Focal Adhesion Kinase Controls Cellular Levels of p27/Kip1 and p21/Cip1 through Skp2-Dependent and...

MOLECULAR AND CELLULAR BIOLOGY, June 2006, p. 4201–4213 Vol. 26, No. 110270-7306/06/$08.00�0 doi:10.1128/MCB.01612-05Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Focal Adhesion Kinase Controls Cellular Levels of p27/Kip1 andp21/Cip1 through Skp2-Dependent and -Independent Mechanisms

Patrick Bryant, Qingxia Zheng, and Kevin Pumiglia*Center for Cell Biology and Cancer Research, Albany Medical College, Albany, New York 12208

Received 19 August 2005/Returned for modification 7 October 2005/Accepted 13 March 2006

Endothelial cell proliferation is a critical step in angiogenesis and requires a coordinated response to solublegrowth factors and the extracellular matrix. As focal adhesion kinase (FAK) integrates signals from bothadhesion events and growth factor stimulation, we investigated its role in endothelial cell proliferation.Expression of a dominant-negative FAK protein, FAK-related nonkinase (FRNK), impaired phosphorylationof FAK and blocked DNA synthesis in response to multiple angiogenic stimuli. These results coincided withelevated cyclin-dependent kinase inhibitors (CDKIs) p21/Cip and p27/Kip, as a consequence of impaireddegradation. FRNK inhibited the expression of Skp2, an F-box protein that targets CDKIs, by inhibitingmitogen-induced mRNA. The FAK-regulated degradation of p27/Kip was Skp2 dependent, while levels ofp21/Cip were regulated independent of Skp2. Skp2 is required for endothelial cell proliferation as a conse-quence of degrading p27. Finally, knockdown of both p21 and p27 in FRNK-expressing cells completelyrestored mitogen-induced endothelial cell proliferation. These data demonstrate a critical role for FAK in theregulation of CDKIs through two independent mechanisms: Skp2 dependent and Skp2 independent. They alsoprovide important insights into the requirement of focal adhesion kinase for normal vascular development andreveal novel regulatory control points for angiogenesis.

Angiogenesis is a highly coordinated process required fornormal development and in response to injury (15). A keycomponent of angiogenesis is the tightly regulated prolifera-tion of endothelial cells (25). Loss of normal cell cycle regu-lation likely contributes to the abnormal vasculature present inmany disease states, including vasculopathies, cancer, cardio-vascular disease, and proliferative retinopathies. Soluble growthfactors such as vascular endothelial growth factor (VEGF) andbasic fibroblast growth factor (bFGF) have been shown toinduce endothelial cell proliferation. However, growth factorengagement alone is insufficient to promote endothelial cellgrowth, which also requires integrin attachment to the extra-cellular matrix (ECM) and proper cytoskeletal organization(26, 65). This notion is supported by the fact that endothelialcells grown in a saturating amount of bFGF proliferate in afibronectin concentration-dependent manner (36). Similarly,growth factors are capable of promoting cell growth in endo-thelial cells plated on fibronectin, but not laminin (52). A largebody of work, particularly in fibroblasts, has examined the roleof extracellular signal-regulated kinase (ERK) in mediatingthe joint signal transduction from receptor tyrosine kinases(RTKs) and the ECM. However, in endothelial cells, alter-ations in the composition of the extracellular matrix can inhibitproliferation despite robust activation of ERK signaling (32).This piece of data implies that additional regulatory moleculesare involved in the control of endothelial cell growth mediatedby growth factors and integrins.

Soluble growth factors promote the transition through theG1 phase of the cell cycle by inducing the formation of cyclin

D-cdk4/6 and cyclin E-cdk2 complexes (78). Formation ofthese complexes results in activation of these enzymes andsubsequent phosphorylation of the retinoblastoma (Rb) pro-tein. The hyperphosphorylated form of Rb is no longer capableof forming inhibitory complexes with E2F transcription factors,resulting in the accumulation of important cell cycle proteinssuch as cyclin A (20, 81). The G1 cyclin-cdks are regulatedby the activity of specific cyclin-dependent kinase inhibitors(CDKIs). The CDKIs consist of two families: the Cip/Kip fam-ily, including p21/Cip1, p27/Kip1, and p57/Kip2; and the INK4family, including p15, p16, p18, and p19 (69). During growthfactor-induced transition through the G1 phase of the cellcycle, the levels of p27 and p21 become down-regulated,thereby allowing increased CDK activity, hyperphosphoryla-tion of Rb, and the release of transcriptionally active E2F.However, the levels of both p27 and p21 remain elevated inquiescent cells under serum-free conditions as well as in cellsthat are maintained in suspension (64, 87). This led us tohypothesize that down-regulation of these CDKIs requiressignals from both mitogens and the extracellular matrix inorder to promote endothelial cell proliferation and thatfocal adhesion kinase (FAK) was an excellent candidate toprovide such a signal.

Focal adhesion kinase is a non-receptor tyrosine kinase thatis localized at focal adhesion sites. FAK is a signal integratorcapable of relaying signals from soluble growth factors andcytokines, mechanical stimuli, as well as integrin engagement.Integrin binding has been shown to induce FAK phosphoryla-tion in numerous cell types, resulting in dramatic effects on theactin cytoskeleton, cell migration, and proliferation. Growthfactors, including VEGF, have also been shown to rapidlyinduce tyrosine phosphorylation of FAK (2). This suggests thatFAK may be a critical signaling component involved in theregulation of angiogenesis. This notion is further supported by

* Corresponding author. Mailing address: Center for Cell Biologyand Cancer Research, Albany Medical College, 47 New Scotland Ave.,Albany, NY 12208. Phone: (518) 262-6587. Fax: (518) 262-5696. E-mail:[email protected].

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the evidence that the FAK knockout mouse displays an em-bryonic lethal phenotype at day E8.5 to 9 as a result of numer-ous abnormalities, including a poorly developed vasculature(34). Recent data using conditional knockout of FAK in en-dothelial cells have provided evidence that the FAK null phe-notype is due to direct effects on the vascular endothelium(67). The loss of FAK in endothelial cells appears to inhibit theability of endothelial cells to survive, migrate, and proliferate.An earlier study, using microinjection, demonstrated a role forFAK in the migration and proliferation of primary endothelialcells; however, no mechanism was defined (27). We sought todetermine the mechanism by which FAK regulates endothelialcell proliferation.

