Mechanistic Analysis of a DNA Damage-Induced, PTEN ...waldmanlab.us/download/MCB 2011.pdfpeptide spectrum matches (PSMs) was facilitated using SEQUEST with a 30- ppm mass tolerance

MOLECULAR AND CELLULAR BIOLOGY, July 2011, p. 2756–2771 Vol. 31, No. 130270-7306/11/$12.00 doi:10.1128/MCB.01323-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Mechanistic Analysis of a DNA Damage-Induced, PTEN-DependentSize Checkpoint in Human Cells�

Jung-Sik Kim,1 Xuehua Xu,3 Huifang Li,1 David Solomon,1,2

William S. Lane,4 Tian Jin,3 and Todd Waldman1*Department of Oncology1 and Tumor Biology Training Program,2 Lombardi Comprehensive Cancer Center, Georgetown University

School of Medicine, Washington, DC 20057; Chemotaxis Signal Section, Laboratory of Immunogenetics, National Institute ofAllergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland 208523; and Mass Spectrometry and

Proteomics Resource Laboratory, FAS Center for Systems Biology, Harvard University,Cambridge, Massachusetts 021384

Received 19 November 2010/Returned for modification 29 December 2010/Accepted 20 April 2011

Following DNA damage, human cells undergo arrests in the G1 and G2 phases of the cell cycle and asimultaneous arrest in cell size. We previously demonstrated that the cell size arrest can be uncoupledfrom the cell cycle arrest by mutational inactivation of the PTEN tumor suppressor gene. Here we showthat the cell size checkpoint is inducible by DNA-damaging chemotherapeutic agents as well as by ionizingradiation and is effectively regulated by PTEN but not by its oncogenic counterpart, PIK3CA. Mutationalanalysis of PTEN and pharmacological inhibition of Akt revealed that modulation of Akt phosphorylationis unnecessary for cell size checkpoint control. To discover putative PTEN regulators and/or effectorsinvolved in size checkpoint control, we employed a novel endogenous epitope tagging (EET) approach,which revealed that endogenous PTEN interacts at the membrane with an actin-remodeling complex thatincludes actin, gelsolin, and EPLIN. Pharmacological inhibition of actin remodeling in PTEN�/� cellsrecapitulated the lack of size checkpoint control seen in PTEN�/� cells. Taken together, these resultsprovide further support for the existence of a DNA damage-inducible size checkpoint that is regulated bya major tumor suppressor, and they provide a novel Akt-independent mechanism by which PTEN controlscell size.

A major focus of modern cancer research has been to de-termine the role of tumor suppressor gene pathways in theregulation of cell cycle arrest. The molecular mechanisms thatenforce these cell cycle arrests are termed checkpoints and areenforced by several of the most commonly mutated tumorsuppressors, including p53 and p16INK4a. The study of check-point-dependent cell cycle arrest has focused primarily on theG1/S and G2/M cell cycle transitions. However, these arrestsare almost invariably accompanied by a third, simultaneousarrest—an arrest in cell size.

The relationship between cell size arrest and the more con-ventional cell cycle arrests has not been investigated thor-oughly, despite the fact that cancer cells are often aberrantlyregulated in size. This phenotype is manifested in several clin-ical presentations, such as the formation of giant cells in manytumor types and the presence of unusually enlarged cells intumor types such as hamartomas. Therefore, determination ofthe genetic and biochemical mechanisms that enforce cell sizecheckpoints is of fundamental importance in cancer biology.

Of the known tumor suppressor genes, the PTEN gene hasbeen the most convincingly implicated in the control of mam-malian cell size. Inherited mutations of PTEN cause a variety

of related cancer predisposition syndromes collectively re-ferred to as PTEN hamartoma syndrome (23), in which tumorsare composed of enlarged cells. In Drosophila melanogaster,PTEN-deficient cells in the eye and wing are enlarged (16, 18,24, 46). Additionally, cells and organs from conditional PTENknockout (KO) mice are often oversized (2, 7, 14, 32). Forexample, tissue-specific deletion of PTEN in the mouse brainresults in the formation of enlarged cells, leading to macro-cephaly (2).

Human cells with targeted deletion of PTEN also have anotable size phenotype (34). After treatment with gamma ir-radiation, PTEN�/� cells arrest in the G1 and G2 phases of thecell cycle and simultaneously stop increasing in size. In con-trast, otherwise isogenic PTEN�/� cells also undergo cell cyclearrest but do not arrest their cell size. As such, PTEN�/� cellsarrested in either the G1 or G2 phases of the cell cycle contin-uously enlarge, ultimately reaching �20 times the size of theirPTEN-proficient counterparts before detachment and death.Based on these data, we have proposed that PTEN controls adistinct radiation-induced cell size checkpoint that can be un-coupled from the radiation-induced G1 and G2 cell cycle ar-rests.

The mechanistic basis for the role of PTEN in cell sizecontrol remains mostly obscure. In mice, the large-cell pheno-type is dependent on PDK1 and mTOR and independent ofS6K (4, 5, 31). As most PTEN phenotypes are thought to occurvia regulation of Akt activation, the effects of PTEN on cellsize control are assumed to be dependent on this pathway aswell. This assumption is based, in part, on the fact that the Akt

* Corresponding author. Mailing address: Lombardi Comprehen-sive Cancer Center, Georgetown University School of Medicine, 3970Reservoir Road, N.W., NRB E304, Washington, DC 20057. Phone:(202) 687-1340. Fax: (202) 687-7505. E-mail: [email protected].

� Published ahead of print on 2 May 2011.

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kinase mTOR plays a known role in cell size regulation (21).However, whether Akt is an important effector of the PTENcell size phenotype in mammalian cells has not been directlytested, due in part to technical difficulties in genetically inhib-iting all three Akt isoforms simultaneously.

Examination of the cell size phenotypes of PTEN deficiencyand the underlying molecular basis has substantial implicationsfor understanding cell and cancer biology. Control of cell sizehas been almost entirely ignored from a mechanistic perspec-tive, yet cell size is arguably one of the most obvious andimportant phenotypes in all of mammalian biology. Finally,although generally overlooked, an arrest in cell size is a criticalcomponent of cell cycle arrest. As most current anticanceragents function, at least in part, by inducing checkpoint-depen-dent cell cycle arrest, understanding the molecular basis of theaccompanying cell size arrest will likely have implications forfurthering our understanding of the molecular basis of cancertherapy. Here we describe investigations of the PTEN-depen-dent cell size checkpoint in human cells.

