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Differential requirement for MEK Partner 1 in DU145 prostate cancer cell migration
© Park et al; licensee BioMed Central Ltd. 2009
- Received: 16 July 2009
- Accepted: 23 November 2009
- Published: 23 November 2009
ERK signaling regulates focal adhesion disassembly during cell movement, and increased ERK signaling frequently contributes to enhanced motility of human tumor cells. We previously found that the ERK scaffold MEK Partner 1 (MP1) is required for focal adhesion disassembly in fibroblasts. Here we test the hypothesis that MP1-dependent ERK signaling regulates motility of DU145 prostate cancer cells. We find that MP1 is required for motility on fibronectin, but not for motility stimulated by serum or EGF. Surprisingly, MP1 appears not to function through its known binding partners MEK1 or PAK1, suggesting the existence of a novel pathway by which MP1 can regulate motility on fibronectin. MP1 may function by regulating the stability or expression of paxillin, a key regulator of motility.
- Focal Adhesion Kinase
- Focal Adhesion
- DU145 Cell
- Wound Edge
Prostate cancer is the most commonly diagnosed cancer in men. While in situ disease usually responds well to treatment, progression to an androgen-hypersensitive, metastatic state is often fatal . Aberrant signaling through the ERK pathway correlates with progression in some models , although oncogenic mutations in Ras or Raf occur infrequently in prostate cancer [3, 4]. Rather, prostate cancer commonly displays misregulation of cell-matrix adhesion signaling, including alterations in integrin expression and upregulation of Focal Adhesion Kinase (FAK) and p21-Activated Kinase (PAK) [5, 6]. We have previously shown that PAK1 can activate MEK1 independently of Raf in model systems  suggesting that this alternative pathway to ERK activation may function in tumor cells lacking oncogenic mutations in Ras and Raf. Indeed, active PAK1 has been shown to stimulate ERK-dependent proliferation of breast cancer cells , and is constitutively active in DU145 prostate cancer cells .
MEK Partner 1 (MP1) is a putative scaffolding protein that binds MEK1 and ERK . MP1 also binds to the late endosomal protein p14 , and the p14-MP1-MEK1 complex regulates trafficking and degradation of the EGFR . Thus MP1/p14 provides a means of colocalizing the endocytosed EGFR and its downstream effectors during proliferation stimulated by EGF. MP1 and p14 also play important roles in cell morphogenesis and migration. MP1 and/or p14 are required for efficient activation of MEK and ERK, and control focal adhesion and actin dynamics during adhesion to fibronectin . Since ERK is known to regulate focal adhesion disassembly [14, 15], these observations implicate MP1-scaffolded ERK in focal adhesion dynamics. However, MP1 also binds active PAK1 , itself a regulator of adhesion disassembly [13, 16, 17], consistent with alternative mechanisms for its involvement in the regulation of cytoskeletal and adhesion dynamics . Elevated PAK activity is associated with altered growth and migratory properties in a number of tumor types, including prostate cancer . We therefore investigated whether MP1 influences ERK and/or PAK signaling during migration of DU145 prostate cancer cells.
EGF (Invitrogen) and UO126 were from Calbiochem. Gridded coverslips were from Bellco; glass-bottomed 35 mm microwell dishes were from MatTek Cultureware. Both were coated with fibronectin as described .
DU145 were maintained in RPMI-1640 containing 10% fetal bovine serum (FBS, Invitrogen).
Cells (4.5 × 105 cells/100 mm dish plated two days prior to transfection) were transfected with 100 nM control siRNA (AGAGAGUAGUAGAGACAAUCGUCUG), MP1 Stealth siRNA (CAACACAGGACUAAUUGUCAGCCUA) or p14 Stealth siRNA (UUAAGAUGCCGCCACUUGGGUGAGG) (Invitrogen) using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions. Experiments were performed 72 hours post-transfection.
