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Originally published In Press as doi:10.1074/jbc.M610010200 on April 25, 2007

J. Biol. Chem., Vol. 282, Issue 25, 18407-18417, June 22, 2007
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Serine 64 Phosphorylation Enhances the Antiapoptotic Function of Mcl-1*

Shogo Kobayashi{ddagger}, Sun-Hee Lee§, Xue W. Meng§, Justin L. Mott{ddagger}, Steven F. Bronk{ddagger}, Nathan W. Werneburg{ddagger}, Ruth W. Craig||, Scott H. Kaufmann§, and Gregory J. Gores{ddagger}1

From the {ddagger}Division of Gastroenterology and Hepatology, the §Department of Molecular Pharmacology and Experimental Therapeutics, and the Division of Oncology Research, Mayo Clinic College of Medicine, Rochester, Minnesota 55905 and the ||Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755

Received for publication, October 25, 2006 , and in revised form, April 25, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mcl-1 is an antiapoptotic Bcl-2 family member that is highly regulated and when dysregulated contributes to cancer. The Mcl-1 protein is phosphorylated at multiple sites in response to different signaling events. Phosphorylations at Thr163 (by ERK) and Ser159 (by glycogen-synthase kinase 3beta) have recently been shown to slow and enhance, respectively, Mcl-1 protein turnover. Phosphorylation is also known to be stimulated at other, as-yet uncharacterized sites in the G2/M phase of the cell cycle. Using an S peptide-tagged Mcl-1 T163A mutant, Ser64 was identified as a novel Mcl-1 phosphorylation site by mass spectrometry. Immunoblotting demonstrated that phosphorylation at this site was maximal in cells in G2/M phase, was enhanced by tumor necrosis factor-{alpha}-related apoptosis-inducing ligand (TRAIL) treatment, was blocked by inhibitors of CDK (but not ERK or glycogen-synthase kinase 3beta), and was stimulated in vitro by CDK 1, CDK2, and JNK1. The half-life of a nonphosphorylatable S64A Mcl-1 mutant was indistinguishable from that of the wild type polypeptide. In contrast, this mutant failed to protect cells from TRAIL-mediated apoptosis, whereas reconstitution with the phosphomimetic S64E Mcl-1 mutant rendered cells TRAIL-resistant. This anti-apoptotic phenotype of the S64E Mcl-1 mutant was also associated with enhanced binding to the proapoptotic proteins Bim, Noxa, and Bak. A pharmacological CDK inhibitor that reduced Ser64 phosphorylation also sensitized cells to TRAIL cytotoxicity. Collectively, these observations not only identify G2/M-associated phosphorylation at Ser64 as a critical determinant of the antiapoptotic activity of Mcl-1 but also elucidate a novel mechanism by which CDK1/2 inhibitors can enhance the effectiveness of the cytotoxic cytokine TRAIL.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Bcl-2 family member Mcl-1 (myeloid cell leukemia 1) promotes cell viability at a critical upstream point in the apoptotic cascade (1). Mcl-1 and other antiapoptotic members of this family (Bcl-2, Bcl-xL, A1, and Bcl-w) interrupt cell death signals by binding to pro-apoptotic family members (e.g. Bim, Noxa, and others in the case of Mcl-1) (2-5). A distinctive characteristic of Mcl-1 is its short half-life (~2-4 h in various cell types) (6-8), which probably relates to the presence of a long proline-, glutamic acid-, serine-, and threonine-rich (PEST)2 region upstream of the Bcl-2 homology domains (9-11).

Alterations that affect Mcl-1 expression can have severe, potentially detrimental consequences. Conditional deletion of Mcl-1 results in bone marrow failure in mice caused by apoptosis of precursor cells (12-14). Conversely, forced Mcl-1 overexpression predisposes hematopoietic cells to malignant transformation (15). In addition, enhanced Mcl-1 expression has been observed in multiple human cancers, often in association with poor prognosis, disease recurrence, or drug resistance (7, 16-21). Thus, the mechanisms that regulate Mcl-1 play an important role both in controlling cell survival in normal tissues and in preventing the emergence of tumors.

Mcl-1 expression is regulated at the transcriptional and post-translational levels. The transcription factors SRF/ETS, STAT3, cAMP-responsive element-binding protein, and PU.1 have been found to regulate Mcl-1 gene expression in various cell types (22-26). At the post-translational level, Mcl-1 is targeted for proteasomal degradation after ubiquitylation by an E3 ligase termed MULE (11). This susceptibility of Mcl-1 to degradation is phosphorylation-dependent. In particular, ERK-mediated phosphorylation of Mcl-1 at Thr163 within the PEST region prolongs the Mcl-1 half-life (6). In contrast, following Thr163 phosphorylation, Ser159 phosphorylation by GSK-3beta reportedly enhances Mcl-1 ubiquitylation and degradation (8). Consistent with the latter results, Akt (cellular homolog of v-Akt/protein kinase B) inhibition, which activates GSK-3beta, results in Mcl-1 down-regulation (7). Mcl-1 is also down-regulated by inhibitors of CDK9 that diminish its synthesis (20, 27, 28).

Mcl-1 phosphorylation is also known to be stimulated at other, as-yet unidentified sites during the G2/M phase of the cell cycle (29). This phosphorylation is prominent in cells accumulating in G2/M phase upon exposure to microtubule disrupting agents and is also seen in synchronized cells passing through this phase. Bcl-2, the prototypic member of this family, likewise undergoes phosphorylation at multiple sites, one of which is stimulated during the G2/M phase (30-35). Phosphorylation of Bcl-2 can affect its stability and/or antiapoptotic function, with differing effects being reported in different systems. For example, phosphorylation of Bcl-2 at Thr69, Ser70, and Ser87 was associated with both enhanced antiapoptotic activity and slowed turnover in cells (35). In the case of Mcl-1, JNK-mediated phosphorylation on Ser121 has been reported to sensitize cells to oxidative stress-induced cell death apparently without affecting turnover (36). Thus, phosphorylation may affect the function as well as its turnover of Mcl-1.