MATERIALS AND METHODS

Tissue culture. HCT116 PTEN�/�, PTEN�/�, PIK3CAWT/KO, andPIK3CAKO/mut cells were created using human somatic cell gene targeting andwere described previously (28, 34). HCT116FLAG-PTEN/FLAG-PTEN cells werecreated by endogenous epitope tagging (EET) and described in a previous study(27). The glioblastoma multiforme (GBM) cell lines U87MG and SNB19 wereobtained from ATCC and cultured as recommended.

Antibodies. Primary antibodies were obtained from Cascade Bioscience(PTEN clone 6H2.1), Calbiochem (p53 clone DO-1), Cell Signaling [p-Akt S473,p-Akt T308 clone C31E5E, Akt, p-FoxO1(T24)/3a(T32), FoxO1 clone C29H4,FoxO3a clone 75D8, p-4E-BP1 clone 236B4, p-mTOR S2448, mTOR clone7C10, p-TSC2 T1462, TSC2 clone D93F12, p-p70S6K S371, p-p70S6K T389,PARP clone 19F4], Santa Cruz Biotechnology (�-actin clone 1A4, �-actin cloneC4, �-actin clone 1-24, actin clone I-19, gelsolin clone C20, PTEN N19), ZymedLaboratories (E-cadherin clone HECD-1), Bethyl Laboratories (EPLIN A300),Neomarkers (�-tubulin clone DM1A), and Sigma (FLAG).

Flow cytometry. Cells were fixed in 70% ethanol and stained in phosphate-buffered saline (PBS) containing 0.1% Triton X-100, 50 �g/ml RNase, and 50�g/ml propidium iodide. DNA content was measured on a FACSort flow cytom-eter (Becton Dickinson), and data were analyzed using ModFit software (VeritySoftware House). At least 20,000 cells were analyzed for each sample.

Irradiation. Subconfluent cell monolayers were irradiated using a J. L.Shepard Mark I 137Cs irradiator at �2 Gy/min.

Cell sizing. Cells were trypsinized in 0.5 ml, added to 0.5 ml of serum-containing medium, and further diluted in 10 ml of Isoton II. Cell diameters weredetermined using a Multisizer III Coulter Counter (Beckman Coulter). At least10,000 cells were counted for each measurement.

Immunoblotting and immunoprecipitation. Protein lysates for direct Westernblotting were prepared in radioimmunoprecipitation (RIPA) buffer. Nuclear andcytoplasmic lysates used for FLAG purification were prepared using a modifi-cation of Dignam’s nondetergent lysis method, described in reference 27 andreferences therein. Protein concentrations were determined using the bicin-choninic assay (Pierce).

For FLAG affinity purification, �-FLAG M2 beads (Sigma) were washed oncewith Tris-buffered saline (TBS) (150 mM NaCl) and then incubated with resus-pended protein lysates derived from parental or FLAG epitope-tagged cells.Samples were incubated with rotation at 4°C for 1 h. Beads were then washedthree times in TBS and packed into a Poly-Prep chromatography column (Bio-Rad). Bound proteins were eluted with 100 ng/�l 1 FLAG peptide (Sigma).Fractions were concentrated by trichloroacetic acid (TCA) precipitation, resus-pended in sample buffer, and separated by SDS-PAGE. Subcellular fractionswere prepared using a ProteoExtract native membrane protein extraction kit(Calbiochem).

PTEN protein complex purification and mass spectrometry. PTEN immuno-precipitation was performed on protein lysates derived from HCT116 parentalcells and their FLAG-PTEN-modified derivatives. Equivalent amounts of totalprotein were immunoprecipitated from both cell lines using FLAG-M2 beads,and bound proteins were eluted via competition with 1 FLAG peptide. Eluted

proteins were then concentrated by TCA precipitation, separated by SDS-PAGE, and stained with GelCode blue stain reagent (Thermo Scientific). Afterdestaining, the two gel lanes were divided into seven sections, reduced, carboxy-amidomethylated, and digested with trypsin in gel. To identify proteins specifi-cally present in immunoprecipitates from FLAG-PTEN-modified cells, theresulting peptides from each section were subjected to microcapillary reverse-phase high-pressure liquid chromatography (HPLC) directly coupled to thenanoelectrospray ionization source of a ThermoFisher LTQ-Orbitrap XL Veloshybrid mass spectrometer (liquid chromatography-tandem mass spectrometry[LC/MS-MS]). The Orbitrap repetitively surveyed an m/z range from 395 to1,600, while data-dependent MS-MS spectra on the 10 most abundant ions ineach survey scan were acquired in the linear ion trap. MS-MS spectra wereacquired with relative collision energy of 30% and a 2.5-Da isolation width, andrecurring ions were dynamically excluded for 60 s. Preliminary evaluation ofpeptide spectrum matches (PSMs) was facilitated using SEQUEST with a 30-ppm mass tolerance against the human subset of the Uniprot Knowledgebase.With a custom version of the Harvard Proteomics Browser Suite (Thermo Sci-entific), PSMs were accepted with a mass error of 3.0 ppm and score thresholdsto attain an estimated false discovery rate of �1% using a reverse decoy databasestrategy.

Site-directed mutagenesis. Site-directed mutagenesis was performed using theQuikchange Kit (Stratagene) using PAGE-purified oligonucleotides (IntegratedDNA Technologies) to introduce the indicated mutations.

Lentiviruses. The pHR-SIN-PTEN was a gift from Nick Leslie. Constructs forstable depletion of gelsolin and EPLIN were obtained from Open Biosystems. Anegative-control construct in the same vector system (pLKO.1) was obtainedfrom Addgene. The lentiviral helper plasmids pHR�CMV8.2�R and pCMV-VSV-G were also obtained from Addgene. All plasmids were prepped, and theirintegrities were confirmed by restriction analysis. The integrity of each shorthairpin RNA (shRNA insert) was confirmed by sequencing. Lentiviral packagingand infection were performed as described previously (28).

Confocal microscopy. Cells were fixed with 3.7% formaldehyde for 10 min atroom temperature and further permeabilized with 3.7% formaldehyde–0.1%Triton X-100 for 10 min at room temperature. After being washed with PBSthree times, actin filaments were labeled and visualized with Alexa-phalloidin(Molecular Probes) using a Zeiss LSM 510 Meta with a 63 Zeiss PLAN Apoobjective.