MATERIALS AND METHODS

Cell culture. Human umbilical vein endothelial cells (HUVECs) and humandermal microvessel endothelial cells (HDMVECs) from pooled donors werepurchased from VEC Technologies (Troy, NY). Cells were cultured as previouslydescribed (50). Experiments were conducted in the presence of serum-freeMCDB-131, and stimulation was performed with the indicated mitogen or com-plete growth medium containing 20% fetal bovine serum, 1% penicillin/strepto-mycin, 10 �g/ml heparin, and 60 �g/ml endothelial cell growth supplement(Becton-Dickinson), often referred to for brevity in the text and figures as“serum.”

Western blotting. Western blot analysis was conducted using the followingantibodies: anti-FAK from Upstate, Lake Placid, NY; anti-FAK Y397, anti-FAKY861, antipaxillin, anti-paxillin Y118, and anti-cyclin D1 from BioSource,Camarillo, CA;, anti-ERK2, anti-phospho-ERK, and anti-phospho-Elk fromSanta Cruz Biotechnology, Santa Cruz, CA;, anti-phospho-RbS795 and anti-phospho-JNK from Cell Signaling, Beverly, MA; anti-p21/Cip1 CP74 from Neo-Markers, Fremont, CA; anti-p27/Kip1 from BD Pharmingen, San Diego, CA;and anti-Skp2 from Zymed Laboratories, Cambridge, United Kingdom. Westernblotting was performed under the conditions previously detailed (49).

Labeling of actin cytoskeleton. HUVECs were seeded at 5.6 � 104 into 35-mmdishes containing coverslips coated with 0.2% gelatin. Cells were allowed toadhere and spread overnight and were then placed in serum-free MCDB-131 for16 h. The cells were then washed with 1� phosphate-buffered saline (PBS) andthen fixed with 3.7% formaldehyde for 15 min. Cells were permeabilized with0.1% Triton X with 1% bovine serum albumin in PBS for 30 min. Filamentousactin was then labeled with Texas red-phalloidin (Molecular Probes) for 30 minas previously described (49). The coverslips were then washed three more timeswith 1� PBS and mounted on glass slides with Vectashield 4�,6�-diamidino-2-phenylindole (DAPI) mounting medium (Vector Labs). Images were obtainedwith a fluorescence microscope equipped with a digital camera.

Measurement of cell surface area. Cells were plated exactly as described forthe actin cytoskeleton labeling. Cells were allowed to adhere and spread over-night and then were transduced with adenovirus constructs expressing greenfluorescent protein (GFP) (Ad.GFP), FAK-related nonkinase (FRNK;Ad.FRNK), or FRNKS (Ad.FRNKS) in serum-free MCDB-131 for 16 h with amultiplicity of infection (MOI) of 10. The cells were then washed with 1� PBSand fixed with 3.7% formaldehyde. The cells were photographed under bright-field settings. The cell surface area of each cell was then traced, and the surfacearea was calculated using Image-Pro Plus software. An average of 50 cells wascalculated per treatment, and the average surface area was calculated andgraphed in Microsoft Excel.

Generation of recombinant adenovirus. Most viruses described in this studywere constructed using the AdEasy adenoviral system (28). GFP-FRNK wasgenerously provided by Allen Samarel (Loyola University Medical Center); Skp2and the F-box Skp2 mutant adenoviruses were from Mark Bond (University ofBristol). The GFP-expressing adenovirus was from Q-Biogene (Carlsbad, CA).GFP-FAK and GFP-FAKY397F were generated by subcloning the coding regionof FAK (with or without the point mutation at amino acid 397) in frame with the3�-coding region of enhanced GFP (EGFP) into a modified pShuttle vector.Recombinant adenoviruses were generated as previously described (28) andwere used at an MOI of between 10 and 20.

Measurement of DNA synthesis. HUVECs were transduced with Ad.GFP,Ad.FRNK, Ad.FRNKS, Ad.GFP-FAK, Ad.GFP-FAKY397F, Ad.WT (wildtype)-Skp2, or Ad.�F-box Skp2 under serum-free conditions. After 24 h, com-plete growth medium, VEGF, or FGF was added as a mitogenic stimulus.

Measurement of DNA synthesis by [3H]thymidine and bromodeoxyuridine(BrdU) incorporation was performed as previously described (50).

Real-time PCR. Total RNA was extracted from 2.5 � 105 HUVECs pertreatment with an RNeasy kit and QiaShredder (QIAGEN). First-strand cDNAwas synthesized from equal amounts of total RNA using iQ reverse transcriptase(Bio-Rad) according to the manufacturer’s instructions. Real-time PCR wasperformed with primers chosen to extend products under 200 bp with the fol-lowing characteristics: (i) melting temperature of 58°C; (ii) a product that spansone intron boundary; and (iii) GC content between 40 and 60% and no predictedprimer dimer formation. The primers selected are as follows: Skp2, forward,5�-CCCACGGAAACGGCTGAAGA-3�, and reverse, 5�-CGCTAGGCGATACCACCTCTTACAA-3�; p27/KIP1, forward, 5�-TGCAACCGACGATTCTTCTACTCAA-3�, and reverse, 5�-CAAGCAGTGATGTATCTGATAAACAAGGA-3�); and p21/CIP1, forward, 5�-CGATGCCAACCTCCTCAACGA-3�, andreverse, 5�-TCGCAGACCTCCAGCATCCA-3�. Real-time PCR was then per-formed on 1 �l of cDNA along with 5 �M as a final concentration for eachprimer combined with iQ SYBR Green Supermix (Bio-Rad) in a MyiQ real-timedetection system (Bio-Rad). Each 20-�l reaction was amplified in thin-wall PCRtubes under the following conditions: 95°C for 2 min followed by 50 cycles of95°C for 15 s and 72°C for 15 s. Melt-curve analysis was performed followingamplification by increasing the temperature in 0.4°C increments starting at 65°Cfor 85 cycles of 10 s each. The presence of a single PCR product was verified bythe presence of a single melting temperature peak and by ethidium bromidestaining following electrophoresis on agarose gels. The relative abundance ofeach gene’s message was normalized against actin in each sample (forwardprimer, 5�-TACCTCATGAAGATCCTCACC-3�; reverse, 3�-TTTCGTGGATGCCACAGGAC-5�) and calculated as 2��CT of Skp2, p27, or p21 � �CT of actin, where CT

represents the threshold cycle for each transcript, as previously described (54). Inthe figures, error bars indicate the standard error of at least three independentexperiments. Control experiments run in the absence of reverse transcriptasedemonstrated no product amplification. Correct products were also verified bysize following electrophoresis on agarose gels.