The present studies were performed to identify additional phosphorylation sites in Mcl-1 distinct from the Thr163 and Ser159 sites that influence turnover. These studies demonstrated that Mcl-1 phosphorylation on Ser64, which is catalyzed by CDK1, CDK2, or JNK1 in vitro, is increased in the G2/M phase of the cell cycle and contributes to the antiapoptotic activity of Mcl-1 by enhancing its binding to proapoptotic Bcl-2 family members rather than altering its expression. Consistent with these results, a CDK inhibitor that blocked Ser64 phosphorylation also sensitized cells to TRAIL-mediated apoptosis. Thus, although phosphorylation at Thr163/Ser159 regulates Mcl-1 turnover, a distinct pathway involving phosphorylation at Ser64 regulates its anti-apoptotic function.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—The CDK inhibitor SU9156 (37) was from Alexis (San Diego, CA). The MEK inhibitors (U0126 and PD98059), the p38 MAPK inhibitor SB220025, the JNK inhibitor SP600125, the GSK-3beta inhibitor BIO (GSK inhibitor IX, (2'Z,3'E)-6-bromoindirubin-3'-oxime), the CDK 2/5 inhibitor PNU 112455A (N4-(6-aminopyrimidin-4-yl)-sulfanilamide), the CDK inhibitors olomoucine and roscovitine, and the proteasome inhibitor MG-132 were from Calbiochem (La Jolla, CA).

Cell Lines and Culture—The human cholangiocarcinoma cell line KMCH-1 was cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin G (100,000 units/liter), streptomycin (100 mg/liter), and gentamicin (100 mg/liter) as described previously (38). The KMCH cell line stably transfected with Mcl-1-shRNA (designated KMCH-shMcl-1) was cultured in medium supplemented with 400 mg/liter G418 as previously described (20).

In Situ Phosphorylation Assay—Polypeptides were recovered after radiolabeling based on S peptide-S protein interactions (39). KMCH cells, and KMCH cells stably transfected with S peptide-tagged versions of wild type, T163A, and S64A Mcl-1 were incubated with or without [32P]phosphate (50 µCi/ml) plus MG-132 (10 µM) for 6 h. At the end of the labeling, the cells were washed in PBS and lysed in lysis buffer consisting of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mM {alpha}-phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM Na3VO4, 100 mM NaF, 20 nM microcystin, and 1% Triton X-100. After the protein concentration was estimated using the Bradford method (40), aliquots containing 1 mg of protein in 1 ml of lysis buffer were incubated with 50 µl of S-protein agarose beads (Novagen, La Jolla, CA) at 4 °C overnight. After at least six washes with lysis buffer, the samples were released from the beads by boiling for 5 min in 50 µl of Laemmli sample buffer (Bio-Rad) containing 5% 2-mercaptoethanol. The samples were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Radioactivity was determined by autoradiography using a phosphorimaging device (Molecular Dynamics, Sunnyvale, CA) at room temperature for 3-6 days. Thr163- and Ser64-specific phosphorylations were detected by immunoblotting using phospho-epitope-specific antibodies as described below.

Phosphorylation Site Analysis—KMCH cells stably transfected with S peptide-tagged T163A Mcl-1 were cultured in 10-cm dishes. After the cells were solubilized in lysis buffer, S peptide-tagged T163A Mcl-1 was recovered on S protein-agarose as previously described (39). The tagged polypeptide was released into SDS sample buffer, resolved by SDS-PAGE, and stained with Coomassie Brilliant Blue (Bio-Rad) in 20% methanol and 7.5% acetic acid. The Mcl-1 band was excised and subjected to tryptic digestion followed by quadruple time-of-flight tandem mass spectrometry analysis at the Taplin Biological Mass Spectrometry Facility, Harvard Medical School.

Immunoblotting—The cells were directly lysed for 30 min on ice with lysis buffer and centrifuged at 14,000 x g for 15 min at 4 °C. After protein concentration was determined by the Bradford assay (40), aliquots containing 30 µg of protein were resolved by 10% SDS-PAGE, transferred to nitrocellulose membrane, and blotted with primary antibodies as described below. Secondary antibodies conjugated to Alexa Fluor 680 (Molecular Probes, Carlsbad, CA) or IRDyeTM 800 (Rockland Immunochemicals, Gilbertsville, PA) were incubated at a dilution of 1:2000. Fluorescent images were captured by an Odyssey fluorescent imager (Licor Biosciences, Lincoln, NE), and their intensities were quantitated using Odyssey software, version 1.2.

Primary antibodies used at the following dilutions were obtained from the indicated suppliers: polyclonal rabbit anti-Mcl-1 antibody (S19) at 1:1000, polyclonal goat anti-beta-actin antibody (C11) at 1:2000, monoclonal mouse anti-Bcl-2 antibody (clone C2) at 1:100, and polyclonal goat anti-caspase 8 large subunit antibody (T16) at 1:100 from Santa Cruz Biotechnology (Santa Cruz, CA); monoclonal mouse anti-Mcl-1 antibody (clone 22) at 1:1000 from BD Pharmingen (Franklin Lakes, NJ); monoclonal mouse anti-Bcl-xL antibody (clone 2H12) at 1:200 from BIOSOURCE (Carlsbad, CA); polyclonal goat anti-Bid antibody (AF860) at 1 µg/ml fromR&D Systems (Minne-apolis, MN); monoclonal rat anti-BimS antibody (14A8) at 1:500 from Chemicon (Temecula, CA); polyclonal rabbit anti-Bax antibody at 2 µg/ml and polyclonal rabbit anti-Bak NT at 2 µg/ml from Upstate%20Biotechnology">Upstate Biotechnology, Inc. (Lake Placid, NY); polyclonal goat anti-DR5 antibody at 1:200 and polyclonal rat anti-cFLIP antibody at 1:200 from Alexis (San Diego, CA); and polyclonal rabbit anti-DR4 antibody at 1:500 (Axxora, San Diego, CA). Monoclonal mouse anti-S peptide antibody was generated as previously described (39). Affinity-purified rabbit antiserum recognizing the Mcl-1 Thr163 phospho-epitope generated by Cell Signaling Technology (Beverly, MA) was used at 1:500.


Figure 1
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  		FIGURE 1.
S peptide-tagged Mcl-1 T163A is phosphorylated in situ. KMCH cells stably transfected with S peptide-tagged wild type or T163A mutant Mcl-1 were incubated with 50 µCi/ml [32P]phosphate and MG-132 (10 µM) for 6 h. Whole cell lysates were prepared, and S peptide-tagged Mcl-1 was enriched from 1000 µg of protein aliquots by affinity purification using S protein-agarose beads. S peptide-tagged Mcl-1 was removed from the beads by boiling in Laemmli sample buffer and resolved by SDS-PAGE. The proteins were transferred to nitrocellulose membranes, and 32P was identified by autoradiography. Immunoblot (IB) analysis was employed to identify Mcl-1 and Mcl-1 Thr163 phosphorylation using specific antisera. Note the absence of signal for the untransfected, parent KMCH cells, and Mcl-1 phosphorylation despite mutation of Thr163.