RESULTS

PTEN is required for the cell size arrest induced by bothionizing radiation and DNA-damaging chemotherapeuticdrugs. Treatment of human cells with ionizing radiation (IR)and DNA-damaging chemotherapeutics leads to senescence-like cell cycle arrest (11). During this cell cycle arrest, cells alsostop increasing in size and mass (i.e., stop growing). We havepreviously shown that PTEN-deficient cells undergo a normalsenescence-like cell cycle arrest after treatment with IR but failto arrest in size (34). As such, we have proposed that PTENregulates a novel, radiation-induced cell size checkpoint.

Our initial work focused exclusively on IR as an inducer ofthe PTEN-dependent cell size checkpoint (reference 34 andFig. 1A). In an effort to demonstrate the generalizability of thisphenotype, we tested whether DNA-damaging chemothera-peutic drugs also induce the PTEN-dependent cell size check-point. HCT116 PTEN�/� and PTEN�/� cells previously cre-ated by human somatic cell gene targeting were treated withthe topoisomerase II inhibitor doxorubicin (0.2 �g/ml) for 6days, a course of doxorubicin that induces senescence-like cellcycle arrest in HCT116 cells and does not cause apoptosis (56).The cell size profiles of treated cells were then measured usinga Multisizer III, a specialized Coulter Counter designed tomeasure cell size (Fig. 1A). The cell cycle profiles were alsomeasured using flow cytometry (Fig. 1B). Two independentlyderived isogenic clones of each genotype were tested to avoidthe possibility of clone-specific artifacts. HCT116 PTEN�/�

VOL. 31, 2011 MECHANISTIC ANALYSIS OF A CELL SIZE CHECKPOINT 2757

cells arrested at an average volume of 33,100 �m3. In contrast,otherwise isogenic HCT116 PTEN�/� cells continued to en-large and eventually arrested at an average volume of 52,900�m3. As previously demonstrated for IR, this size phenotypewas not secondary to a more primary effect on the cell cycle, as

the flow cytometry profiles of doxorubicin-treated HCT116PTEN�/� and PTEN�/� cells were indistinguishable (Fig. 1B).Phase-contrast micrographs of doxorubicin-induced enlarge-ment of PTEN�/� cells are depicted in Fig. 1C.

To confirm and extend these results, we repeated these ex-

FIG. 1. DNA-damaging chemotherapeutics induce a PTEN-dependent cell size checkpoint in human cells. (A) HCT116 PTEN�/� andHCT116 PTEN�/� cells created by human somatic cell gene targeting were measured using a Multisizer III during exponential growth or 6 daysafter treatment with IR (6 Gy), doxorubicin (0.2 �g/ml), and etoposide (5 �g/ml). (B) Cells treated as described above were stained with propidiumiodide and studied by flow cytometry. (C) HCT116 PTEN�/� cells treated as described above were imaged using phase-contrast microscopy.

2758 KIM ET AL. MOL. CELL. BIOL.

periments with the topoisomerase II inhibitor etoposide. Wepreviously demonstrated that this dose of etoposide inducessenescence-like cell cycle arrest in HCT116 cells without con-comitant apoptosis (56). After 6 days of treatment, HCT116PTEN�/� cells arrested at an average volume of 42,000 �m3

(Fig. 1A), whereas otherwise isogenic HCT116 PTEN�/� cellscontinued to enlarge and eventually arrested at an averagevolume of 89,300 �m3. As with IR and doxorubicin, the sizephenotype was not secondary to a more primary effect on cellcycle, as the flow cytometry profiles of etoposide-treatedHCT116 PTEN�/� and PTEN�/� cells were indistinguishable(Fig. 1B). Micrographs of etoposide-induced enlargement ofPTEN�/� cells are depicted in Fig. 1C. Taken together, thesedata, which were obtained using two different topoisomerase IIinhibitors, demonstrate that PTEN controls a size checkpointthat is inducible not only by IR but also by several commonlyused DNA-damaging chemotherapeutic drugs.

Restoration of size checkpoint control in PTEN�/� cells vialenti-PTEN infection. Despite the use of multiple indepen-dently derived PTEN�/� and PTEN�/� clones, it remained aformal possibility that differences in cell size following DNAdamage may stem from clone-specific artifacts unrelated toPTEN. To investigate this possibility, we tested whether ecto-pic reexpression of PTEN restored cell size checkpoint controlto HCT116 PTEN�/� cells. We obtained a lenti-PTEN con-struct (gift from Nick Leslie), created infectious lentivirus, andinfected HCT116 PTEN�/� cells as described in Materials andMethods. Infection of PTEN�/� cells with lenti-PTEN but notwith the lentiviral vector alone led to reexpression of PTENprotein in these cells (Fig. 2A).

Next, infected cells were exposed to 6 Gy IR and culturedfor 6 days before cell size determination using a Multisizer III.As expected, HCT116 PTEN�/� cells infected with the lenti-viral vector alone were unable to a undergo cell size arrest andenlarged dramatically to a postirradiation average cell volumeof 69,100 �m3 (Fig. 2B). In contrast, infection of HCT116

PTEN�/� cells with lenti-PTEN led to a virtually completerestoration of cell size checkpoint control, as evidenced by apostirradiation average cell volume of 10,700 �m3 (Fig. 2B).These data provide formal confirmation of the role of PTEN incell size checkpoint control. Furthermore, these findings vali-date lenti-PTEN as a reagent that can restore the cell sizecheckpoint to PTEN�/� cells.

