphorylation remained in the nucleus with lower LKB1 levels in response to ONOO-. These data imply that LKB1 phosphorylation mainly occurs in the nucleus. Mutation of LKB1 serine 428 with alanine (LKB1 S428A) attenuated PTEN phosphorylation at Ser380/Thr382/383 in vitro (Fig. 5B, lane 4 versus lane 3), suggesting that LKB1 Ser428 phosphorylation was required for PTEN phosphorylation at Ser380/Thr382/383 in vitro. Because the phosphorylation of PTEN within the C-terminal tail is reported to increase its stability (31, 32), these data suggest that LKB1 may stabilize PTEN via increased phosphorylation at its C terminus.

To further examine the role of LKB1 phosphorylation in ONOO--induced Akt inhibition, LKB1-deficient HeLa S3 cells were transiently transfected with WT LKB1 or phosphorylation-defect LKB1 mutants (LKB1-S428A). Overexpression of wild type LKB1 but not LKB1-S428A increased PTEN phosphorylation at Ser380/Thr382/383 but inhibited Akt Ser473 phosphorylation in response to ONOO- (Fig. 5C). In contrast, overexpression of LacZ or LKB1-S428A exhibited increased Akt phosphorylation when exposed to ONOO-. Taken together, these results suggest that ONOO- via LKB1 Ser428 phosphorylation caused a PTEN-dependent Akt inhibition.


Figure 3
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  		FIGURE 3.
ONOO- induces the translocation of LKB1 from the nucleus to the cytoplasm. A, in vitro kinase assays with recombinant PKC{zeta} in the presence or absence of recombinant LKB1. Representative Western blots for phosphorylated PKC{zeta}-Thr410 and LKB1-Ser428 from four separate experiments are shown. B, representative Western blots of PKC{zeta} in nuclear and cytosolic fractions obtained from ONOO--exposed HUVECs (n = 3; Figure 3, p < 0.01 compared with control). C, immunocytochemical staining of LKB1 in ONOO--exposed HUVECs. D, LKB1-null A549 cells were transiently transfected with wild type LKB1 (WT) or inactive LKB1 (S428A) prior to ONOO- exposure. LKB1 was detected by immunocytochemistry, and nuclear and cytosolic fractions were analyzed for LKB1 by Western blot. (n = 3; Figure 3, p < 0.01 compared with control (Ctrl)).

 


Figure 4
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  		FIGURE 4.
PTEN negatively regulates Akt in response to ONOO-. A, PTEN activity in HUVECs exposed to ONOO- (n = 5; Figure 4, p < 0.01 compared with control). B, PTEN null MDA-MA-468 cells were exposed to varying concentrations of ONOO- for 15 min. Phosphorylation of PKC{zeta}, LKB1, and Akt was analyzed by Western blot. C, MDA-MA-468 cells were transiently transfected with phosphatase-dead mutant PTEN (C124S) or LacZ prior to ONOO- exposure (5 µM, 15 min). Lysates were analyzed for phosphorylated Akt-Ser473, Akt, and PTEN. Representative blots are shown from three separate experiments.

 
PKC{zeta} Participates in LKB1-PTEN Signaling—We next determined whether PKC{zeta} was required for ONOO--enhanced LKB1 phosphorylation at Ser428 and consequent Akt inhibition by PTEN. Because Ser428 phosphorylation was required for LKB1 nucleus export (Fig. 3D), we first determined whether PKC{zeta} could mediate LKB1 nucleus export. As shown in Fig. 6 (A and B), ONOO- markedly increased the amount of LKB1 in the cytosol, whereas it lowered LKB1 in the nucleus. Interestingly, inhibition of PKC{zeta} with PKC{zeta}-PS abolished ONOO--enhanced nucleus export of LKB1 (Fig. 6, A and B), suggesting that PKC{zeta} was required for ONOO--enhanced LKB1 nucleus export. LKB1 increased PTEN phosphorylation at Ser380/Thr382/383 in vitro (Fig. 6C, lane 3 versus lane 1). Further, PKC{zeta} significantly enhanced LKB1-mediated PTEN phosphorylation (Fig. 6C, lane 4 versus lane 3) in vitro.