 
Production and Purification of Rabbit Polyclonal Anti-Ser(P)64-Mcl-1 Phospho-specific Antiserum—A 15-amino acid peptide (Mcl-1 amino acids 57-71) with a phosphoserine at position 64 and an N-terminal cysteine was synthesized by the Mayo Clinic Proteomics Core. After conjugation to keyhole limpet hemocyanin, the peptide was utilized to immunize rabbits according to standard procedures. To permit affinity purification of serum, the immunizing peptide and its nonphosphorylated counterpart were individually conjugated to SulfoLink-Sepharose (Pierce) according to the supplier's protocol. Immune serum was then passed sequentially over columns containing the unphosphorylated and phosphorylated peptides; and purified antibodies were eluted from the second column with glycine (pH 2.2). Protein-containing fractions were dialyzed into PBS with 0.02% sodium azide and concentrated using Centriprep YM-30 centrifugal filters (Millipore, Bedford, MA). Purified antibodies were stored at -80 °C.

Dot blot analysis was employed to examine the specificity of the affinity-purified antisera for the phosphorylated peptide. Bovine serum albumin-conjugated peptides with phosphorylated or unphosphorylated serine 64 were serially diluted into a dot blot apparatus, and detection was performed as described above under "Immunoblotting."

Detection of Mcl-1 Phosphorylation at Ser64—Because the phospho-specific anti-Mcl-1 (Ser64) antibody generated does not yield a reliable signal on immunoblot of total protein lysates, enrichment of Mcl-1 was necessary. For analysis of the endogenous Mcl-1 phosphorylation, Mcl-1 was immunoprecipitated from parental KMCH cells using a polyclonal anti-Mcl-1 antibody (S19), followed by immunoblot analysis for total (S19) or Ser64-phosphorylated Mcl-1. For KMCH cells transfected with the S peptide-tagged Mcl-1 mutants (see below), protein S-agarose purification was used to recover tagged Mcl-1 prior to immunoblot analysis.

In Vitro Phosphorylation Assays—Plasmid encoding GST-Mcl-1 was generated by subcloning Mcl-1 lacking the transmembrane domain into the commercially available pGEX-4T-1 vector (GE Healthcare, Piscataway, NJ). BL21 cells transformed with the plasmid were cultured in 10 ml of Circlegrow (Q-BIOgene, Carlsbad, CA) plus ampicillin (100 µg/ml) until the optical density at 600 nm reached 0.8-1.5. The cells were then induced with 1 mM isopropyl beta-D-1-thiogalactopyranoside for 3 h at 37 °C. The harvested cells were lysed with BugBuster® (Novagen), benzonase, lysozyme, and protease inhibitor mixture II (Calbiochem, La Jolla, CA) for 20 min and transferred into a glutathione-agarose column under gravity flow. After washing with GST bind/wash buffer (Novagen), the bound protein was eluted with glutathione (Novagen). The elution buffer was exchanged to the corresponding kinase buffer (New England Biolabs, Ipswich, MA), and the protein was concentrated using Centriprep YM-30 centrifugal concentrators (Millipore, Bedford, MA). The protein concentration was measured by the Bradford assay (40). In vitro kinase assays were performed by incubating GST-Mcl-1 protein with CDC2 (CDK1) plus cyclin B or CDK2 plus cyclin A (New England Biolabs, Ipswich, MA) in the presence of 1 mM ATP for 30 min at 30 °C. After the reaction was terminated with SDS sample buffer, the samples were resolved by 10% SDS-PAGE, transferred to nitrocellulose, and probed with anti-Ser(P)64 Mcl-1 antiserum to detect the phosphorylated species. Equal loading of GST-Mcl-1 was verified by blotting for total Mcl-1.

For JNK phosphorylation studies, BL21 cells were lysed as above and clarified by centrifugation. Glutathione-agarose (100 µl of 50% slurry) was then added to 1 ml of supernatant and mixed end-over-end at 4 °C overnight. The glutathione-agarose bound to GST-Mcl-1 was harvested by centrifugation, washed six times in bind/wash buffer, and rinsed one time with kinase buffer. GST-Mcl-1 still bound to glutathione-agarose was used as a substrate for JNK1{alpha}1 (Cell Signaling Technologies, Beverly, MA) or JNK2{alpha}2 (Calbiochem, La Jolla, CA) under conditions suggested by the respective suppliers. After 30 min at 30 °C, the reaction was stopped by the addition of SDS sample buffer. The samples were boiled and resolved by 12% SDS-PAGE, transferred to nitrocellulose, and probed with anti-Ser(P)64 Mcl-1 antiserum to detect the phosphorylated species. Equal loading of GST-Mcl-1 was verified by blotting for total Mcl-1.

Site-directed Mutagenesis—pcDNA3.1 containing the full-length Mcl-1 cDNA has been previously described (41). A 15-amino acid S peptide tag (KETAAAKFERQHMDS) with GA linker (total 28 amino acids) was inserted at the N terminus of Mcl-1 essentially as described (39). Plasmid encoding the S pep-tide-tagged Mcl-1 was then subjected to site-directed mutagenesis to generate S peptide-tagged T163A, S64A, and S64E mutants using a QuikChange II XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) following the supplier's instructions. To allow expression of the S peptide-tagged variants in cells expressing Mcl-1 shRNA, the wild type and mutant tagged Mcl-1 cDNAs were rendered shRNA-resistant by silent mutation of the third nucleotides in four of the targeted codons to the sequence cgggactggctagttaaac using the same site-directed mutagenesis kit. Finally, for stable transfection, the cDNAs were subcloned into pcDNA 3.1 Hygro(+) (Invitrogen). All of the plasmids were subjected to automated sequencing to verify the described mutation and confirm that no additional mutations were present. These plasmids were prepared for transfection using a plasmid miniprep kit (Bio-Rad).


Figure 2
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  		FIGURE 2.
Identification of Ser64 as a new Mcl-1 phosphorylation site. A, collision-induced fragmentation spectrum of peptide (m/e ratio 1296.2 (doubly charged)) extending from Glu47 to Arg74. The left panel is the sequence of the peptide and the masses of fragments expected from sequential fragmentation at each end. Singly charged fragments that were unequivocally identified in the original spectrum are indicated in red and blue. The putative phosphorylated serine is denoted by S*. Note that the size of fragments containing phosphoserine (e.g. b1-18, b1-19, y1-14, y1-15, y1-18, and y1-19) would be 80 atomic mass units smaller if the peptide were not phosphorylated. B, sequence alignment of predicted Mcl-1 polypeptides in the region corresponding to Ser64. Corresponding sequences of Mcl-1 extending from 61 to 70 for human, dog, and cat and from 63 to 72 for rodents (mouse and rat), respectively, are shown. Note the corresponding serine in red is conserved in human, dog, and cat, whereas the negatively charged glutamate exists in the rodent.