PTEN regulates size checkpoint control in GBM cells thatcontain naturally occurring mutations of PTEN. We nexttested whether human cancer cell lines with naturally occurringmutations of PTEN were deficient in the DNA damage-induc-ible size checkpoint. For these experiments, we focused ourstudies on glioblastoma multiforme (GBM) cell lines, sincemutations and deletions of PTEN are common in GBM(�40%; reference 57). In particular, we studied two differentPTEN-deficient human GBM cell lines: U87MG cells, whichharbor a 49-bp deletion that leads to a frameshift mutation andan absence of PTEN protein expression (37), and SNB19 cells,which harbor an insertion of two T residues in exon 7 leadingto a frameshift mutation and a complete absence of PTENprotein expression (25). Both cell lines harbor loss of hetero-zygosity of the remaining wild-type allele of PTEN. Initially,U87MG and SNB19 cells were infected with lenti-PTEN orwith vector alone, and expression of PTEN was confirmed byWestern blotting (Fig. 3A). Infected cell lines were thentreated with doxorubicin or etoposide and cultured for 5 days.The resultant cell size was then measured using a Multisizer III(Fig. 3B to D). Of note, IR was not used in any of theseexperiments, since GBM cell lines are notoriously radioresis-tant. Cells infected with lentiviral vector alone continued toenlarge after treatment with doxorubicin and etoposide. Incontrast, cells infected with lenti-PTEN arrested in size, re-flecting restoration of cell size checkpoint control. This sizephenotype was not due to differences in polyploidization be-tween PTEN-proficient and PTEN-deficient cells. Expressionof PTEN in U87MG cells appeared to rescue U87MG cells

FIG. 2. Reconstitution of the cell size checkpoint in PTEN�/� cells via infection with lenti-PTEN. HCT116 PTEN�/� cells were infected withlentiviral vector alone (pHR-SIN) or with lenti-PTEN (pHR-SIN-PTEN). (A) Three days after infection, cells were harvested in RIPA buffer andstudied by Western blotting to document expression of PTEN protein. (B) Infected cells were treated with 6 Gy IR and cultured for 6 days, andtheir sizes were measured using a Multisizer III.


from doxorubicin- and etoposide-induced cytotoxicity (com-pare the fragmented cell populations in the Multisizer datadepicted in Fig. 3B). This result is consistent with previousobservations that PTEN expression protects cells from DNAdamage-induced cytotoxicity (34). Taken together, these datageneralize our previous findings and demonstrate that twodifferent GBM cell lines with naturally occurring PTEN mu-tations are deficient in PTEN-dependent size checkpoint con-trol.

While these data are intriguing, neither doxorubicin noretoposide is used clinically for treatment of GBM, and there-fore, these data have questionable clinical relevance. To de-termine whether PTEN may modulate cell size control inGBM cells arrested by a more clinically relevant chemothera-peutic drug, we tested temozolomide, an alkylating agent thatis a standard-of-care upfront treatment for GBM. SNB19 cellsthat had been preinfected with either lentiviral vector alone orlenti-PTEN were treated with temozolomide and then cultured

FIG. 3. Restoration of cell size checkpoint control in PTEN-deficient human GBM cell lines via infection with lenti-PTEN. U87MG and SNB19cells were infected with lentiviral vector alone and lenti-PTEN. (A) Infected cells were then harvested in RIPA buffer 3 days after infection andstudied by Western blotting to document expression of PTEN protein. Infected U87MG cells (B) and SNB19 cells (C) were treated withdoxorubicin (0.2 �g/ml) and etoposide (5 �g/ml) and cultured for 5 days, and their sizes were measured using a Multisizer III. (D) Quantificationof size differences induced by doxorubicin and etoposide in cells infected with lentiviral vector alone or lenti-PTEN. (E) SNB19 cells were treatedwith temozolomide (100 �M) and cultured for 5 days, and their sizes were measured using a Multisizer III.


for 5 days. The sizes of these treated cells were measured usinga Multisizer III (Fig. 3E). Cells infected with lentiviral vectoralone continued to enlarge after treatment with temozolomide. Incontrast, cells infected with lenti-PTEN arrested in size, reflectingrestoration of cell size checkpoint control. These data implicatePTEN in the control of GBM cell size arrest that was induced bya clinically relevant chemotherapeutic drug.

Oncogenic PIK3CA fails to efficiently modulate cell sizecheckpoint control. We wondered whether abrogation of theradiation-induced cell size checkpoint was a generalizable fea-ture of activation of PI3K signaling. To test this, we studiedPIK3CA gene-targeted derivatives of HCT116 cells, which har-bor an endogenous heterozygous oncogenic mutation in the

catalytic domain of PIK3CA (H1047R). Human somatic cellgene targeting technology was used to create derivatives ofHCT116 cells in which either the mutant allele or the wild-typeallele of PIK3CA had been deleted (28). Parental HCT116cells and derivatives lacking either the wild-type or mutantallele of PIK3CA were treated with 6 Gy IR and examined6 days after irradiation (Fig. 4). In contrast to HCT116PTEN�/� cells, each of the three otherwise isogenic PIK3CAgene-targeted cell lines was able to efficiently arrest its cell size(Fig. 4A), despite the ability of oncogenic PIK3CA to regulatethe phosphorylation state and activity of Akt in these cells (Fig.4B). These data indicated that unlike PTEN, PIK3CA appearsnot to be involved in regulation of the IR-induced cell size

FIG. 4. Inability of PIK3CA to efficiently regulate cell size checkpoint control. (A) HCT116 parental cells and PTEN and PIK3CA gene-targeted derivatives lacking either the wild-type allele of PIK3CA (PIK3CAKO/mut) or the oncogenic allele (PIK3CAWT/KO) were treated with 6Gy IR and cultured for 6 days, and their sizes were measured using a Multisizer III. (B) Cells were harvested in RIPA buffer 24 h after treatmentwith DNA-damaging agents, and Western blotting was performed with the indicated antibodies.


checkpoint. In addition, these results suggested that the abilityof PTEN to regulate intracellular levels of PIP2 and PIP3 is notits only biochemical activity required for cell size checkpointcontrol.

The lipid phosphatase activity of PTEN is necessary for cellsize checkpoint control. The fact that lenti-PTEN was able torestore cell size checkpoint control to PTEN-deficient humancells provided us with an experimental system for testing the

FIG. 5. Mutational analysis of the PTEN-dependent cell size checkpoint. (A) Eleven different missense mutations were engineered intolenti-PTEN using site-directed mutagenesis, as described in Materials and Methods. The constructs were then packaged into infectious lentivirusand used to infect HCT116 PTEN�/� cells. WT, wild type; V, vector. (B) Cells were harvested 3 days after infection in RIPA buffer and studiedby Western blot analysis to evaluate levels of PTEN, p-Akt (S473), and total Akt protein. (C) Cells were treated with 6 Gy IR and cultured for6 days, and their sizes were measured using a Multisizer III. (D) Tumor type of origin, previously determined lipid phosphatase activity withreference, and ability to regulate p-Akt and the cell size checkpoint are shown. The three mutant proteins that were deficient in the cell sizecheckpoint are denoted with boxes.