It was interesting to determine whether PKC{zeta} increased LKB1-dependent PTEN phosphorylation in intact cells. To this end, PKC{zeta} in HUVECs was inhibited by transfecting PKC{zeta}-specific siRNA. As shown in Fig. 6D, transfection of PKC{zeta} siRNA, but not scrambled siRNA, markedly reduced PKC{zeta} expression and PKC{zeta} phosphorylation in HUVECs, implying that PKC{zeta} siRNA effectively reduced PKC{zeta} in HUVECs. Importantly, like PKC{zeta}-PS, PKC{zeta} siRNA, but not scrambled siRNA, abolished ONOO--enhanced phosphorylation of both LKB1 and PTEN. In parallel, PKC{zeta} siRNA, but not scrambled siRNA, abolished ONOO--enhanced inhibition on Akt-Ser473 phosphorylation (Fig. 6E). Taken together, these results further implied that PKC{zeta} might be required for LKB1-mediated but PTEN-dependent Akt inhibition in HUVECs.

PKC{zeta}-LKB1-PTEN-Akt Axis Is Operated in ONOO--induced Endothelial Cell Apoptosis—The fate of cell survival and cell death is determined by the balance of cell death/survival signals. Both LKB1 and PTEN function as tumor suppressors, whereas Akt is considered a major kinase for survival. Because PKC{zeta} activation increased both LKB1 and PTEN but suppressed Akt, we reasoned that the PKC{zeta}-LKB1-PTEN-Akt axis triggered by ONOO- might be involved in ONOO--induced endothelial cell apoptosis (33). Exposure of HUVECs to Sin-1 (1 mM, 18 h), a ONOO- donor, markedly induced the phosphorylation of PKC{zeta}, LKB1, and PTEN, whereas it markedly attenuated Akt phosphorylation (Fig. 7A). In addition, Sin-1 lowered cell viability by 2-fold (Fig. 7B) and increased apoptosis, as measured by histone-associated DNA fragmentation (Fig. 7C). Interestingly, the addition of PKC{zeta}-PS or uric acid, a potent ONOO- scavenger, abolished Sin-1-enhanced apoptosis and the reduction of cell viability in HUVECs (Fig. 7, B and C).


Figure 5
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  		FIGURE 5.
LKB1 Ser428 phosphorylation is necessary for LKB1-mediated Akt inhibition. A, HUVECs were exposed to ONOO-, and phosphorylated LKB1-Ser428 was detected by Western blot (n = 3; Figure 5, p < 0.01 versus control). B, in vitro kinase assays showing the effect of phosphorylated LKB1 on PTEN phosphorylation. Phosphorylation of PTEN-Ser380/Thr382/383 or LKB1-Ser428 was analyzed by Western blot. Similar results were obtained in three independent experiments. C, HeLa S3 cells were transfected with wild type LKB1 (WT), LKB1 phosphorylation mutant (S428A), or LacZ prior to ONOO- exposure (25 µM, 15 min), and the lysates were analyzed for LKB1, Akt, and phosphorylated Akt-Ser473, LKB1-Ser428, and PTEN-Ser380/Thr382/383 by Western blot (n = 3; Figure 5, p < 0.01 compared with WT).

 


Figure 6
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  		FIGURE 6.
PKC{zeta} participates in ONOO--induced LKB1-PTEN signaling to inhibit Akt. A and B, HUVECs were treated with PKC{zeta}-specific inhibitors (PKC{zeta}-PS) prior to ONOO- exposure, and the cytosolic translocation of LKB1 was analyzed by Western blot (n = 3; Figure 6, p < 0.01 compared with control; {dagger}, p < 0.01 compared with ONOO-). C, in vitro kinase assay showing LKB1-mediated PTEN-Ser380/Thr382/383 phosphorylation in the presence or absence of recombinant PKC{zeta}. Similar results were obtained in three separate assays. D and E, HUVECs were treated with PKC{zeta}-PS (10 µM, 30 min) or transfected with 100 nM PKC{zeta} specific siRNA (48 h) before ONOO-exposure (5 µM, 15 min). The lysates were analyzed by Western blot for phosphorylated and total PKC{zeta} (D) or for Akt and phosphorylated forms of Akt, PTEN, and LKB1 (E). Representative blots are shown (n = 3; Figure 6, p < 0.01 compared with control; {dagger}, p < 0.01 compared with ONOO-).