 
Generation of KMCH Stable Transfectants—Transfection was performed using Lipofectamine (Invitrogen). Stably transfected clones were selected in medium containing 1200 mg/liter G418 or both G418 plus 200 mg/liter hygromycin. Individual colonies were isolated using cloning rings, subcloned, and screened by immunoblotting. Established clones were maintained in medium supplemented with 400 mg/liter G418 and/or 200 mg/liter hygromycin.

Mcl-1 Half-life Estimation—KMCH cells stably expressing shRNA to the endogenous Mcl-1 and stably expressing S pep-tide-tagged Mcl-1 constructs (described above) were treated for a total of 4 h with vehicle or TRAIL. During this treatment, 20 µg/ml cycloheximide was added for 0-120 min. At the end of the incubation, floating cells were collected by centrifugation added to adherent cells, and lysed, followed by the addition of SDS sample buffer in preparation for SDS-PAGE and transfer to nitrocellulose. After blots were probed with anti-Mcl-1 and anti-actin antibodies, the signals were detected and quantified using an Odyssey fluorescent imager as described above. Exponential trend lines from three independent experiments were calculated using Microsoft Excel (Microsoft Office, Microsoft, Redmond, WA). Half-life was determined by the formula t1/2 = ln(2)/{lambda}, where {lambda} is the decay constant.

Determination of Mcl-1 Binding Partners—KMCH cells expressing short interfering RNA-resistant S peptide-tagged mutant (S64A or S64E) and short interfering RNA against endogenous Mcl-1 were solubilized in lysis buffer containing 1% CHAPS in place of Triton X-100. Tagged Mcl-1 was pulled down using S protein-agarose. Pelleted proteins were solubilized in SDS sample buffer, clarified by centrifugation, separated by electrophoresis, and probed using antibodies to the indicated Bcl-2 family members.


Figure 3
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  		FIGURE 3.
CDKs and JNK1 mediate Mcl-1 Ser64 phosphorylation. A, dot blot analysis of affinity-purified antiserum obtained from a rabbit immunized with keyhole limpet hemocyanin-conjugated peptide containing phosphorylated Ser64. Dot blot analysis was performed using serial dilutions of the bovine serum albumin-conjugated peptide. P, Ser64 phosphorylated peptide. UP, corresponding unphosphorylated peptide. B, to visualize Ser64 phosphorylation of endogenous Mcl-1, the proteasome inhibitor MG132 (10 µM) was used to stabilize Mcl-1. The cells were lysed in lysis buffer containing 1% CHAPS, and Mcl-1 was immunoprecipitated using a commercial anti-Mcl-1 antibody pulled down with protein G/A beads. Bound proteins were analyzed by immunoblot for total Mcl-1 and with the anti-Ser(P)64 antiserum. Because the immunoprecipitating and immunoblotting primary antibody were both generated in rabbit, there is increased background staining compared with experiments using S-tag enrichment of tagged Mcl-1. C, CDK inhibitors reduce Mcl-1 phosphorylation at Ser64. Wild type and S64A S peptide-tagged Mcl-1 constructs were stably transfected into KMCH cells. The cells were treated with the indicated inhibitors for 4 h. The cell lysates were enriched by S protein-agarose bead affinity purification, and the proteins were resolved by SDS-PAGE and subjected to immunoblot analysis with anti-Ser(P)64 anti-serum. The blot was reprobed with antiserum that recognizes total Mcl-1 as a control. As a negative control, the proteins were also isolated from cells stably transfected with the S peptide-tagged Mcl-1 S64A mutant. The inhibitors are as follows: CDK1/CDK2 inhibitor SU9516 (SU, 10 µM); MEK inhibitors PD98059 (PD, 50 µM) and U0126 (U0, 10 µM); p38 inhibitor SB220025 (SB, 20 µM); JNK inhibitor SP600125 (SP, 20 µM); GSK inhibitor BIO (10 µM); CDK2/CDK5 inhibitor PNU112455A (PNU, 100 µM); and the less selective CDK inhibitors olomoucine (OLO, 100 µM) and roscovitine (ROS, 20 µM). The ratio of the phospho-Mcl-1 (Ser64) signal to total Mcl-1 signal is indicated below the blots. D, in vitro phosphorylation assay of GST-Mcl-1 using CDK1/cyclin B, CDK2/cyclin A, JNK1{alpha}1, or JNK2{alpha}2. Purified GST-Mcl-1 protein and the respective kinases were incubated in buffer containing 1 mM ATP at 30 °C for 30 min. The samples were resolved by SDS-PAGE, and Ser64 phosphorylation was detected by immunoblot analysis as indicated above. Veh, vehicle.

 
Measures of TRAIL-induced Cytotoxicity in KMCH Cells—After treatment with TRAIL as indicated in the various figure legends, apoptosis was quantitated by identifying the characteristic nuclear changes of apoptosis (i.e. chromatin condensation and nuclear fragmentation) using the nuclear binding dye DAPI and fluorescence microscopy. To assess changes in caspase 3/7 activity, cells plated in 96-well plates were assayed using the commercially available Apo-ONE homogenous caspase 3/7 assay (Promega, Madison, WI), which measures cleavage of profluorescent rhodamine 110-conjugated bis-(N-benzyloxycarbonyl-L-aspartyl-L-glutamyl-L-valyl-aspartic acid amide) to liberate rhodamine 110, according to the supplier's instructions using excitation and emission wavelengths of 498 and 521 nm, respectively.

Cell Synchronization and Cell Cycle Analysis—KMCH cells stably transfected with S peptide-tagged Mcl-1 were arrested in mitosis by treatment with nocodazole (40 ng/ml) and at the G1/S boundary by treatment with aphidicolin (10 µM) for 16 h. Confirmation of the cell cycle effects of the drugs was obtained by flow cytometric analysis. Briefly, after treatment, trypsinized cells were collected, washed with PBS twice, fixed with 50% ethanol, rinsed with PBS, treated with RNase A (100 µg/ml) at 37 °C for 10 min, and stained with propidium iodide (100 µg/ml) for 5 min. Cell cycle distribution was assessed using a FACSan (Becton Dickinson, Mountain View, CA). The Modfit LT for Mac version 3.0 (Verity Software House, Topsham, ME) was used to estimate the percentage of cells in various phases of the cell cycle.