FIG. 6. Mutations in the amino terminus of PTEN uncouple control of Akt phosphorylation from control of cell size. Nine different missensemutations were engineered into the amino terminus of PTEN using site-directed mutagenesis, as described in Materials and Methods. Theconstructs were then packaged into infectious lentivirus and used to infect HCT116 PTEN�/� cells. Of note, the data for two of the mutations,�1-15 and �1-25, are described in the text but are not depicted in this figure. (A) Cells were harvested 3 days after infection in RIPA buffer andstudied by Western blot analysis to evaluate levels of PTEN, p-Akt (S473), p-Akt (T308), and total Akt protein. WT, wild type; V, vector. (B) Cellswere treated with 6 Gy IR and cultured for 6 days, and their sizes were measured using a Multisizer III. (C) Ability of proteins to regulate p-Aktand cell size checkpoint is shown. The six mutant proteins that were proficient in regulating the cell size checkpoint but deficient in regulating p-Aktare denoted with boxes.


effect of PTEN mutations on cell size checkpoint control. Ini-tially, we used site-directed mutagenesis to introduce 11 dif-ferent tumor-derived mutations into the known functional do-mains of PTEN (Fig. 5A; also described in Materials andMethods). The origins of the mutations and their previouslydetermined effects on PTEN lipid phosphatase activity arelisted in Fig. 5D. The constructs were then packaged intoinfectious lentivirus and used to infect HCT116 PTEN�/�

cells. Western blotting was performed to confirm expression ofPTEN and to measure the effects of mutant PTEN proteins onmodulation of p-Akt (Fig. 5B). In addition, infected cells were

treated with 6 Gy IR and cultured for 6 days. The cell size wasthen measured using a Multisizer III (Fig. 5C).

Three of the 11 mutations are known to disrupt the lipidphosphatase activity of PTEN (D24Y, C124S, and G129E). Asexpected, these mutants were unable to downregulate levels ofp-Akt in PTEN-deficient cells (Fig. 5B and D). Similarly, thesethree mutant proteins were completely unable to restore sizecheckpoint control to HCT116 PTEN�/� cells (Fig. 5C and D).Based on these data, we concluded that the lipid phosphataseactivity of PTEN is necessary for effective PTEN-dependentcell size checkpoint control.

FIG. 7. Pharmacological inhibition of Akt fails to restore the cell size checkpoint to PTEN�/� cells. (A) HCT116 PTEN�/� cells were treatedwith 0.5, 1, or 2 �M MK2206 and 10, 50, and 100 �M LY294002 for 2 h, harvested in RIPA buffer, and studied by Western blotting. (B) HCT116PTEN�/� cells were pretreated with 2 �M MK2206 for 1 h, irradiated (6 Gy), cultured in the presence of MK2206 for 3 days, and measured usinga Multisizer III.


The PTEN Y138L mutant is deficient in protein phospha-tase activity but retains wild-type lipid phosphatase activity (9).Therefore, this mutation is particularly useful for evaluatingthe effect of protein phosphatase activity on PTEN-relatedphenotypes. As expected, PTEN Y138L downregulated thep-Akt levels in HCT116 PTEN�/� cells similarly to wild-typePTEN (Fig. 5B). Furthermore, PTEN Y138L efficiently re-stored cell size checkpoint activity to HCT116 PTEN�/� cells(Fig. 5C). Therefore, we concluded that the protein phospha-tase activity of PTEN is dispensable for the control of the DNAdamage-inducible cell size checkpoint.

Mutations in the amino terminus of PTEN uncouple lipidphosphatase activity and cell size regulation from control ofAkt phosphorylation. Of the 11 mutations tested, PTEN Y16Cwas particularly intriguing. This mutant protein, which waspreviously reported to have wild-type lipid phosphatase activ-ity, restored cell size checkpoint control to HCT116 PTEN�/�

cells similarly to wild-type PTEN but failed to downregulatep-Akt levels (Fig. 5B and C and reference 15). This dichotomysuggests that the ability of PTEN to modulate p-Akt levels isnot required for cell size checkpoint control.

Next, we created an additional seven missense mutationsand two deletions in the amino terminus of PTEN. The bio-chemical and phenotypic properties of several of these muta-tions have been previously reported (10). These nine addi-tional mutant proteins were tested for their abilities to regulatelevels of p-Akt and for their abilities to regulate the DNAdamage-inducible size checkpoint (Fig. 6A to C). Of the ninemutations, �1-15 and �1-25 were defective in both assays andwere therefore not evaluated further. As with PTEN Y16C,each of the additional seven missense mutations in the amino

terminus of PTEN restored cell size checkpoint control toHCT116 PTEN�/� cells similarly to wild-type PTEN (Fig. 6B).However, PTEN R11A, R14A, F21A, L23F, and L25A wereeach deficient in their ability to downregulate the levels ofp-Akt in HCT116 PTEN�/� cells (similar to results for PTENY16C, most likely due to the deleterious effects of these mu-tations on the expression of PTEN). Taken together, thesedata provide strong evidence that the Y16C mutation is not anoutlier and that missense mutations in the amino terminus ofPTEN uncouple the ability to control the radiation-inducedcell size checkpoint from the ability to regulate p-Akt levels.

Pharmacological inhibition of Akt kinase activity fails torestore size checkpoint control to HCT116 PTEN�/� cells.Since the Akt pathway has been previously implicated in thecontrol of cell size, our mutational analysis data that suggestedthat Akt was not a required effector of the PTEN-dependentcell size checkpoint were surprising. To more directly test thehypothesis that Akt activity is unnecessary for cell size check-point control, we employed MK2206, a recently developedsubmicromolar pharmacological inhibitor of all Akt isoforms(5 nM for Akt1, 12 nM for Akt2, and 65 nM for Akt3) that iscurrently in phase II clinical trials (39). MK2206 is an allostericAkt inhibitor that inhibits the folding of Akt proteins and,therefore, abolishes the ability of Akt to be recruited to theplasma membrane and be activated by phosphorylation.