 
LKB1 is thought to function as a tumor suppressor through its ability to negatively regulate the Akt signaling pathway. Thus, we determined whether LKB1 was required for endothelial apoptosis caused by Sin-1. Overexpression of LKB1 in fibrosarcoma cells induces cell death (8). Accordingly, introduction of WT LKB1 into LKB1-null A549 cells sensitized these cells to the effects of Sin-1 (Fig. 7D). In contrast, no significant difference existed between the apoptosis of A549 cells transfected with LacZ, WT, or mutants alone (data not shown). Active LKB1 appeared to be required for the induction of apoptosis by Sin-1, because neither of the two LKB1 inactive mutants (LKB1-S428A or LKB1-D194A) accentuated Sin-1-induced apoptosis in HUVECs (Fig. 7D).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we have provided evidence in support of the hypothesis that ONOO- activates the PKC{zeta} -> LKB1 -> PTEN signaling axis, which induces apoptosis through suppression of Akt phosphorylation and Akt activity. We have shown that ONOO- significantly elevates the levels of phosphorylated PKC{zeta} and induces PKC{zeta} nuclear import, which then phosphorylates LKB1 at Ser428. LKB1, in turn, phosphorylates PTEN at Ser380/Ther382/383. Phosphorylation of PTEN at these sites increases its stability and leads to an accumulation of phosphatase activity, which appears to inhibit Akt signaling and induce apoptosis in response to ONOO-. Indeed, ONOO- increased Akt phosphorylation in LKB1-deficient HeLa S3 cells (Fig. 2B) because PTEN phosphorylation requires LKB1. This is further confirmed in PTEN-deficient MDA-MB-468 cells because ONOO- also increased Akt phosphorylation in these cells. Because ONOO- increased Akt phosphorylation in cells lacking LKB1 or PTEN and LKB1 activates Akt in vitro assays, these results can only be explained by LKB1-dependent PTEN activation. Enhanced ONOO- formation plays a causal role in the development of a variety of cardiovascular diseases (24, 34), and our results might have uncovered a novel mechanism by which reactive nitrogen species inhibits Akt and causes apoptosis.


Figure 7
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  		FIGURE 7.
PKC{zeta} mediates ONOO--induced apoptosis in HUVECs. A, HUVECs were exposed to 1 mM Sin-1 overnight. Phosphorylated and total PKC{zeta}, LKB1, PTEN, and Akt were analyzed by Western blot. Representative blots are shown from three separate experiments. B, viability in uric acid (UA)- or PKC{zeta}-PS-pretreated cells exposed to Sin-1 (1 mM) (n = 6; Figure 7, p < 0.01 compared with control; {dagger}, p < 0.01 compared with Sin-1). C, apoptosis, as assessed by levels of cytosolic DNA fragments (n = 3; Figure 7, p < 0.01 compared with control; {dagger}, p < 0.01 compared with Sin-1). D, apoptosis of LKB1-deficient A549 cells transfected with wild type LKB1 (WT), inactive LKB1 (S428A or D194A), or LacZ (control) for 24 h prior to Sin-1 exposure (1 mM) (n = 6; Figure 7, p < 0.01 compared with LacZ/Sin-1).