Statistical Analysis—Statistical analysis was performed using a Student's t test from at least three independent experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Mcl-1 Is Phosphorylated at Ser64—Previous studies have suggested that phosphorylation of Mcl-1 on Thr163 and Ser159 can influence the half-life and, therefore, the expression level and antiapoptotic effects of this polypeptide. To assay for Mcl-1 phosphorylation at additional sites, we initially compared the metabolic labeling of Mcl-1 by [32P]phosphate in cells expressing S peptide-tagged versions of wild type or T163A Mcl-1. Because phosphorylation of Thr163 is required for Ser159 phosphorylation, neither Thr163 nor Ser159 can be phosphorylated in the T163A mutant (8), effectively allowing us to abrogate phosphorylation at both sites with one mutation. When the tagged polypeptides were recovered from cells by affinity purification on S protein-agarose (39), resolved by SDS-PAGE, and subjected to autoradiography as well as immunoblotting (Fig. 1), Thr(P)163-Mcl-1 epitope-specific antiserum specifically labeled wild type Mcl-1 but not the T163A mutant, whereas 32P labeled both species. These results suggested that Mcl-1 is phosphorylated on one or more residues in addition to Thr163 and Ser159.

To further identify these phosphorylation sites, microgram quantities of unlabeled S peptide-tagged Mcl-1 163A were affinity-purified from exponentially growing cells, subjected to SDS-polyacrylamide gel electrophoresis, and excised from the gel. Trypsin digestion followed by quadruple time-of-flight tandem mass spectrometry identified a previously unknown phosphorylation site Ser64 (Fig. 2A). Further analysis demonstrated that Ser64, which is within a "poor" PEST sequence upstream from the PEST sequence that contains Thr163 and Ser159, is conserved through a wide range of species (Fig. 2B) including dog and cat. Interestingly, the rodent Mcl-1 (both mouse and rat) contains an acidic glutamic acid at this residue, which could function as a constitutive phosphomimetic. In addition, Ser64 is a serine-proline site that conforms to several consensus kinase motifs (scansite.mit.edu/motifscan_seq.phtml and www.cbs.dtu.dk/services/NetPhos/). Given this information, we focused our efforts on the potential role of Ser64 phosphorylation in Mcl-1 regulation.


Figure 4
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  		FIGURE 4.
Mcl-1 phosphorylation at Ser64 is cell cycle-dependent. KMCH cells stably transfected with wild type S peptide-tagged Mcl-1 were treated with either 50 ng/ml nocodazole (NOC) or 10 µM aphidicolin (APH) for 16 h. Control cells were left untreated. After staining with propidium iodide, the cells were examined by flow cytometry, and the percentages of cells in different phases of the cell cycle were determined (A and B). C, blots were probed for Ser(P)64 Mcl-1, and total Mcl-1 after S peptide-tagged Mcl-1 was affinity-purified from control, nocodazole-treated, or aphidicolin-treated cells.

 
CDK- and JNK1-induced Ser64 Phosphorylation in Vitro—To facilitate further analysis, we generated and characterized a phosphoepitope-specific antiserum against the peptide surrounding Ser64. As illustrated in Fig. 3A, dot blot analysis demonstrated that affinity-purified aliquots of this serum recognized the phosphorylated Ser64 peptide against which it was raised much more avidly than the corresponding unphosphorylated polypeptide. This serum was then utilized to determine whether Ser64 is phosphorylated in situ and to assess the effects of various kinase inhibitors.

Initial experiments with this serum examined whether endogenous Mcl-1 was phosphorylated at Ser64 under basal conditions and in the presence of the proteasome inhibitor MG132, which increases cellular levels of Mcl-1 (Fig. 3B). Based on computer analysis suggesting that Mcl-1 might be phosphorylated on Ser64 by GSK3beta, ERK, p38 MAPK, JNK, CDK5, and/or CDK1, cells were treated with inhibitors of these kinases and harvested for immunoblotting. To avoid an effect of these inhibitors on Mcl-1 transcription, the studies were performed in KMCH stably transfected with Mcl-1 shRNA and shRNA-resistant S peptide-tagged Mcl-1, which was under the control of a cytomegalovirus promoter and therefore not subject to the usual transcriptional regulation of the Mcl-1 promoter. Analysis of the immunoblots demonstrated a >20-fold stronger signal with the anti-Ser(P)64 antiserum when cells were expressing wild type Mcl-1 as compared with S64A Mcl-1 (Fig. 3C), confirming the specificity of the serum in the setting of whole cell lysates. Moreover, this analysis revealed that exposure to the CDK inhibitor SU9516 (which is known to inhibit CDKs 1, 2, and 5) substantially diminished Ser64 phosphorylation (Fig. 3C). A reduction in phosphorylation was also observed with the JNK inhibitor (SP600125). In contrast, inhibitors of MEK1/2 (PD98059 and U0126), p38 MAPK (SB220025), and GSK3beta (BIO), did not inhibit phosphorylation. PNU112455A, which inhibits predominantly CDK5, like-wise did not diminish Ser64 phosphorylation. To confirm the effects of the CDK inhibitor SU9516 on Ser64 phosphorylation, phosphoimmunoblot analysis was also performed in cells treated with the less selective CDK inhibitors olomoucine and roscovitine. Treatment with these agents also reduced Ser64 phosphorylation, with roscovitine being more potent than olomoucine, consistent with their known affinities for CDKs (42).


Figure 5
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  		FIGURE 5.
Mcl-1 half-life is unaltered by S64A or S64E mutation. KMCH cells stably transfected with a Mcl-1 shRNA were further stably transfected with S peptide-tagged wild type (WT), S64A, and S64E Mcl-1 plasmids. The cells were then pretreated with TRAIL or vehicle (Veh, medium) for 4 h total. At selected time points, cycloheximide (CHX, 20 µg/ml) was added to block protein synthesis, as indicated. Whole cell lysates (30 µg of total protein) were prepared for immunoblot analysis using Mcl-1 antisera. Equal protein loading was verified by immunoblot for actin. A, representative immunoblot analysis. Low level expression of the endogenous Mcl-1 is detected as a band running slightly faster than the S peptide-tagged transgene. B, exponential trend lines were generated from signal intensity measurements of three individual experiments. Mcl-1:actin ratios were calculated from these data and normalized to time 0.