Apoptosis assay. Caspase 3/7 activities were measured by Apo-ONE homog-enous caspase 3/7 assay (Promega) as we have previously described (48). A96-well plate coated with 0.2% gelatin was seeded with 2.0 � 104 cells/well.HUVECs were transduced with Ad.GFP or Ad.FRNK, and the following day,serum-free MCDB-131 was added to the wells. Tumor necrosis factor alpha(TNF-�) and alpha interferon were added as a positive control to induce apop-tosis, essentially as described by Alavi et al. (3).

siRNAs. Validated small interfering RNA (siRNA) sequences targeting p21(identification no. 1621) and Skp2 (identification no. 1655) were purchased fromAmbion, Inc., and p27 (catalog no. D-003472-04) was purchased from Dharma-con Research. The control duplex was directed against luciferase and was pur-chased from Dharmacon (catalog no. D-002050-01-05). Electroporation wasused to transduce 125 ng of siRNA into 7.5 � 104 HUVECs resuspended in 75�l of siRNA electroporation buffer (Ambion, Inc.). Cells were plated and al-lowed to recover for 24 h in complete medium prior to any subsequent infectionor manipulation. The results shown are from duplexes indicated above. In allcases, similar results were obtained with additional siRNA sequences obtainedfrom the same vendors but directed against different sequences in the sametarget molecule (data not shown).

Statistical analysis. All data except those from the real-time PCR are repre-sentative experiments; in the case of proliferation experiments, three indepen-dent replicates were conducted in each experiment. Similar results were obtainedin at least three independent experiments. In real-time PCR experiments, datawere pooled from multiple independent experiments. One-way analysis of vari-ance was conducted as appropriate using Statistica software (Tulsa, OK).

RESULTS

FRNK inhibits FAK and paxillin phosphorylation. To testthe role of FAK in growth factor-induced endothelial cell pro-liferation, we chose a genetic approach of inhibiting FAK ac-tivation by expression of the C-terminal portion of FAK re-ferred to as FRNK. This naturally occurring splice variant ofFAK functions as a dominant negative by competitively dis-placing FAK from focal contacts, resulting in reduced tyrosinephosphorylation of FAK as well as the focal adhesion-associ-ated protein paxillin (60). We generated recombinant adeno-viruses which were used to transduce cells, one expressing

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FRNK and one expressing a point mutant of FRNK (C1034S)which is not targeted to the site of focal contacts, these arereferred to as Ad.FRNK and Ad.FRNKS respectively. FRNKSis a useful negative control because it disrupts the focal adhe-sion targeting domain and therefore does not displace FAKfrom focal contacts (70). The equal expression of both proteinswas confirmed by Western blot analysis using a C-terminal-siteFAK antibody. Lysates were then prepared, and expressionlevels were analyzed by Western blotting using a C-terminalFAK antibody (Fig. 1A). The 125-kDa band represents totalendogenous FAK contained in the cells, and FRNK is detect-able at 41 kDa. The FRNKS is shifted as a result of a triple-hemagglutinin tag present on this construct. To characterizethe effect of FRNK expression on endothelial cells, we mea-sured FAK activation following treatment with VEGF on theY397 autophosphorylation site and the Src-dependent phos-phorylation site Y861 (2, 24). Expression of FRNK reducedboth Y397 and Y861 below the basal level and inhibited the

ability of VEGF to induce FAK Y397 and Y861 phosphor-ylation (Fig. 1B). Similar results were seen when completegrowth medium was used as a stimulus in place of VEGF (see,for example, Fig. 5B). The observed inhibition was dependenton targeting FRNK to focal adhesions, as FRNKS failed toalter the phosphorylation states of either tyrosine residue.Since FAK can phosphorylate paxillin at Y118 (51), we exam-ined the effect of FRNK on paxillin phosphorylation to assessthe effects on signaling downstream of FAK. In GFP-express-ing cells, mitogen stimulation resulted in the phosphorylationof paxillin at Y118; however, in GFP-FRNK-expressing cells,phosphorylation of paxillin Y118 was inhibited (Fig. 1C).

FRNK inhibits VEGF- and serum-induced endothelial cellproliferation. We next directly examined the role of FAK inVEGF-induced cell proliferation. Expression of FRNK inhib-ited HUVEC [3H]thymidine incorporation in response toVEGF almost completely, while FRNKS was without effect(Fig. 2A). This result was confirmed by BrdU incorporation toensure that the decrease in [3H]thymidine incorporation wasdue to a defect in DNA synthesis and not a loss of cell number(Fig. 2B). Given the considerable evidence of endothelial cellheterogeneity, we also tested the effect of interfering with FAKsignaling in primary microvessel endothelial cells. We foundthe requirement for FAK signaling was not specific to largevessel endothelial cells as FRNK also inhibits proliferation ofhuman dermal microvessel endothelial cells in response toVEGF (Fig. 2C). To determine if this effect was specific forVEGF signal transduction or was a more universal requirementof proliferation, we also tested the effect of FRNK expression onS-phase entry induced by complete growth medium containingserum. This is a much stronger mitogenic stimulus with an arrayof diverse receptor systems likely to be activated in response. Asshown in Fig. 2D, FAK signaling was required for S-phase entryand DNA synthesis, even in the presence of serum-containinggrowth medium. Identical inhibitory results were seen with bFGFas a mitogenic stimulus (data not shown). Taken together, thesedata indicate that FAK is a critical global regulator of en-dothelial cell proliferation and that these effects are notdependent on either a specific growth factor or endothelialcell type.

FRNK does not disrupt cell spreading or viability in adher-ent cells. In order to ensure that the FRNK-expressing cellswere not apoptotic as has been reported in rat cardiomyocytesand FAK�/� cells (30, 33), we transduced HUVECs withAd.GFP and Ad.FRNK and measured apoptosis using acaspase 3/7activity assay. The expression of GFP and FRNKshowed equivalent and low levels of caspase 3/7 activity. Incontrast, the positive control treatment of TNF-� and alphainterferon greatly increased caspase 3/7 activity (Fig. 3A).

Expression of FRNK has been reported to inhibit spreadingand migration in many cell types, including human brain mi-crovessel endothelial cells (HBMECs), particularly when cellsare detached into suspension and replated (6). As previousstudies have shown that disruption of the actin cytoskeletonand cell shape can influence cell cycle progression (17, 32), wetested the effects of FRNK expression on cell shape, spreading,and cytosleletal organization in adherent endothelial cells.GFP- and FRNK-transduced cells were equally well spread, asdetermined by calculation of the total cell surface area (Fig.3B). We analyzed actin staining in cells that expressed either

FIG. 1. FRNK expression inhibits FAK and paxillin phosphory-lation. HUVECs were transduced with Ad.GFP, Ad.FRNK, orAd. FRNKS for 16 h in serum-free MCDB-131 and then treated with50 ng/ml VEGF for 10 min, and whole cell lysates were collected andanalyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresisand immunoblotting using antibodies to total FAK (A) and phosphor-ylation-specific antibodies to Y397 and Y861 (B). HUVECs weretransduced with Ad.GFP or Ad.GFP-FRNK for 16 h in serum-freeMCDB-131 and then treated with serum for 10 min and analyzed withtotal paxillin and Y118 paxillin antibodies (C). pPaxillin, phosphory-lated paxillin.