Initially, we examined the effectiveness of MK2206 in regu-lating the activation state of Akt. HCT116 PTEN�/� cells weretreated with MK2206 or LY294002 for 2 h, and then proteinlysates were prepared and analyzed by Western blotting. Asdepicted in Fig. 7A, MK2206 treatment led to a dramaticreduction in levels of p-Akt at both S473 and T308, as well as

FIG. 8. Expression of myr-Akt fails to abrogate the DNA damage-induced cell size checkpoint in HCT116 PTEN�/� cells. HCT116 PTEN�/�

cells were transiently transfected with myr-Akt expression vector or vector alone. (A) 48 h after transfection, cells were harvested in RIPA bufferand levels of p-Akt and total Akt were measured by Western blotting. (B) Cells were treated with 6 Gy IR, cultured for 3 days, and measured usinga Multisizer III.


of the Akt substrate p-FoxO1/3a. These effects were morepronounced than the effects of LY294002 and occurred atsubstantially lower concentrations. In contrast, MK2206 hadlittle (if any) effect on the phosphorylation state of members ofthe mTOR pathway, consistent with data that have been re-ported by Samuels et al. (45). Next, we tested whether inhibi-tion of activated Akt using MK2206 restored cell size controlto HCT116 PTEN�/� cells. HCT116 PTEN�/� cells weretreated with 6 Gy IR in the presence or absence of 2 �MMK2206 and cultured for 3 days in the presence of drug, whichled to no overt toxicity. Cell size was then measured using aMultisizer III. Pharmacological inhibition of Akt failed to re-store cell size checkpoint control to PTEN-deficient cells (Fig.7B). To further confirm that Akt was not involved in theradiation-induced cell size checkpoint, HCT116 PTEN�/�

cells were transiently transfected with a myr-Akt expressionconstruct (Fig. 8). Despite expression of p-Akt (as documentedby Western blot in Fig. 8A), there was no effect on the integrityof the radiation-induced cell size checkpoint (Fig. 8B). Takentogether, these data confirm that Akt is not a required PTENeffector for cell size checkpoint control.

Identification of novel putative PTEN effectors via endoge-nous epitope tagging (EET). Since the ability of PTEN toregulate Akt phosphorylation is unnecessary for regulation ofthe PTEN-dependent cell size checkpoint, we sought to iden-tify novel effectors of this checkpoint. In particular, we hypoth-esized that PTEN interacts with one or several PIP2- or PIP3-regulated proteins in order to regulate cell size checkpointcontrol.

Since identification of PTEN-interacting proteins hasproven to be very difficult due, in part, to issues of ectopicoverexpression (3), we developed a new technology (27),termed endogenous epitope tagging (EET). This techniqueallows us to efficiently add a short epitope tag to the endoge-nous allele of genes in cultured human cells in order to avoidectopic overexpression of epitope-tagged transgenes while stillexploiting high-efficiency affinity reagents for protein complexpurification. In a proof-of-principle experiment for this ap-proach, we described the creation of HCT116 cell lines inwhich the amino termini of both PTEN alleles were modifiedwith the addition of a 1 FLAG tag (27). Here, we employedthese cells to identify novel PTEN-interacting proteins.

Purification and mass spectrometric identification of PTEN-interacting proteins are described in detail in Materials andMethods. In brief, protein lysates were prepared fromHCT116FLAG-PTEN/FLAG-PTEN cells and an equivalent numberof negative-control HCT116 parental cells and applied to aFLAG-M2 affinity column, and bound proteins were elutedusing 1 FLAG peptide. The proteins were separated by SDS-PAGE (Fig. 9A), and the protein composition of the eluentswas determined using tandem mass spectrometry. Proteinspresent specifically in FLAG immunoprecipitates fromHCT116FLAG-PTEN/FLAG-PTEN cells but not in immunoprecipi-tates from HCT116 parental cells are listed in Fig. 9B.

As expected, the endogenous FLAG-PTEN fusion proteinwas the most prominent differentially immunoprecipitatedprotein. Other proteins that were present specifically in immu-noprecipitates from FLAG-PTEN cells included actin and itsremodeling proteins gelsolin and EPLIN. Actin was sufficientlyabundant to be visible in the Coomassie brilliant blue (CBB)-

stained gel (Fig. 9A). Notably, gelsolin is regulated by PIP2

(50).Endogenous PTEN interacts and colocalizes with an endoge-

nous PIP2-regulated actin depolymerization complex. To con-firm these putative endogenous interactions, immunoprecipi-tation and Western blot analyses were performed. PTEN wasimmunoprecipitated from FLAG-PTEN cells using FLAG-M2beads, and Western blotting was performed with antibodies forgelsolin, EPLIN, and the three major actin isoforms. As de-picted in Fig. 10A and 10B, immunoprecipitation of endoge-nous PTEN led to coimmunoprecipitation of endogenous�-actin, �-actin, gelsolin, and EPLIN. Subcellular fractionationexperiments demonstrated that the plasma membrane was theonly cellular compartment in which each of these proteins waspresent, suggesting that the interactions were likely to occur at

FIG. 9. Identification of novel putative PTEN-interacting proteinsusing endogenous epitope tagging (EET). HCT116 cells in which bothendogenous alleles of PTEN have been modified via the addition ofamino-terminal 1 FLAG tags (HCT116FLAG-PTEN/FLAG-PTEN) havebeen previously described (27). HCT116 parental (�/�) andHCT116FLAG-PTEN/FLAG-PTEN (F/F) cells were cultured, nondetergentlysis was performed, and FLAG immunoprecipitation was performed,all as described in Materials and Methods. (A) Immunoprecipitateswere concentrated by TCA precipitation, separated by SDS-PAGE,and visualized after staining with Coomassie brilliant blue (CBB).PTEN and a coimmunoprecipitating protein later determined to beactin are denoted with arrows. As expected, several nonspecific immu-noprecipitating proteins are also present in both lanes. (B) The gellanes depicted in panel A were subjected to high-resolution massspectrometry, as described in Materials and Methods. Proteins withpeptide counts of �4 in immunoprecipitates from epitope-tagged cellsbut absent in immunoprecipitates from parental cells are listed.


the cell membrane (Fig. 10C). Subsequent immunoprecipita-tion and Western blot analyses of subcellular fractions con-firmed that these interactions occur at the plasma membrane(Fig. 10D). These experiments also demonstrated that the in-teraction between PTEN, actin, gelsolin, and EPLIN was in-sensitive to oxidation state, a known regulator of PTEN (Fig.10D; reference 35). The interaction between PTEN and actinwas further confirmed by immunoprecipitation (IP)/Westernblotting using anti-PTEN antibodies in genetically unmodifiedHCT116, LN229, and 293T cells (Fig. 10E).