 
PKC consists of a family of serine/threonine kinases that phosphorylate effector proteins leading to multiple outcomes. Among the PKC family, the atypical PKC{zeta} has recently emerged as an important isoform in endothelial cells (26, 35). PKC{zeta} promotes the adhesive phenotype of endothelial cells through generation of reactive oxidants and subsequently, nuclear factor {kappa}B (NF-{kappa}B)-dependent ICAM-1 expression (36). The role of PKC{zeta} in regulating apoptosis has been demonstrated in many cell types in response to multiple stimuli. For example, sustained activation of PKC{zeta} in macrophages and in cardiac myocytes have been reported to cause apoptosis. We previously found that, in HUVECs, PKC{zeta} mediates responses to HOCl, which is known to induce endothelial cell apoptosis (37). Several signaling molecules downstream of PKC{zeta} have been identified, including c-Jun N-terminal kinase (26), FAS ligand (38), and NF-{kappa}B (39). Here, we demonstrated that PKC{zeta} mediated ONOO--induced apoptosis and that it did so through successive activation of LKB1, stabilization of PTEN, and consequent inhibition of Akt. In accordance with our data, previous studies have shown that PKC{zeta} can block Akt Ser473 phosphorylation (40). PKC{zeta} fragments resulting from caspase cleavage have also been shown to promote endothelial apoptosis (26). Although PKC{zeta} can be activated via cytochrome c release-mediated caspase-dependent processing (41–43), we could not exclude that ONOO- may activate PKC{zeta} via co-factor-dependent redox processing (44). Aside from apoptosis, PKC{zeta} exerts an important role in insulin action. Multiple lines of evidence have suggested that insulin-activated PKC{zeta} enhances GLUT4 translocation and glucose uptake in muscle cells (45) and adipocytes (46). Furthermore, insulin-enhanced PKC{zeta} activity is reduced in skeletal muscles of patients with obesity and type 2 diabetes (47). Insulin-stimulated PKC{zeta} activation may impair insulin signaling via insulin receptor substrate-1 serine phosphorylation (48, 49), which contributes to insulin resistance. We have shown that ONOO- activates PKC{zeta}, which activates LKB1 by stimulating LKB1 nuclear export and inhibits Akt. This pathway may represent an important mechanism underlying the development of insulin resistance. Our results, in combination with previous results suggesting a crucial role for PKC{zeta} in regulation of endothelial cell dysfunction (50), suggest that insulin sensitization may be manipulated by targeting this novel signaling pathway.

In the present study, we have also found that ONOO- activates caspase that leads to PKC{zeta} cleavage and activation in endothelial cells. ONOO- may release cytochrome c to activate caspase (43); then activated caspase processes PKC{zeta} at 3 aspartate residues (Asp210, Asp222, and Asp239) and promotes relief of the autoinhibitory state by separating the kinase domain from the pseudosubstrate autoinhibitory sequence (amino acids 116–122) (26, 41, 42, 51). However, we cannot exclude other possibilities. Low concentrations of ONOO- (1–10 µM) are reported to activate PKC{zeta} in a redox-dependent manner (44). ONOO- might activate PKC{zeta} by directly oxidizing the zinc finger of PKC (52). Finally, ONOO- is reported to activate the phosphatidylinositol 3-kinase pathway (53). Because PKC{zeta} is downstream of phosphatidylinositol 3-kinase/PDK1 (54), ONOO- might increase PKC{zeta} activity through the phosphatidylinositol 3-kinase pathway. How PKC{zeta} became activated by ONOO- warrants further investigation.