 
To more directly determine whether Ser64 can be phosphorylated by a CDK, an in vitro assay was performed by incubating GST-Mcl-1 with CDK1/cyclin B or CDK2/cyclin A (Fig. 3D). As assessed by Ser64 phospho-immunoblot analysis, both CDK1 and CDK2 directly phosphorylated Mcl-1 at Ser64. In addition, JNK1 but not JNK2 phosphorylated GST-Mcl-1 under the conditions employed in vitro (Fig. 3D). Taken together, these data suggest that CDKs and JNK1 have a role, either direct or indirect, in Mcl-1 phosphorylation at Ser64.

Cell Cycle-dependent Ser64 Phosphorylation in Intact Cells—The observations using CDK inhibitors and recombinant CDK enzymes suggested that Ser64 might be phosphorylated in a cell cycle-related manner (43-45). To examine this possibility, the cells were synchronized at the G1/S boundary by aphidicolin or in G2/M by nocodazole prior to immunoblot analysis for Ser64 phosphorylation (Fig. 4, A and B). The results showed that Ser64 was phosphorylated to a greater extent in G2/M phase than at the G1/S boundary (Fig. 4C). This was consistent with the previous observation of a distinct phosphorylation stimulated in G2/M phase (29) and with the above findings concerning a role for CDKs. Thus, Mcl-1 is likely phosphorylated at Ser64 in a CDK/cell cycle-dependent manner.

Ser64 Phosphorylation Fails to Alter Mcl-1 Half-life—Because phosphorylation at other sites can regulate Mcl-1 half-life (see the Introduction), we next assessed the effect of Ser64 phosphorylation on Mcl-1 half-life. KMCH cells stably transfected with S peptide-tagged wild type Mcl-1, S peptide-tagged Mcl-1 S64E, or S peptide-tagged Mcl-1 S64A were employed for these studies. When protein synthesis was inhibited with cycloheximide and S peptide-tagged Mcl-1 levels were assessed in whole cell lysates by immunoblotting, neither the half-life of S peptide-tagged S64A nor S64E Mcl-1 was significantly different from that of the wild type protein (Fig. 5). The half-lives of wild type and mutant Mcl-1 proteins were also not significantly different from each other during treatment with the death ligand TRAIL. Thus, mutation of Ser64 to a non-phosphorylatable or a phosphomimetic amino acid does not alter Mcl-1 half-life despite its location within the PEST region.

Ability to Phosphorylate Ser64 Is Required for Mcl-1-mediated Protection from TRAIL-mediated Apoptosis—Mcl-1 inhibits apoptosis by binding to BH3-only proteins and/or Bak (2-5). Therefore, we initially examined binding of S peptide-tagged S64A and S64E Mcl-1 to various Bcl-2 proteins (Fig. 6); wild type Mcl-1 was not employed for these studies because of its variable phosphorylation status (Fig. 3). No binding by either mutant to Bcl-2, Bax, or Bid was observed (not shown). The phosphomimetic S64E mutant preferentially bound to Noxa, BimEL, and perhaps Bak (Fig. 6), suggesting that Ser64 phosphorylation might enhance the antiapoptotic function of Mcl-1 by altering its binding partners.

Next, we examined the effect of Ser64 phosphorylation on the antiapoptotic function of Mcl-1. We chose to induce apoptosis using the death ligand TRAIL because Mcl-1 contributes to TRAIL resistance in many cell types, including the human cholangiocarcinoma cell line used for this study (20, 46-48). KMCH cholangiocarcinoma cells that were stably transfected with shRNA to diminish endogenous Mcl-1 and render them TRAIL-sensitive (20) were subsequently transfected with shRNA-resistant cDNAs encoding S64A, S64E, T163A, or wild type Mcl-1. The resulting stable clones used for this analysis expressed similar levels of wild type or mutant S peptide-tagged Mcl-1 (Fig. 7A, top panel). These clones also expressed similar levels of other apoptotic mediators, including Bcl-2, Bcl-xL, Bid, Bim, Bax, Bak, death receptors 4 and 5, cFLIP, and caspase 8 (Fig. 7A). As previously reported, KMCH sh-Mcl-1 cells were quite sensitive to TRAIL-mediated apoptosis (Fig. 7, B and C). Although transfection with wild type or T163A Mcl-1 made these cells resistant to TRAIL-mediated apoptosis, transfection with the S64A mutant did not (Fig. 7, B and C). In contrast, stable transfection with the phosphomimetic S64E mutant conveyed TRAIL resistance (Fig. 7, B and C).


Figure 6
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  		FIGURE 6.
Comparative binding of Mcl-1 S64A and Mcl-1 S64E to proapoptotic Bcl-2 family members. After KMCH-shMcl-1 cells that stably express S peptide-tagged Mcl-1 (S64A and S64E) were lysed, S peptide-tagged Mcl-1 was enriched by binding to S protein-agarose. Mcl-1 binding partners in these pull-downs were then analyzed by immunoblot analysis. Immunoblot for total Mcl-1 shows efficient pull-down from both cell lines. No signal was detected for Bid, Bax, or Bcl-2 (not shown). Western blot signals were quantified and the ratio of the signal from S64E pull-down to S64A pull-down is presented in the column to the right of the blots.

 
Because the wild type Mcl-1 was as effective in preventing apoptosis as the S64E mutant, we surmised that either wild type Mcl-1 (Ser64) was constitutively phosphorylated or phosphorylation was induced during TRAIL treatment. Indeed, treatment with TRAIL was associated with enhanced phosphorylation of wild type Mcl-1 at Ser64 (Fig. 7D). Basal Ser64 phosphorylation of Mcl-1 was again decreased with both a JNK inhibitor (SP600125) and a CDK inhibitor (SU9156). Phosphorylation of Mcl-1 at Ser64 in TRAIL-treated cells was also decreased by both inhibitors with the least Ser(P)64-Mcl-1 detected in SU9156-treated cells in the presence of TRAIL (Fig. 7E).