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GFP or GFP-FRNK in order to visualize cellular cytoskeletonarchitecture. We found both GFP and GFP-FRNK-expressingcells contain numerous and well-formed actin stress fibers withFRNK localized at their termini. Interestingly, there appear to

be fewer membrane ruffles in FRNK-expressing cells and thestress fibers are more pronounced (Fig. 3C). We also examinedthe cellular morphology under serum-stimulated conditionsand found the two cell populations to be nearly indistinguish-

FIG. 2. FRNK expression inhibits mitogen-induced endothelial cell proliferation. HUVECs (A, B, and D) or HDMVECs were serum starved andtransduced with Ad.GFP, Ad.FRNK, or Ad.FRNKS prior to stimulation. The cells were then incubated with 50 ng/ml VEGF (or complete growthmedium; Fig. 3D) for 16 h. Cells were then pulsed with [3H]thymidine (H3 counts; A, C, and D) for an additional 3 h prior to scintillation counting. Cellspulsed with BrdU were visualized with an anti-BrdU antibody. BrdU-positive cells were quantified, and the data were graphed as the percentage ofpositive cells compared to total cell number (B).

FIG. 3. Expression of FRNK does not induce apoptosis or disrupt cell spreading. (A) HUVECs were transduced with Ad.GFP or Ad.FRNK-GFPand analyzed at 48 h postinfection for the presence of caspase activity using a fluorescent substrate (Apo-ONE; Promega). Apoptosis was induced witha combination of TNF and alpha interferon (INF-�), as a positive control. RFLU, relative fluorescence units. (B) Adherent HUVECs were plated andtransduced with Ad.GFP, Ad.FRNK-GFP, or Ad.FRNKS for 16 h under serum-free conditions. The total average cell surface area was calculated usingImage-Pro Plus (Media Cybernetics) software. (C) Adherent HUVECs were placed in serum-free MCDB-131, infected with Ad.GFP or Ad.GFP-FRNKfor 16 h, and then washed with 1� PBS and fixed with 3.7% formaldehyde. Cells were stained with Texas red-phalloidin and DAPI, and images of GFP,actin stress fibers, and nuclear staining were captured under �100 magnification. White arrows indicate localization of GFP-FRNK at focal adhesion sites.



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able (data not shown). These data are consistent with resultsrecently reported for human pulmonary artery endothelial cellsexpressing FRNK (31). Together, these results support theconclusion that under the conditions employed, FRNK expres-sion in HUVECs does not disrupt cell adhesion and spreadingor dramatically alter cell shape.

FAK Y397 phosphorylation is required but not sufficient forendothelial cell proliferation. A previous study in fibroblastsfound that expression of FAK is sufficient to stimulate prolif-eration and that phosphorylation of tyrosine 397, the site ofautophosphorylation, is critical for this effect (84). To furtherinvestigate the role of FAK in endothelial cell proliferation, weexamined the effect of overexpressing wild-type FAK. We alsoinvestigated the role of the Y397 residue that regulates theactivation of FAK (51) and binding of Src, as FRNK expres-sion inhibits the ability of Y397 to be phosphorylated. Wecreated GFP-WT-FAK and GFP-Y397F-FAK adenovirusesand examined their ability to inhibit proliferation in responseto mitogens. These viruses were titrated to achieve similarexpression levels (Fig. 4). Expression of WT-FAK was notsufficient to induce proliferation in endothelial cells, nor did ithave any deleterious or additive effect on the stimulatedgrowth response. Similar results were observed even at expres-sion levels at least 5 times as high as those shown, where FAK397 phosphorylation was superphysiological (data not shown).In contrast, expression of the Y397F mutant resulted in anearly complete inhibition of the mitogen-induced BrdU in-corporation (Fig. 4). These data implicate signal transductiondownstream of FAK 397 phosphorylation as being necessarybut not sufficient for endothelial cell proliferation.

Expression of FRNK does not inhibit ERK signaling butprevents serum-induced degradation of the CDKIs p27 andp21. FAK has been shown to play an important role in theregulation of ERK activation and cyclin D1 expression in othercell types (84, 85); therefore, we tested the effect of FRNKexpression on ERK phosphorylation and cyclin D1 accumula-tion in response to mitogens. Serum induced the phosphory-lation of ERK and JNK; however, expression of FRNK did notdisrupt these signals (Fig. 5A and B). The nuclear transcriptionfactor Elk, an ERK substrate, was also examined since integrinsignaling has been shown to be required for ERK nucleartranslocation (4). However, the expression of FRNK did notinterfere with VEGF-induced Elk phosphorylation (Fig. 5A).Consistent with these findings, the induction of cyclin D inresponse to serum was unaltered by FRNK expression.

The ECM has been shown to alter the expression levels ofp27 and p21 in some cell types. We therefore examined theeffect of FRNK on p27 and p21 expression levels in endothelialcells. We found the levels of both p27 and p21 are elevated inquiescent cells in serum-free media. Upon the addition ofmitogens, the levels of these inhibitors are significantly re-duced. FRNK expression strongly inhibited the degradation ofboth p27 and p21 in response to serum (Fig. 5C), such thatCDKI levels remained high. Since several reports have docu-

FIG. 4. Expression of an Y397F mutant of FAK also inhibits endo-thelial cell proliferation. HUVECs were serum starved and transducedwith Ad.GFP, Ad.GFP-Y397F-FAK, or Ad.GFP-WT-FAK prior to stim-ulation. The cells were then incubated with complete growth medium for16 h. Cells were then pulsed with BrdU and visualized with an anti-BrdUantibody and recorded with a fluorescence digital camera. BrdU-positivecells were quantified, and the data were graphed as the percentage ofpositive cells compared to total cell number.