Cells were typically lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 5 mM EDTA, 50 mM NaF) supplemented with 1 μg/mL Leupeptin, 3 mM Benzamidine, 10 nM MicrocystinLR, 100 μM orthovanadate, 5 mM NaPPi, and 1 mM PMSF. Equal amounts of lysate protein were used for immunoblots. Proteins were detected using the following antibodies: MEK1 phospho-S298 ; MP1 ; MEK1 phospho-S218/S222 (Sigma); phosphoERK1/2 (clone12D4; Upstate) ERK1/2 phospho-T202/Y204 (Cell Signaling); paxillin (Biosource); PAK1 N-20 (Santa Cruz); pS141 PAK1-3 (Invitrogen). p14 antibody #5933 was raised against the peptide sequence Ac-CLKAKAQALVQYLEEPLTQVAAS-OH and affinity purified using standard techniques. Blots were imaged on an LAS-1000 Plus Intelligent Dark Box (Fujifilm) using Image Reader software (Fujifilm); the same software was used for quantification of immunoblot results.
Adhesion/EGF stimulation assay
Cells were plated on fibronectin essentially as described . For EGF stimulation, cells were washed in serum-free (SF) medium then fed SF medium for 2 hours prior to stimulation.
Cells were trypsinized and collected in SF medium containing 1 mg/mL soybean trypsin inhibitor (Sigma), pelleted, suspended in SF medium and incubated at 37°C for two hours. 2 × 106 cells were allowed to form confluent monolayers on fibronectin-coated grid-etched coverslips for 5-7 hours at 37°C. Cells were treated with 1 μg/mL Mitomycin C (Sigma) and either 25 μM UO126 or DMSO vehicle for an additional 3 hours. The medium was aspirated and the coverslip was scratched 4 times with a 200 μL tip to form a # wound pattern. Loose cells were removed with SF medium; cells were fed fresh SF medium containing 1 μg/mL of Mitomycin C and either 25 μM UO126 or the equivalent volume of DMSO, with or without 1% FBS or 10 ng/mL EGF as indicated. Wounds were photographed in 6 different locations at zero-hour time point, and then incubated at 37°C for 5 hours (transfected cells) or 10 hours (untransfected cells) at which time the same locations were photographed. All photographs were taken with an Olympus IX81 microscope equipped with Slidebook software (Intelligent Imaging Innovations) and the area covered by migrating cells was calculated using ImageJ software (NIH). Parallel cultures were processed for immunoblotting.
Immunofluorescence was carried out essentially as described  using a Leica DMRA2 microscope equipped with Slidebook software. The number and size of vinculin-positive focal adhesions at the wound edge was calculated using Slidebook software.
2× 106 cells were plated on 35 mm FN-coated glass-bottomed Petri dishes in SF medium with 1 μg/mL Mitomycin C and allowed to adhere for 5-7 hours. Confluent monolayers were wounded and imaged starting 10-20 min post wounding and again after 2 hours of migration. All cells were imaged for 5 minutes at 1 frame per second on an Olympus IX81 microscope equipped with Slidebook software. Kymographs were generated using ImageJ software.
Wound-healing assays were repeated in triplicate and statistical analysis was performed on the pooled results. A general linear model was constructed and linear contrast was used to perform pairwise comparisons of the means for the combined data set. Tukey's Honestly Significant test was used for post-hoc pairwise comparisons of migration under experimental conditions to that of migration under SF conditions and to each other. ANOVA on pooled data indicated significant differences within the data set.
ERK activity is required for growth factor-, but not fibronectin-stimulated, DU145 migration
MP1/P14 is required for migration of DU145 cells on fibronectin
MP1 depletion decreases membrane activity and peripheral focal adhesions in migrating cells
To examine focal adhesions in these assays, a monolayer was wounded and fixed after cells had migrated for 5 hrs. Control- and MP1 siRNA-transfected cells display adhesions of similar total number and size distribution (Fig. 3B, C). However, MP1 siRNA-transfected cells have ~50% fewer adhesions at the wound edge (Fig. 3B; defined as adhesions occurring between the cell periphery and the cortical actin ring, not shown; vinculin spots occurring at sites of cell-cell contact were not counted as focal adhesions). This suggests that early nucleation events required for adhesion formation at the wound edge occur at a lower frequency in cells depleted of MP1/p14, or that turnover of this population of adhesions is more pronounced in cells lacking MP1/p14.