Pharmacological Inhibition of Mcl-1 Ser64 Phosphorylation Enhances TRAIL Sensitivity—Collectively, the results in Fig. 7 indicate that inability to phosphorylate Ser64 diminishes the antiapoptotic function of Mcl-1. As an extension of these observations, we predicted that pharmacological inhibition of Ser64 phosphorylation would also diminish the antiapoptotic function of Mcl-1. To test this possibility, we examined the effect of combining SU9516, which we already demonstrated inhibits Mcl-1 Ser64 phosphorylation (Figs. 3 and 7), with TRAIL. For these experiments, parental KMCH cells were treated for 4 h with 10 µM SU9516, a time and concentration that does not diminish total cellular Mcl-1 levels (Fig. 8A). As indicated using two different assays (Fig. 8), treatment with SU9516 sensitized the cells to TRAIL-induced apoptosis. In contrast, the ERK pathway inhibitor PD98059 and the CDK5 inhibitor PNU112455A, which do not affect Ser64 phosphorylation, did not affect TRAIL sensitivity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the present study demonstrate that Mcl-1 can be phosphorylated on Ser64 and establish that the inability to phosphorylate this site diminishes the antiapoptotic function of Mcl-1 without altering the half-life of the protein. Further analysis is consistent with the interpretation that CDK1 and 2, and JNK1 can directly phosphorylate this site in vitro. Moreover, pharmacological CDK inhibitors and a JNK inhibitor reduce Mcl-1 Ser64 phosphorylation. The most potent CDK inhibitor was also found to enhance the cytotoxicity of the antineoplastic protein TRAIL in cells in which Mcl-1 plays a major role in resistance. These observations have potentially important implications for current understanding of Mcl-1 regulation and function.

Three previous Mcl-1 phosphorylation sites have been identified. Phosphorylation of Thr163 by p42/44 MAPK is thought to enhance the antiapoptotic function of Mcl-1 by prolonging the half-life of the polypeptide (6). However, Thr163 phosphorylation also renders Mcl-1 a substrate for GSK-3beta-mediated phosphorylation of Ser159 (8), and this dual phosphorylation then facilitates Mcl-1 ubiquitylation, presumably by MULE and/or other E3 ligases, which accelerates Mcl-1 degradation by the proteasome (11). JNK-mediated Ser121 phosphorylation reduces the antiapoptotic function of Mcl-1 against cell death by oxidative stress (36).

To identify additional phosphorylation sites, we performed tandem mass spectrometry on tryptic digests of S peptide-tagged Mcl-1 T163A, a mutant that cannot be phosphorylated on either Thr163 or Ser159. This analysis revealed an additional phosphorylation site at Ser64, which is within the upstream regulatory half of the Mcl-1 protein. Our subsequent data suggest that Ser64 is phosphorylated in a manner that can be regulated by the cell cycle. Although Ser64 was within a potential consensus recognition motif for several kinases, phosphorylation at this site in situ was inhibited by the CDK inhibitors SU9516, olomoucine, and roscovitine but not by the GSK3beta inhibitor BIO or the MEK inhibitors PD098059 or U0126. Both CDK1 and CDK2 were capable of directly phosphorylating Mcl-1 at Ser64 under cell-free conditions. Phosphorylation at this site was greater in cells in G2/M phase as compared with G1/S phase of the cell cycle, which is also consistent with a role for CDK1 and/or CDK2. We recognize that kinase inhibitors may potentially have nonspecific effects, therefore the identification of CDK1 and 2 as Mcl-1 Ser64-phosphorylating enzymes is correlative but consistent with the observed cell cycle-dependent phosphorylation.

Because Ser64 is adjacent to Pro65, this site might also be a substrate for proline-directed kinases, such as JNK. Indeed, JNK1 phosphorylates Ser64 in vitro (Fig. 3D), and the JNK path-way inhibitor SP600125 decreased basal Ser64 phosphorylation in culture (Fig. 3C). Moreover, TRAIL treatment, which is known to activate JNK in some model systems, induces Ser64 phosphorylation (Fig. 7D).


Figure 7
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  		FIGURE 7.
Ability to phosphorylate Ser64 is required for Mcl-1-mediated protection from TRAIL-induced apoptosis. KMCH cells were stably transfected with a Mcl-1 shRNA to knock down endogenous Mcl-1. These cells were further stably transfected with shRNA-resistant (sh) S peptide-tagged wild type (WT), S64A, S64E, and T163A Mcl-1 plasmids. A, whole cell lysates (30 µg of protein) were resolved by SDS-PAGE and subjected to immunoblot analysis for the indicated polypeptides. Equal protein loading was verified by blotting for actin. Note that the relative levels of Bcl-2, Bcl-xL, Bid, Bim, Bax, Bak, DR4, DR5, caspase 8, and cFLIP were similar among the clones. B and C, the cell lines described above were incubated in the presence or absence of TRAIL (5 ng/ml) for 4 h. Apoptosis was evaluated by DAPI staining and fluorescence microscopy (B). Caspase 3/7 activity was measured as described under "Experimental Procedures." (C). D, the effect of TRAIL treatment (5 ng/ml) on Mcl-1 phosphorylation was determined after 0-6 h of treatment. After enrichment on S protein-agarose beads, the proteins were probed by immunoblot for total Mcl-1 and Ser(P)64-Mcl-1. E, cells overexpressing S peptide-tagged WT Mcl-1 were pretreated for 1 h with SP600125 (SP, 20 µM) or SU9516 (SU, 10 µM) followed by TRAIL treatment (5 ng/ml) for 4 h. Enrichment on S protein-agarose was followed by SDS-PAGE and immunoblot for Ser(P)64-Mcl-1 or total Mcl-1. Ratios of the band intensity of phosphorylated over total Mcl-1 are indicated. Apoptosis and caspase 3/7 activity data are expressed as averages ± standard error from four individual experiments. *, p < 0.001.

 
Depending on the site modified, Mcl-1 phosphorylation has previously been shown to either prolong or shorten the half-life of Mcl-1 (6, 8). However, the half-life of the nonphosphorylatable Mcl-1 S64A mutant was virtually identical to that of wild type Mcl-1 in a human cholangiocarcinoma cell line (Fig. 5). Although Ser64 is within the upstream regulatory portion of Mcl-1 (amino acids 1-176 of human Mcl-1), it lies in a sequence categorized as a "poor" PEST sequence (by PESTFIND), whereas Thr163 and Ser159 are in a region containing strong potential PEST sequences (residues 104-176) (6). This might explain why phosphorylation of Ser64 fails to affect the half-life of Mcl-1. Consistent with these observations, immunoblot analysis of affinity-purified Mcl-1 also did not detect any difference in the amount of ubiquitinylation for wild type Mcl-1 versus the S64A mutant (data not shown).

S64E Mcl-1, which binds increased amounts of Noxa (Fig. 6), also has the same half-life as wild type and S64A Mcl-1 (Fig. 5B). Noxa has previously been reported to bind Mcl-1 (4, 49) and target it for degradation in certain model systems (2). The unaltered half-life of S64E Mcl-1 suggests that the effect of Noxa on Mcl-1 degradation might be cell type- and context-specific.