FIG. 5. Expression of FRNK does not inhibit ERK signaling butdoes prevent serum-induced degradation of the CDKIs p27 andp21. (A) Serum-starved HUVECs were transduced with Ad.GFP,Ad.FRNK, or Ad.FRNKS and then stimulated with complete growthmedium for 16 h and probed with antibodies specific for pJNK, pERK,pElk, and cyclin D1. (B) Serum-starved HUVECs were transducedwith Ad.GFP or Ad.GFP-FRNK prior to stimulation with completegrowth medium for 16 h. Cell lysates were made after 16 h and probedwith antibodies specific for FAK Y397, p27, p21, pRb S795, and ERK2.



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mented the ability of these CDKIs to inhibit the activity of thecyclin-CDK complexes (75), we also examined the phosphory-lation status of the Rb protein. Consistent with the failure ofp27 and p21 to be degraded by serum in the presence ofFRNK, the phosphorylation of Rb at S795 was also inhibited(Fig. 5C). These data suggest that FAK signaling can controlthe levels of cyclin-dependent kinase inhibitors with corre-sponding effects on CDK substrate phosphorylation and sub-sequent cell cycle progression.

FRNK does not alter the mRNA expression of p27 or p21.The levels of both p27 and p21 can be controlled throughseveral different mechanisms, including transcriptional regula-tion and mRNA stability (23, 42, 44, 66). To investigate themechanism of FAK-regulated p27 and p21 expression, we usedquantitative real-time PCR to measure the mRNA levels incells expressing GFP (control) or GFP-FRNK. We found stim-ulation with growth medium produced a modest decrease in p27mRNA levels; however, this decrease was unaffected by the dis-ruption of FAK signaling (Fig. 6A). Analysis of p21 mRNA re-vealed a slight elevation following mitogen stimulation; however,no significant changes in the mRNA levels of p21 were detectedin mitogen-stimulated cells expressing GFP compared to thoseexpressing GFP-FRNK (Fig. 6B). Similar results were seen byNorthern blotting (data not shown). Another possibility was that

FAK might be modulating the expression of these proteinsthrough proteasome-mediated degradation (12). To test this pos-sibility, we treated cells with inhibitors to the 26S proteasome.Treatment of asynchronously growing cells with the inhibitorslactacystin and MG-132 resulted in a robust increase in the pro-tein levels of both p27 and p21, suggesting that there is significantturnover of these proteins that is normally mediated throughproteasomal degradation (Fig. 7A).

FIG. 7. FRNK inhibits mitogen-induced expression of Skp2 proteinand mRNA. Asynchronously growing HUVECs were treated with theproteasome inhibitors lactacystin and MG-132 as shown for 24 h, and thenlysates were analyzed with antibodies specific to p27 and p21. Westernblot analysis was then performed to analyze the levels of Skp2 proteinexpression after the addition of complete growth medium for 16 h (B).HUVECs were treated as in panel B, except mRNA was collected insteadof protein using an RNeasy kit (QIAGEN). The mRNA was then re-versed transcribed, and real-time PCR was performed using primers toSkp2 (C). Data are plotted as mean � standard error. *, statisticallysignificant difference from GFP; #, statistically significant difference fromGFP treated with serum (P 0.05) (n � 5).

FIG. 6. FRNK does not alter the mRNA expression of p27 or p21.Real-time PCR was performed on HUVECs expressing GFP (control)or GFP-FRNK for 16 h in serum-free media, at which time completegrowth medium was added for an additional 16 h to the cells asindicated. The mRNA was then reversed transcribed and analyzed byquantitative real-time PCR using primers specific to p27 (A) and p21(B) with a Bio-Rad real-time PCR machine (A and B). Data arenormalized to the control value and are reported as percentage ofincrease � standard error of three experiments. *, significantly differ-ent from serum-free paired controls (P 0.05). NS, nonsignificantdifference from serum-stimulated GFP controls.



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This piece of data prompted us to examine mechanismsinvolved in the degradation of p27 and p21. Both p27 and 21can be targeted for proteasomal destruction by the F-box pro-tein Skp2 (13, 21, 77). This led us to investigate a potential roleof Skp2 in mediating the FAK-dependent degradation of p27and p21. The level of Skp2 was typically undetectable in qui-escent cells, consistent with the high levels of p27 and p21observed. The addition of serum for 16 h resulted in an in-crease in Skp2 (Fig. 7B) levels that parallels the decrease in thelevels of p27 and p21 (Fig. 5C). Expression of GFP-FRNKmarkedly inhibited the expression of Skp2 (Fig. 7B), correlat-ing well with the corresponding failure of p27 and p21 to bedegraded (Fig. 5C).

The expression of Skp2 is also highly regulated and can beimpacted at both the level of transcription, as well as by post-transcriptional regulation through the anaphase-promotingcomplex/cyclosome (APC/C) (9, 35, 80). In order to ascertainhow Skp2 expression was being regulated in our system, wefirst examined the levels of Skp2 mRNA using real-time PCR.Interestingly, the addition of growth medium for 16 h resultedin an approximate fourfold increase in the level of Skp2mRNA. In contrast, induction of Skp2 mRNA was completelyinhibited in FRNK-expressing cells (Fig. 7C). These data im-plicate the levels of mRNA as a critical control point regulatedby FAK signaling.

Skp2 regulates the FAK-dependent degradation of p27 butnot p21. To determine if Skp2 regulates the FAK-dependentproteasomal degradation of both p27 and p21, HUVECs weretransduced with Ad.GFP or Ad.WT-Skp2 and made quiescentin serum-free media. The cells were then treated with serumfor 16 h, and the levels of p27 and p21 were analyzed byimmunoblotting. After 16 h of serum stimulation, the levels ofp27 and p21 significantly decreased compared to those in qui-escent cells, as previously shown in Fig. 5. Skp2 expressionresulted in decreased p27 levels in quiescent cells as well as incells coexpressing GFP-FRNK, but failed to induce the degra-dation of p21 (Fig. 8A). To further test the role of Skp2 in thedegradation of p27 and p21, a similar experiment was per-formed with cells expressing a dominant-negative form of Skp2(�Fbox-Skp2). The mutant form of this protein while still bind-ing p27 could no longer form a complex with Skp1-CUL1,thereby preventing ubiquitination of the target protein (73).Expression of dominant-negative Skp2 inhibited the serum-induced degradation of p27. However, unlike FRNK, expres-sion of dominant-negative Skp2 did not inhibit serum-inducedp21 degradation (Fig. 8B). These data provide evidence thatSkp2 is required for the majority of p27-mediated degradation.The ability of expressed Skp2 to rescue the FRNK-inhibited p27degradation suggests that the induction of Skp2 is likely a funda-mental control process regulated by FAK. In contrast, the degra-dation of p21 is independent of Skp2 expression, indicating thatfocal adhesion kinase can affect cyclin-dependent kinase inhibitorlevels through at least two separate mechanisms.