Depletion of MP1/p14 modestly inhibits PAK activity
Paxillin is largely absent in DU145 cells depleted of MP1/p14
To extend these observations, we asked whether PAK phosphorylation of the cytoskeletal linker/adapter paxillin was regulated by MP1/p14. Paxillin has been shown to play essential roles in focal adhesion dynamics, membrane protrusion and motility [14, 27–29]. PAK phosphorylates paxillin on S273, and phosphorylation of this site is required for stabilizing membrane protrusion . Surprisingly, the abundance of paxillin was substantially reduced in cells depleted of MP1/p14 (~60% reduction, Fig. 4B), suggesting that paxillin is poorly expressed or unstable in cells lacking MP1/p14, or that it resides in a compartment not extracted by our lysis buffer. To exclude the latter possibility, we prepared extracts in SDS-PAGE sample buffer which is expected to solubilize all cellular proteins. The abundance of paxillin was similarly reduced (by ~60%) in SDS-PAGE buffer extracts of DU145 cells transfected with MP1 siRNA, while expression of other focal adhesion markers vinculin, talin and zyxin was unaffected by knock-down of MP1/p14 (Fig. 4B). Reduction of paxillin levels is unlikely to result from off-target effects of the MP1 siRNA since similar results were obtained with an independent p14 siRNA (Fig. 4C). These data indicate that the MP1 and/or p14 are required to maintain normal paxillin protein levels.
MP1/p14 regulated signaling events
MP1/p14 is essential for ERK activation during adhesion of fibroblasts to fibronectin, and in this context, depletion of MP1/p14 causes an apparent stabilization of focal adhesions . We therefore predicted that depletion of MP1/p14 would cause inhibition of fibronectin-stimulated ERK activation and stabilization of focal adhesions in DU145 cells, and thus inhibit cell motility. To our surprise, we found that migration of DU145 cells on fibronectin does not require acute MEK activity but does require MP1/p14. Conversely, migration stimulated by growth factors requires acute MEK activity, but does not require MP1/p14. Furthermore, MP1/p14 depletion does not significantly inhibit ERK phosphorylation in response to fibronectin or growth factor stimulation. These data indicate that there are separable MEK activity- and MP1/p14-dependent pathways leading to DU145 motility in response to serum growth factors and fibronectin respectively.
Because Group I PAKs have multiple roles in cell motility  and PAK1 binds MP1 , we reasoned that MP1 might function through PAK to regulate fibronectin-stimulated DU145 migration. However, depletion of MP1 caused only modest differences in PAK autophosphorylation or PAK phosphorylation of MEK1; phosphorylation of the PAK-dependent substrate cofilin was also unaffected by depletion of MP1 (data not shown). These data suggest that PAK is not a major effector for MP1 in DU145 cell motility.
MP1/p14 is required for maintenance of paxillin levels
Surprisingly, we found that paxillin was largely absent from DU145 cells depleted of MP1/P14 while expression of other focal adhesion proteins was unaffected. Future experiments will determine whether this loss of paxillin results from decreased mRNA or protein synthesis, or increased turnover of paxillin protein.
Depletion of MP1/p14 influences membrane dynamics and focal adhesion abundance
We have found that depletion of MP1/p14 results in a loss of paxillin, decreases membrane protrusion, and reduces the number of focal adhesions at the wound edge. Paxillin serves as a signaling nexus at focal adhesions and in the leading edge of migrating cells. In particular, the GIT1-PIX-PAK complex is stabilized by PAK phosphorylation of paxillin, leading to increased localized Rac activation, membrane protrusion, and migration . This complex localizes to a population of dynamic focal complexes at the leading edge [14, 24], suggesting that it may be important for stabilizing membrane protrusion. Alternatively, paxillin may liberate p190A RhoGAP from p120 RasGAP at the leading edge of motile cells . Locally released p190A RhoGAP is free to stimulate hydrolysis of GTP on Rho allowing membrane protrusion. Interestingly, while the loss of paxillin correlates with a failure of DU145 to migrate on fibronectin, it does not preclude serum-stimulated motility.
Our data are the first to indicate that MP1/P14 has important functional roles outside the ERK pathway. These observations suggest that MP1/p14- and paxillin-dependent and -independent pathways for motility may be operational in DU145 cells in a context-specific manner.
This work was supported by Department of Defense Prostate Cancer Research Program Predoctoral Traineeship Award W81XWH-05-1-0591 (E.R.P.) and National Institutes of Health RO1 GM068111 (A.D.C.). We thank Dr. Becky Worthylake for her assistance with the imaging experiments and Dr. Wayne Orr (LSUHSC, Shreveport) for helpful discussions.
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