Another important finding in the present study is the regulation of LKB1, a tumor suppressor, by PKC{zeta}. Human LKB1 is a serine-threonine kinase of 433 amino acids that contains both a kinase domain and a nuclear localization signal in its N-terminal region (55). Germline mutations of the LKB1 gene underlie the cancer-prone disorder Peutz-Jeghers syndrome. The majority of Peutz-Jeghers syndrome missense mutations is located in the region encoding the kinase domain and abolish enzymatic activity, disrupting all functions attributed to LKB1. The C-terminal region of LKB1 consists of 124 residues and contains several post-translational modifications. Five phosphorylation sites have been identified: two residues are autophosphorylation sites (Thr336 and Thr402) and three others (Ser325, Thr363, Ser428) are phosphorylated by upstream kinases (55). In addition, LKB1 has been shown to undergo farnesylation at a cysteine residue located in the C-terminal region (Cys430 in human LKB1). The C-terminal region may also possibly serve as a regulatory domain mediating dynamic interactions with several classes of proteins and promoting subcellular targeting. In the present study, we have provided evidence that PKC{zeta} phosphorylates LKB1 at Ser428, resulting in increased LKB1-PTEN interaction and subsequently, Akt inhibition. PTEN-LKB1 interaction plays an essential role in Akt inhibition. This is best demonstrated by the finding that Akt is not inhibited by ONOO- in PTEN-deficient cells (MDA-MB-468 cell line). Similarly, ONOO- activated Akt in LKB1-deficient cells (HeLa S3 or A549 cells), implying that LKB1 is required for the inhibitory effects of ONOO- on Akt. Transfection of wild type, but not mutant, LKB1 restored the effects of ONOO- on Akt. Indeed, recombinant PKC{zeta} significantly phosphorylated LKB1 at Ser428, and substitution of Ser428 with alanine, like kinase-dead LKB1 mutants, abolished ONOO--induced Akt inhibition. Finally, pharmacological or genetic inhibition of PKC{zeta} abolished the effects of ONOO-, suggesting that PKC{zeta} lies upstream of LKB1 and that Ser428 located in the C-terminal part of LKB1 might play a crucial role in regulating Akt activity. However, we cannot exclude the possibility that PKC{zeta} might regulate LKB1-AMP-activated protein kinase interactions through post-translational modifications at other sites in LKB1.

The first order rate constants for ONOO- decomposition measured over a pH range from 4 to 8.5 indicates that the pKa is 7.49 ± 0.06 at 37 °C, and the half-life of ONOO- is 1.9 s at pH 7.4 (23). Therefore, most exogenously added ONOO- will be quenched before reaching its cellular targets. Although there is no way to quantify how much ONOO- added was taken up by the cells, we believe this concentration of ONOO- is likely pathologically relevant and can be generated within tissues. Although NO· is normally present at 1–20 nM levels in biological tissues and up to 100–150 nM levels in stimulated vessels, NO· concentrations of 0.1 µM up to several µM may occur in pathological states of ischemia or inflammation (56–58). In post-ischemic tissues, such as the heart, it has been shown that the levels of Formula production measured by spin trapping of the vascular effluent are 0.2–1.0 µM; and in the presence of PMNs, this Formula generation is further increased (56–58). Therefore, it is very likely that submicromolar to micromolar levels of NO· Formula, and ONOO- are formed in stimulated or post-ischemic tissues. In cultured cells, the rate of ONOO- production in macrophages is estimated to be as high as 50–100 µM per min (59). Thus, the PKC{zeta}-LKB1-PTEN-Akt axis we have described here might be implicated in many pathological conditions including hypoxia-reoxygenation, diabetes, and atherosclerosis.

In summary, PKC{zeta} potently phosphorylates LKB1 at Ser428, and LKB1 Ser428 phosphorylation is required for ONOO--induced Akt inhibition, which might play a crucial role in cell survival and insulin signaling. We conclude that PKC{zeta} acts upstream of LKB1-dependent, PTEN-mediated Akt inhibition.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants HL079584, HL074399, HL080499, and HL089920, a research award from the American Diabetes Association, a research award from the Juvenile Diabetes Research Foundation, a grant from Oklahoma Center for Advancement of Science and Technology, and funds of the Travis Endowed Chair in Endocrinology, University of Oklahoma Health Sciences Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: BSEB 325, Section of Endocrinology and Diabetes, Dept. of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104. Tel.: 405-271-3974; Fax: 405-271-3973; E-mail: ming-hui-zou{at}ouhsc.edu.

2 The abbreviations used are: PKC, protein kinase C; GSK, glycogen synthase kinase; HUVEC, human umbilical vein endothelial cell; ONOO-, peroxynitrite; PKC{zeta}-PS, PKC{zeta} pseudosubstrate peptides; Sin-1, 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride; siRNA, small interference RNA; WT, wild type. Back



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