Since p21 levels were unaffected by Skp2, we considered ahypothesis alternate to impaired proteasomal degradation thatmight be regulated by FRNK expression. We observed that p21mRNA levels actually increase in response to mitogens in bothcontrol and FRNK-expressing cells: one possibility is that p21mRNA translation is blocked in serum-treated control cellsand that this postulated translational repression is inhibited in

FIG. 8. Skp2 regulates the FAK-dependent degradation of p27 butnot p21. HUVECs were transduced with Ad.GFP, Ad.GFP-FRNK,Ad.WT-Skp2, or Ad.GFP-FRNK and Ad.WT-Skp2 combined in se-rum-free MCDB-131. The HUVECs were then treated with serum for16 h and probed for p27, p21, and ERK (A). HUVECs were trans-duced with Ad.GFP or Ad.�F-box Skp2 treated the same as in panelA and then probed for p27, p21, and ERK2 (B). HUVECs in completegrowth medium were transduced with Ad.GFP or Ad.GFP-FRNK for6 h, treated with 10 �M lactacystin for the indicated times, and thenlysed and probed for p21 protein levels (C). The net intensity of eachband was quantified using a Kodak digital camera along with densi-tometry software (D).



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cells expressing FRNK. To test this, control cells and GFP-FRNK-expressing cells, in the presence of serum, were treatedwith lactacystin and examined for p21 protein expression overa time course. As starting mRNA levels are similar (see Fig. 6),we reasoned that in the absence of proteasome activity, proteinaccumulation over time would be an estimate of translationalcapacity. This experiment predicts that if FRNK-expressingcells do prevent a translational repression of p21 typically in-duced by mitogens; higher levels of p21 should be observed inthe FRNK-expressing cells compared to in the control. Wefollowed p21 protein accumulation over a 36-h time course andfound that p21 protein levels were nearly identical under bothconditions (Fig. 8C). The net intensity of the p21 expressionwas quantified by digital imaging to verify that the changes inp21 expression were numerically similar between control andFRNK-expressing cells (Fig. 8D). Notably, there was no addi-tive effect of FRNK and lactacystin on p21 accumulation at anytime point, arguing against any mechanisms regulated byFRNK that were independent of protein degradation. Thesedata, in conjunction with our other findings, are most con-sistent with the notion that the FAK-dependent regulationof p21 occurs at the level of protein turnover mediated bythe 26S proteasome and not at the level of transcription ortranslation.

Skp2 regulates endothelial cell proliferation through mod-ulation of p27. Based on the sensitivity of Skp2 message andprotein levels to alterations in FAK signaling, we next investi-gated the requirement of Skp2 in endothelial cell proliferationusing siRNA-mediated knockdown of Skp2. We found thatknockdown of Skp2 with siRNA prevented serum-induceddegradation of p27 similar to that seen with the mutant Skp2(Fig. 9A). Furthermore, the knockdown of Skp2 expressioncompletely inhibited the ability of cells to proliferate in re-sponse to serum (Fig. 9B). As Skp2 has also been shown toregulate other proteins important for proliferation, includingcyclin E1 and myc (38, 55), we used siRNA targeting thecombination of Skp2 and p27 to determine if p27 is the keyprotein degraded by Skp2 for proliferation of endothelialcells. The addition of p27 siRNA prevented accumulation ofp27 in quiescent cells as well as cells cotreated with Skp2siRNA (Fig. 9A). Knockdown of p27 completely restored pro-liferation in cells that were treated with serum but lacked Skp2protein (Fig. 9B). This finding suggests that p27 is the principaltarget of Skp2 in regulating the G1/S-phase transition in endothe-lial cells.

Knockdown of p21 and p27 restores the ability of FRNK-expressing endothelial cells to proliferate. Although p21 andp27 are known to be important inhibitors of growth, paradox-ically, Cip/Kip proteins have also been shown to be requiredfor the assembly of the cyclin D-cdk complex (19). As an initialexperiment, we investigated the role of the CDKI proteins onnormal endothelial cell growth control. We found that siRNAduplexes targeted to p21 or p27 specifically knocked down theprotein in question without affecting the other, and the knock-down lowered the level of both proteins to levels at or belowthose seen in serum-treated cells (Fig. 10A). Knockdown ofp21, p27, or the combination of both proteins, was insufficientto promote growth in quiescent cells, underscoring the impor-tance of other mitogen-induced events such as the induction ofcyclin D. Likewise, knockdown of the proteins with the addi-

tion of mitogen did not significantly increase the percentage ofcells entering S phase (Fig. 10B). These data demonstrate thatknockdown of the CDKIs per se does not confer a proliferativeadvantage (or disadvantage) in the context of otherwise nor-mal cellular signaling.

Our data suggested that alterations in FAK signaling controlthe levels of two important CDKIs, p21 and p27, through twoindependent mechanisms. However disruption of FAK signal-ing could have additional effects on cell cycle proteins such ascyclin E, cdk2, or other inhibitors like p57/Kip2 and INK4family members. We used siRNA knockdown of both p21 andp27 to test the role of these proteins in mediating the inhibitionof cell proliferation seen following expression of GFP-FRNK.We found that siRNA-mediated repression of both p21 andp27 completely restored entry into S phase in response tomitogen stimulation in cells expressing FRNK (Fig. 10C). No-tably, individual knockdown of either p21 or p27 displayedintermediate responses, suggesting both CDKIs have growth-suppressive effects (data not shown).

FIG. 9. Skp2 regulates endothelial cell proliferation through mod-ulation of p27. HUVECs were electroporated with 150 to 200 ng ofsiRNA targeted toward luciferase (control), Skp2, or p27 and thenallowed to adhere overnight. The following day, the cells were placedin serum-free MCDB-131 for 16 h and then treated with completegrowth medium for an additional 16 h. A portion of the cells plated in35-mm dishes were then lysed and probed for Skp2, p27, or ERK2 (A).A second portion of the cells plated in a 24-well dish were pulsed withBrdU and analyzed for the percentage that were BrdU positive (B).