Next, immunofluorescence was performed to determinewhether PTEN and actin colocalize in human cells. A lentivirusthat expresses green fluorescent protein (GFP) GFP-PTENwas generated and used to infect HCT116 PTEN�/� cells.Infected cells were then fixed and stained with Alexa-conju-gated phalloidin, which binds to and stains actin filaments.Cells were then imaged with fluorescence microscopy (Fig. 11).As previously reported, the majority of GFP-PTEN was dif-fusely present in the cytoplasm and the nucleus, with a minoritypresent at the plasma membrane. While GFP alone did not

FIG. 10. Interaction of endogenous PTEN with an actin remodeling complex. (A and B) HCT116 parental (�/�) and HCT116FLAG-PTEN/FLAG-PTEN

(F/F) cells were cultured, and nondetergent lysis followed by FLAG immunoprecipitation (M2-IP) was performed. Immunoprecipitates wereconcentrated by TCA precipitation and separated by SDS-PAGE, and Western blotting was performed with the antibodies indicated. (C) Cyto-plasmic, membrane, and nuclear fractions of HCT116FLAG-PTEN/FLAG-PTEN cells were prepared and studied by Western blotting with the antibodiesindicated. (D) Membrane fractions from HCT116 parental (�/�) and HCT116FLAG-PTEN/FLAG-PTEN (F/F) cells were subjected to immunopre-cipitation with FLAG-M2 beads and studied by Western blotting with the antibodies indicated. Immunoprecipitations were performed in thepresence and absence of 1 mM dithiothreitol (DTT) as indicated. (E) HCT116, LN229, and 293T cells were harvested in lysis buffer containing1% NP-40–150 mM NaCl–20 mM Tris-HCl, pH 7.5. One milligram of protein lysate was incubated overnight with 1 �g of PTEN N19 antibodyor 1 �g normal goat IgG and protein A/G agarose beads. Immunocomplexes were washed in lysis buffer and boiled in sample buffer, and Westernblotting was performed.


colocalize with actin, GFP-PTEN and actin colocalized at theplasma membrane (yellow arrows). This colocalization wasseen as a subtle but distinct overlap of GFP and phalloidinstaining. These signals also overlapped with staining on themembrane-associated actin network. These data are consis-tent with the immunoprecipitation and Western blot datadepicted in Fig. 10.

To determine if the interaction between PTEN and actinwas regulated by DNA damage, PTEN and actin colocalizationwas measured by immunofluorescence either in unirradiatedcells or 30 h after irradiation with 6 Gy. DNA damage failed toenhance the degree of colocalization to any measurable extent.Similarly, the presence of tumor-derived mutations R11A,Y16C, F21A (each described in detail in Fig. 6), and G129E inthe GFP-PTEN construct failed to affect the colocalizationbetween PTEN and actin.

Pharmacological inhibition of actin depolymerization abro-gates cell size checkpoint control in PTEN�/� cells. We nextconsidered the possibility that a defect in actin remodelingcould be responsible for the absence of size checkpoint controlin HCT116 PTEN�/� cells. In this case, we would expect thatpharmacological inhibition of actin remodeling in PTEN�/�

cells would be phenotypically equivalent to deletion of PTEN.To test this, we measured the effect of cytochalasin D, a potentinhibitor of actin polymerization, on the cell size checkpoint inHCT116 PTEN�/� and PTEN�/� cells. Cells were pretreatedwith 200 nM cytochalasin D, treated with 6 Gy IR, and thencultured for 3 days. Cell sizes were then measured. Pharma-cological inhibition of actin polymerization abrogated cell sizecheckpoint control in PTEN�/� cells, recapitulating the phe-

notype of PTEN deletion (Fig. 12A and B). Importantly, cy-tochalasin D had no effect on the size of PTEN�/� cells,demonstrating that the effect of the drug on cell size check-point control was specific to PTEN�/� cells. However, deple-tion of gelsolin or EPLIN individually was insufficient to ab-rogate cell size checkpoint control. Taken together, these dataindicate that the postirradiation cell size control defect inPTEN�/� cells is caused by a generalized defect in the abilityto normally regulate actin dynamics.

DISCUSSION

The genetic and biochemical mechanisms that regulate cellsize during cellular proliferation and cell cycle arrest remainmostly obscure. To date, most published work on cell sizecheckpoints has focused on the existence (or lack thereof) of asensing mechanism in the G1 phase of the eukaryotic cell cyclethat halts the cell cycle until the cell has reached sufficient sizeand mass to support cell division (examples in references 6, 12,13, 19, and 52). In the studies presented here, we have focusedour attention on a related but different issue—the mechanismresponsible for ensuring that human cells arrested in the G1 orG2 phases of the cell cycle simultaneously stop increasing insize. We focus specifically on the cell size checkpoint that isenacted during DNA damage-induced arrest.

In the work described in this paper and in a previous pub-lication (34), we identified the PTEN tumor suppressor as arequired effector of this cell size checkpoint. Cells in whichPTEN has been deleted by human somatic cell gene targetingor in which PTEN is inactivated by naturally occurring tumor-derived mutations are unable to normally arrest their cell sizeduring DNA damage-induced cell cycle arrest. Mutationalanalysis of PTEN revealed that the lipid phosphatase activityof PTEN is required for this PTEN-dependent cell size check-point, while the ability of PTEN to modulate Akt phosphory-lation is dispensable for this checkpoint. This conclusion wassubsequently confirmed with the use of Akt inhibitors. Endoge-nous PTEN was shown to interact at the membrane with anactin-remodeling complex that contains actin-remodeling pro-teins, such as gelsolin, a protein known to be regulated by PIP2.Treatment of PTEN�/� cells with cytochalasin D, a potentinhibitor of actin remodeling, led to abrogation of the cell sizecheckpoint. Importantly, this inhibitor produced no effect oncell size control in otherwise isogenic PTEN�/� cells. Takentogether, these data indicate that direct control of actin re-modeling but not control of Akt phosphorylation is requiredfor PTEN-dependent cell size checkpoint control (Fig. 13).