Figure 8
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  		FIGURE 8.
Pharmacological inhibition of Mcl-1 Ser64 phosphorylation enhances TRAIL sensitivity. KMCH cells were incubated in the presence or absence of SU9516 (SU, 10 µM), PD98059 (PD, 50 µM), and PNU112455A (PNU, 100 µM) or vehicle (Veh) with or without TRAIL (1 ng/ml) for 8 h. Apoptosis was evaluated by DAPI staining and fluorescence microscopy (A) and quantitation of caspase 3/7 activity (B). Caspase 3/7 activity was measured as described under "Experimental Procedures. " Note that SU9516, which sensitized KMCH cells to TRAIL, did not alter total Mcl-1 polypeptide levels (inset). All of the data were expressed as averages ± standard error from four individual experiments. *, p < 0.001 versus vehicle.

 
We and others have previously reported that Mcl-1 plays a pivotal role in resistance to TRAIL-mediated apoptosis in certain cell types (7, 20, 48). In the cholangiocarcinoma cells utilized in the present study, depletion of Mcl-1 by shRNA enhances TRAIL sensitivity, and expression of shRNA-resistant wild type Mcl-1 restores TRAIL resistance. Importantly, the phosphomimetic S64E mutant also confers TRAIL resistance, whereas the nonphosphorylatable S64A Mcl-1 mutant does not (Fig. 7). The observation that TRAIL induces phosphorylation of Ser64 (Fig. 7D) likely helps to explain why wild type and S64E Mcl-1 give rise to similar pro-survival activity during exposure to TRAIL. Collectively, these data suggest that Ser64 phosphorylation enhances the antiapoptotic activity of Mcl-1.

Mcl-1 is thought to inhibit apoptosis by sequestering the proapoptotic BH3-only proteins and/or binding the proapoptotic multidomain Bcl-2 protein Bak (5). Consistent with these current concepts, the phosphomimetic S64E Mcl-1 showed enhanced binding to the BH3-only proteins Noxa and Bim as well as increased binding to Bak compared with the S64A mutant protein (Fig. 6). This increased binding occurred without any apparent alteration in the mitochondrial localization of Mcl-1 (data not shown). Although Mcl-1 binding to Bim and Bak and their roles in TRAIL-mediated apoptosis are well established (48, 50), a role for Noxa in TRAIL-mediated apoptosis has not been reported. The current data suggest that S64E Mcl-1 may protect against cell death, in part, by sequestering Noxa. Based on these results, further experiments to examine the role of Noxa and other BH3-only polypeptides in TRAIL-induced apoptosis appear to be warranted.

Mcl-1, in our system, bound to the pro-apoptotic protein Bak, but not to Bax. Mcl-1 has been reported to bind to Bax using the yeast two-hybrid method (51), but in mammalian cells, the results have been controversial. Several authors failed to observe binding (2, 52, 53) consistent with our results, whereas others have reported an interaction (49, 51); one group described an interaction between recombinant Mcl-1 and in vitro translated Bax in a cell-free system but found that Mcl-1 and Bax did not interact in cells (54). The reported difference likely reflects variations between cell types, cell context, and the apoptotic stimulus.

Comparison of Mcl-1 sequences from different species (Fig. 2B) indicates that Ser64 is conserved among humans, cat, and dogs. In mice and rats, on the other hand, this region of the sequence contains two acidic residues, glutamate and aspartate (Fig. 2B), creating an ortholog that could conceivably act like the S64E version of the human polypeptide. Although it is possible that a different serine or threonine in the mouse/rat Mcl-1 might serve the same regulatory function, it is also possible that control of Mcl-1 function by Ser64 phosphorylation is a relatively recent evolutionary addition to the regulation of apoptosis.

Based on the observation that the nonphosphorylatable S64A mutant fails to protect cells as effectively as wild type Mcl-1 (Fig. 7, B and C), we hypothesized that pharmacological inhibition of Mcl-1 phosphorylation might augment the action of apoptotic stimuli that are modulated by Mcl-1. As a preliminary test of this concept, we examined the effect of combining SU9516, which inhibits Mcl-1 phosphorylation at Ser64 (Fig. 3B), with TRAIL, which is strongly affected by Mcl-1 expression in KMCH cells (Fig. 7). As predicted by the analysis of Mcl-1 mutants, SU9516 enhanced TRAIL-mediated apoptosis under conditions where Mcl-1 phosphorylation was diminished but Mcl-1 polypeptide levels were not (Fig. 8). Although the enhancement of TRAIL cytotoxicity by SU9516 might be greater than expected from the proportion of cells in the cell cycle phases where Mcl-1 is maximally phosphorylated by CDKs, perturbations of the cell cycle, as occurs with CDK inhibitors, have previously been shown to sensitize cells to TRAIL, likely accounting for this observation (55). However, because we cannot rule out the possibility that SU9516 is enhancing the cytotoxicity of TRAIL by affecting phosphorylation of some other substrate in place of (or in addition to) Mcl-1, further study with additional inhibitors and additional model systems will be required to establish whether inhibition of Ser64 Mcl-1 phosphorylation is truly an effective strategy for sensitizing tumors that overexpress this apoptotic regulator. Nonetheless, these preliminary observations raise the possibility that inhibition of Mcl-1 phosphorylation using kinase inhibitors might be a way of enhancing TRAIL sensitivity in cancers characterized by Mcl-1 overexpression.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants DK 59427 (to G. J. G.), CA 69008 (to S. H. K.), and CA 57359 (to R. W. C.) and funds from the Mayo and Palumbo Foundations (to G. J. G.) and the Japanese Society for Science Promotion (to S. K.). 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: Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, 200 First St. SW, Rochester MN, 55905. Tel.: 507-284-0686; Fax: 507-284-0762; E-mail: gores.gregory{at}mayo.edu.

2 The abbreviations used are: PEST, proline-, glutamic acid-, serine-, and threonine-rich; CDK, cyclin-dependent kinase; DAPI, 4',6-diamidino-2-phenylindole dihydrochloride; ERK, extracellular signal-regulated kinase; GSK3, glycogen-synthase kinase 3; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; PBS, Dulbecco's calcium- and magnesium-free phosphate-buffered saline; shRNA, short hairpin RNA; TRAIL, tumor necrosis factor-{alpha}-related apoptosis-inducing ligand; STAT, signal transducers and activators of transcription; E3, ubiquitin-protein isopeptide ligase; GST, glutathione S-transferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MEK, MAPK/ERK kinase. Back



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 ABSTRACT
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 RESULTS
 DISCUSSION
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