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DISCUSSION

Our data indicate that FAK signaling is required for thecontrol of CDKI levels, which are normally lowered in re-sponse to growth factors. In contrast to some other cell types,induction of mitogen-activated protein kinase MAPK activitiesand cyclin D proceeds normally. The regulation imposed by

FAK signaling does not occur through changes in the mRNAlevels of these CDKIs, but rather appears to be mediatedthrough regulation of protein turnover. Furthermore, the reg-ulatory mechanisms appear to bifurcate as the regulation ofp27 was shown to be dependent on Skp2 while p21 degradationoccurs independently of Skp2. Our data indicate that the ad-hesion-dependent, mitogen-stimulated accumulation of Skp2mRNA is dependent on FAK and demonstrate that Skp2 iscritical for endothelial cell proliferation. Finally, we demon-strate that the regulation of these two CDKIs is the criticalregulatory event in the inhibition of proliferation by disruptionof FAK signaling, as enforced knockdown of both CDKIs re-stores mitogen-stimulated proliferation.

The coordinated signaling of growth factors and the ECM isbelieved to be critical for the propagation of adherent endo-thelial cells. Supporting this notion is the accumulation ofprogrowth ECM molecules, including a fibronectin-rich inter-stitial matrix, during the remodeling/proliferative stage of an-giogenesis. This is in sharp contrast to the laminin-rich ECMfound in the maturation phase of angiogenesis as the cells formcapillaries and display negative growth properties (62). Onepotential mechanism to account for the ability of particularECM proteins to impart such dramatically different prolifera-tive phenotypes involves signal transduction through distinctintegrins and their ability to form focal adhesions and activateFAK (68).

Focal adhesion kinase is a key integrator of signals fromgrowth factors, the ECM, as well as mechanical stimuli. Inendothelial cells, FAK becomes highly phosphorylated in re-sponse to VEGF, plating on fibronectin, and in response tolaminar flow (2, 40, 45). The ability of FAK to become auto-phosphorylated at Tyr 397 and become activated in response tothese diverse stimuli is dependent on cell adhesion. The de-creases in the levels of the CDKIs p27 and p21 required for cellcycle progression have been reported to be dependent on celladhesion as well as growth factor stimulation. Studies alongwith this one have recently shown that adhesion-dependentdegradation of p27 and p21 can be regulated via proteasomaldegradation (8, 39, 57). Our findings suggest that the abilitiesof adherent cells to degrade p27 and p21 in response to manydiverse stimuli likely converge at the level of FAK signaling.This finding is important because it provides a possible singleexplanation for the numerous and diverse findings that cellshape, cell size, adhesion, and mitogens, as well as mechanicalforces can affect cell proliferation and/or CDKI levels (5, 36,46, 79), since all of these environmental stimuli are also knownto alter FAK signaling.

Phosphorylation of FAK Y397 appears to be a critical de-terminant for the cell to transition successfully from G1 to Sphase. We do not believe this is a response unique to expres-sion of FRNK, as we see identical results when FAK Y397F isexpressed, but not WT-FAK. Moreover, recent data from Shenet al. demonstrate that endothelial cells derived from mice witha floxed FAK allele have defects in BrDU incorporation fol-lowing deletion of the FAK allele with Cre-recombinase (67),underpinning a critical role for FAK in endothelial cell prolif-eration. In preliminary experiments, we have attempted to useoligonucleotide-based siRNA to knock down FAK expression.While we have achieved moderate knockdown of FAK protein(approximately 70% inhibited), effects on FAK 397 phosphor-

FIG. 10. Knockdown of p21 and p27 restores the ability of FRNK-expressing endothelial cells to proliferate. HUVECs were electropo-rated with 150 to 200 ng of siRNA targeted toward luciferase (control),p21, and p27. The cells were then treated exactly as described in thelegend to Fig. 9 and probed for p27 and p21 (A) and for the percentageof BrdU-positive cells (B). HUVECs were first electroporated with thespecified siRNA as in panel A and on the following day during serumstarvation were also transduced with Ad.GFP or Ad.GFP-FRNK. Thenext day, the cells were stimulated with complete growth medium for16 h, at which point they were pulsed with BrdU and the percentage ofBrdU-positive cells was calculated (C).



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ylation have been less dramatic (typically 30 to 50% inhibited).Under these conditions, it is important to note that cell cycleprogression does not appear to be disrupted. Taken together,these observations suggest that the FAK-dependent signallikely represents a checkpoint-like mechanism with a relativelysmall threshold (FAK 397 phosphorylation has to be greaterthan zero but can be less than 50%) in order to trigger CDKIdegradation. Mitogen enhancement of FAK phosphorylationmay not have to take place, but rather sufficient engagementwith the substratum and cellular tension may need to be inplace to maintain a level of FAK phosphorylation as a permis-sive step. Studies are currently under way with vector-basedinducible siRNA technologies to further define the lower limitof FAK expression (and/or 397 phosphorylation), as well asconditions which may alter those limits.

Our data demonstrate how crucial the degradation of theCip/Kip CDKIs is in regulating endothelial growth, as suppres-sion of p27 and p21 was capable of completely restoring pro-liferation in the presence of disrupted FAK signaling. Con-versely, a somewhat surprising finding was that knockdown ofp21 and p27 had no effect on normal endothelial cell prolifer-ation, as previous reports have suggested Cip/Kip proteins arerequired for the assembly of the active cyclin D-cdk complexes(19, 43). Our data would suggest that this function is notcritical in endothelial cells, as knockdown of p27 and p21individually or in tandem did not impede proliferation. Alter-natively, it may be that the levels required for cyclin-cdk com-plex assembly is quite low and below the threshold of ourknockdown.

Different cell systems have evolved multiple mechanisms toregulate CDKIs. Mechanisms described to date include nuclear-cytoplasmic shuttling, transcriptional regulation, and mRNA sta-bilization, as well as proteasomal degradation (7, 11, 14, 37, 41,42, 59). Previous studies have indicated both p27 and p21 canbe regulated through transcriptional mechanisms in endothe-lial cells (23, 74). For instance, the inhibition of FOXO tran-scription factors with RNA interference has been shown todecrease p27 levels, thereby promoting cell proliferation (1,58). While FAK could theoretically alter the activation ofFOXO factors through modulation of phosphatidylinositol-3�-kinase (PI 3�-kinase) signaling, our data would suggest this isnot occurring, as the decrease in p27 mRNA in response tomitogens occurred normally following disruption of FAK sig-naling. Therefore, it is also unlikely that PI 3�-kinase-mediatedchanges in p21 and p27 message are involved, which is inagreement with findings that PI 3�-kinase may not be essentialfor endothelial cell proliferation (50, 82). Moreover, serum-induced suppression of p27 mRNA levels is not sufficient tosubstantially alter p27 protein levels when the induction ofSkp2 is blocked. Likewise, we have shown that loss of Skp2expression completely inhibits endothelial cell proliferation,which requires only the knockdown of p27 to be completelyrestored. Together, these findings underscore the importanceof inducing Skp2 for endothelial cell proliferation.