It was surprising to us that the PTEN-dependent size phe-notype described herein was Akt independent, since there arenumerous reports in the literature of Akt being a central playerin cell size control. In D. melanogaster, activation of Akt leadsto increased cell and organ growth, and regulation of Aktappears to be required for the effects of PTEN on cell andorgan size (41, 55). Akt has also been shown to promote celland organ growth in mice (33, 48), though the presence ofmultiple Akt homologs has complicated testing its epistasiswith PTEN. We do not understand the molecular basis ofthe discrepancies between these types of published studiesand the data presented herein. Possible explanations include(i) mechanistic differences between cell size control during

FIG. 11. Colocalization of PTEN and F-actin. HCT116 PTEN�/�

cells infected with GFP lentivirus or GFP-PTEN lentivirus were fixedand stained as described in Materials and Methods. Alexa 594-phal-loidin-labeled F-actin colocalized with GFP-PTEN but not GFP aloneat the protrusions of the cells and in the magnified actin network(denoted by the yellow arrows). The lower panels of GFP-PTEN andGFP alone are magnified images derived from the squares in the upperpanels. DIC, differential interference contrast.


organismal development and DNA damage-induced cell cy-cle arrest, (ii) mechanistic differences in cell size controlbetween humans, mice, and flies, and/or (iii) the possibilitythat PTEN and Akt function in parallel pathways to controlcell size.

Currently, PTEN is the only known major regulator of theDNA damage-induced cell size checkpoint. It is worth noting,however, that a variety of genes, including the LK6, S6K,TSC1, and TSC2 genes and myc, have been shown to regulatecell size during proliferation (examples in references 1, 17, and29). The fact that many of these genes are cancer related raisesthe important question whether the abrogation of cell sizecheckpoint control is fundamental to neoplastic transforma-tion in a fashion analogous to that of abrogation of the G1 andG2 checkpoints. Clearly, several cytopathological findings thatpresent in PTEN-deficient cancers are likely due to defectivePTEN-dependent cell size checkpoint control. The presence of

giant cells in tumors and the existence of tumor types that arecomposed exclusively of enlarged cells are two such cytopatho-logical presentations.

Despite these findings, whether abrogation of cell sizecheckpoint control actually drives neoplasia is not clear. SinceAkt is thought to be a key effector of PTEN-dependent tumorsuppression but is clearly dispensable for cell size checkpointcontrol in the systems studied here, the cell size checkpointmay not be related to driving neoplasia. However, recent stud-ies have called into question whether Akt is actually a requiredeffector of PI3K pathway-driven oncogenesis (54). Further-more, emerging data suggest that Akt inhibitors may be oflimited clinical utility in tumors driven by mutations in PTEN.Thus, the extent to which Akt is a required effector of PTENtumor suppression is not clear at this time.

How might abrogation of cell size checkpoint control actu-ally drive neoplasia? We hypothesize that the explanation may

FIG. 12. Pharmacological inhibition of actin remodeling leads to abrogation of cell size checkpoint control in PTEN�/� cells. (A) HCT116PTEN�/� and PTEN�/� cells were exposed to 200 nM cytochalasin D, treated with 6 Gy IR, cultured for either 3 or 6 days, and measured witha Multisizer III. (B) Quantification of cell size in irradiated PTEN�/� and PTEN�/� cells treated with either 200 nM cytochalasin D or with vehiclealone.


be related to the eukaryotic cell checkpoint that halts celldivision at the G1 stage of the cell cycle until cells have reachedsufficient size to separate their biomass into two daughter cells.Whereas in normal-sized cells, this checkpoint is vigilant inpreventing cell division and proliferation, in oversized PTEN-deficient cells, this checkpoint may permit cells to enter the cellcycle, contributing to enhanced proliferation and neoplasia.This hypothesis, however, remains experimentally untested.

In addition to demonstrating that Akt is dispensable for cellsize checkpoint control, we identified actin remodeling as acritical PTEN-regulated process that is involved in regulatingcell size control. These findings are consistent with the earlywork of Goberdhan et al., who demonstrated that in D. mela-nogaster, PTEN affects cytoskeletal organization in multiplecell types (18). Here we have identified a physical interactionbetween PTEN and an actin-remodeling complex that includes�-actin, �-actin, and several actin-remodeling proteins, includ-ing gelsolin and EPLIN. This finding raises another unresolvedquestion: which of these proteins interacts directly withPTEN? We speculate that PTEN interacts directly with actinand indirectly with the remodeling proteins, since actin ap-pears to be the most abundant protein (other than PTENitself) in PTEN immunoprecipitates (Fig. 9A). In addition,PTEN contains a domain with homology to tensin, a knownactin-interacting protein. A definitive answer to this questionwill require the ability to recapitulate the interactions withpurified components, and these efforts are ongoing in ourlaboratory.

This newly identified interaction between PTEN and theactin-remodeling complex is reminiscent of the recent work ofvan Diepen et al., who demonstrated that PTEN interacts withmyosin V in neurons (53). These researchers further showedthat this interaction is critical for the ability of PTEN to controlthe size of these neurons. While we did not specifically identifymyosin V as a PTEN-interacting protein in our study, wespeculate that this omission is due to cell type-specific differ-ences in the expression pattern of the myosin V gene. Deter-mination of whether myosin V is part of a larger actin-contain-ing complex in the neurons used in this study will beinteresting. Furthermore, our work is also reminiscent of otherrecent studies that demonstrated that PTEN colocalizes withactin and myosin during chemotaxis in Dictyostelium (42). Our

studies suggest that this reported colocalization may resultfrom direct (or indirect) physical interaction. In addition, Go-ranov et al. have suggested that direct regulation of actin re-modeling may be an important biochemical mechanism foreukaryotic cell size control (19, 20).

In summary, we have identified and evaluated a PTEN-dependent cell size checkpoint in human cancer cells. Currentwork is focusing on better understanding the structural natureof the identified interaction between PTEN and the actin-remodeling complex and evaluating how and why abrogationof PTEN-dependent cell size checkpoint control either directlyor indirectly drives neoplasia.

ACKNOWLEDGMENTS

We thank Nick Leslie for the PTEN lentivirus construct.This work was supported by National Institutes of Health grants

CA115699 and CA143282 to T.W., American Cancer Society grantRPG MGO-112078 to T.W., and the Lombardi Comprehensive Can-cer Center Support grant P30 CA051008. D.S. was supported, in part,by the NIH training grant T32 CA009686.

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