The oncogene Skp2 has been found to be important in theprogression of numerous tumors and, based on our data, iscritical for the growth of primary endothelial cells as well (10,22). We have found that induction of Skp2 mRNA accumula-tion is FAK dependent. This finding provides a biochemicalmechanism for the adhesion-dependent accumulation of Skp2

previously described in fibroblasts (16). This finding may alsohave significant implications in certain cancers such as malig-nant gliomas, melanomas, and ovarian carcinoma (56, 71,86)—all of which maintain elevated levels of FAK phosphory-lation independently of adhesion (29). This could provide amechanism for adhesion-independent expression of Skp2 andcell cycle progression in these tumor types. In endothelial cells,our data would suggest that FAK activation alone is not suf-ficient to stimulate cell cycle proliferation, suggesting a re-quirement for simultaneous activation of growth factor signals.

Currently little is known about the regulation of Skp2, par-ticularly in nontransformed cells, although reports have sug-gested that Skp2 regulation is dependent on Ras pathways (53,63). However, we do not see an inhibition of ERK signaling inFRNK-expressing cells and PI 3�-kinase does not appear to berequired for growth in endothelial cells (50, 82). Consistentwith this, attempts to rescue the FRNK-induced proliferationblock with activated forms of Raf and Akt were ineffective(data not shown). While these data do not discount the possi-bility that Ras-related signaling may impinge on Skp2 regula-tion, they do suggest that FAK-derived signals act as an alter-nate or parallel control mechanism.

Skp2 levels can be regulated by proteasomal degradationthrough the APC/C complex (9, 80). A recent report onsmooth muscle cells suggested that FAK signaling could mod-ulate the levels of Skp2 by controlling protein degradation, asthey saw no alterations in Skp2 mRNA levels (12). In contrast,we find that Skp2 mRNA levels are robustly induced by mito-gens and that this enhancement is FAK dependent. In addi-tion, disruption of FAK signal transduction did not alter thelevels of ectopically expressed Skp2 in endothelial cells, argu-ing against alterations in the protein degradation machinery asbeing the primary mechanism controlling Skp2 levels (data notshown). To date, few studies have investigated the Skp2 pro-moter. To date only the Ets-related transcription factor,GABP, and E2F have been shown to be transcriptional acti-vators of the Skp2 promoter (35, 83). Further study will berequired to determine if these transcription factors are medi-ating the induction of Skp2 mRNA regulated by FAK in en-dothelial cells.

Although the precise downstream signals through whichFAK regulates Skp2/p27 and p21 remain to be clarified, arecent study examining the role of FAK in endothelial perme-ability has found that expression of FRNK inhibits Rac activitywhile simultaneously activating Rho (31). Our data also foundthat expression of FRNK seems to inhibit ruffling while en-hancing stress fibers under resting conditions. The possibilitythat FAK is functioning to regulate small GTPases is intriguingbased on evidence that Rho manipulation can control p27 andthat Rac can modulate p21 (8, 47). However this hypothesisneeds to be approached cautiously, as alterations in Rho andRac activity are known to regulate FAK activation.

We find that FAK signaling is required for the loss of p21protein in response to serum. Previous reports have implicatedSkp2 as being an important component of p21 degradation:largely based on experiments conducted in vitro. Our datasuggest that in primary endothelial cells the regulation of p21appears to be independent of Skp2 expression as (i) enforcedexpression of Skp2 (even at supraphysiological levels) waswithout effect on p21 levels and (ii) expression of an F-box



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mutant which blocks targeting to the Cul1 E3-ligase did notdisrupt the degradation of p21, while completely inhibiting thedegradation of p27. These data are consistent with a report infibroblasts which also suggested regulation of p21 was Skp2independent (72) and with a recent report that indicates thatp21 cannot be ubiquitinated in vivo due to the presence ofN-terminal acetylation (18). These data support the notionthat p21 proteolysis does not necessarily require ubiquitination(66) and may be explained by the ability of p21 to directly bindto the C8-� subunit of the 20S proteasome (76); however,these possibilities remain to be validated in intact cells.

Our data underscore the significance of cell-type-specificsignal transduction. Previous studies investigating FAK andcell cycle progression in fibroblasts have found a requirementfor FAK in the regulation of ERK activation and cyclin Dexpression (85) and a sufficiency of FAK expression to inducecellular proliferation. In contrast, we find ERK activation andcyclin D1 induction proceed normally following disruption ofFAK signaling in endothelial cells. It should be noted that wefind an absolute dependence on FAK for activation of ERK inprimary fibroblasts (data not shown), in agreement with theprevious finding. Similarly, our data reveal a role for FAK inregulating mRNA levels of Skp2, while in smooth muscle cells,FAK affects Skp2 stability, not mRNA levels. These data sug-gest that different cell types have unique cell cycle controlnetworks that could be exploited to develop therapeutics withmore selective antiproliferative actions. In addition, our datahighlight the significance of proteasomal degradation in theproliferation control of endothelial cells. A clinical study usingthe proteasome inhibitor bortezomib for the treatment of mul-tiple myeloma found that the drug also impaired angiogenesis(61). Consistent with this, we have found that treatment ofHUVECs with the proteasome inhibitor MG-132 inhibitsVEGF-induced growth (data not shown). Collectively, ourdata suggest that targeting the proteasomal degradation ofCDKIs may be a useful approach for the treatment of diseasesdependent upon endothelial cell proliferation, such as prolif-erative retinopathies, hemangiomas, and tumor angiogenesis.

ACKNOWLEDGMENTS

This work was supported by CA81419 from the National CancerInstitute and the David E. Bryant Trust for Research in Blindness(K.P.). This work also received support through an institutional pre-doctoral training grant (T32-HL-07194) and an individual predoctoralaward from the American Heart Association (P.B.).

We acknowledge the assistance of Hanqui Zheng for his help inestablishing conditions for siRNA knockdown of the CDKIs. We thankMark Bond (University of Bristol) for generously providing the WT-Skp2 and Skp2 adenoviruses with the F-box deleted; Allen Samareland Maria Heidkamp (Loyola University Medical Center) for theGFP-FRNK adenovirus, and Rebecca Keller (Albany Medical Col-lege) for the FRNK and FRNKS adenoviruses. In addition, we ac-knowledge the support of the Developmental Therapeutics Program ofthe National Cancer Institute for providing VEGF and endothelialcells.

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Focal Adhesion Kinase Controls Cellular Levels of p27/Kip1 and p21/Cip1 through Skp2-Dependent and...

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