Protein kinase Cvarepsilon mediates Stat3Ser727 phosphorylation, Stat3-regulated gene expression, and cell invasion in various human cancer cell lines through integration with MAPK cascade (RAF-1, MEK1/2, and ERK1/2)

Abstract

Protein kinase C epsilon (PKCvarepsilon), a novel calcium-independent PKC isoform, has been shown to be a transforming oncogene. PKCvarepsilon-mediated oncogenic activity is linked to its ability to promote cell survival. However, the mechanisms by which PKCvarepsilon signals cell survival remain elusive. We found that signal transducers and activators of transcription 3 (Stat3), which is constitutively activated in a wide variety of human cancers, is a protein partner of PKCvarepsilon. Stat3 has two conserved amino-acid (Tyr705 and Ser727) residues, which are phosphorylated during Stat3 activation. PKCvarepsilon interacts with Stat3alpha isoform, which has Ser727, and not with Stat3beta isoform, which lacks Ser727. PKCvarepsilon-Stat3 interaction and Stat3Ser727 phosphorylation was initially observed during induction of squamous cell carcinomas and in prostate cancer. Now we present that (1) PKCvarepsilon physically interacts with Stat3alpha isoform in various human cancer cells: skin melanomas (MeWo and WM266-4), gliomas (T98G and MO59K), bladder (RT-4 and UM-UC-3), colon (Caco-2), lung (H1650), pancreatic (PANC-1), and breast (MCF-7 and MDA:MB-231); (2) inhibition of PKCvarepsilon expression using specific siRNA inhibits Stat3Ser727 phosphorylation, Stat3-DNA binding, Stat3-regulated gene expression as well as cell invasion; and (3) PKCvarepsilon mediates Stat3Ser727 phosphorylation through integration with the MAPK cascade (RAF-1, MEK1/2, and ERK1/2). The results indicate that PKCvarepsilon-mediated Stat3Ser727 phosphorylation is essential for constitutive activation of Stat3 and cell invasion in various human cancers.

Figure 1. PKCε associates with Stat3 in human glioblastoma and breast cancer cells. (a): PKCε and Stat3 expression in glioblastoma and breast cancer cells

Cells at 70–80% confluency were homogenized in IP lysis buffer as described in Materials and Methods. 25 µg protein of whole cell lysates were fractionated by SDS-PAGE and immunoblotted (IB) with the indicated antibodies. β-actin was used as a control for gel loading variations. b: Protein quantification (normalized to β-actin) was performed as described in Materials and Methods. c: Association of PKCε with Stat3 in human (i) glioblastoma (T98G and M059K) and (ii) breast (MCF-7 and MDA:MB-231) cancer cell lines. Whole-cell lysates were used for immunoprecipitation (IP) with the indicated antibodies. The immunoprecipitated samples were analyzed by Western blot (IB) using the indicated antibodies. (iii): Whole cell lysate of the indicated cells were incubated with the indicated antibodies alone or in combination with their blocking peptide (1µg/mL) at 4°C for overnight before the immunoprecipitation. The immunoprecipitated samples were analyzed by Western blot (IB) using the indicated antibodies. d: double immunofluorescence indicates localization of PKCε and Stat3 in glioblastoma (T98G) and breast cancer cells (MCF-7 and MDA:MB-231). Localization of PKCε and Stat3 is shown by green and red fluorescence, respectively. Colocalization of PKCε with Stat3 is shown by yellow fluorescence. Images were captured at 20 × Magnification.

Figure 2. PKCε associates with Stat3 in human melanoma, lung, pancreatic, bladder and colon cancer cells. (a): Basal PKCε and Stat3 expression in human melanoma, lung, pancreatic, bladder and colon cancer cells

Cells at 70–80% confluency were homogenized in IP lysis buffer as described in Materials and Methods. 25 µg of whole cell lysates were fractionated by SDS-PAGE and immunoblotted (IB) for individual antibodies. β-actin was used as a control for gel loading variations. b: Protein quantification (normalized to β-actin) was performed as described in Materials and Methods. c: Association of PKCε with Stat3 in human (i) melanoma (MeWo and WM266-4), (ii) lung (H1650), colon (Caco-2), pancreatic (PANC-1) and (iii) bladder (RT-4 and UM-UC-3), cancer cell lines. Whole-cell lysates were used for IP with the indicated antibodies. The immunoprecipitated samples were analyzed by Western blot (IB) using the indicated antibodies. d: Double immunofluorescence localization of PKCε and Stat3 in (i) melanoma (MeWo and WM266-4), (ii) bladder (RT-4) and pancreatic (PANC-1) cancer cells. Localization of PKCε and Stat3 is shown by green and red fluorescence, respectively. Colocalization of PKCε with Stat3 is shown by yellow fluorescence. Images were captured at 20 × Magnification

Figure 3. PKCε mediates phosphorylation of Stat3Ser727, Stat3-regulated genes expression and cell invasion in human cancer cells

Melanoma (WM266-4), glioma (T98G), pancreatic (PANC-1), and lung (H1650) cancer cells were transfected with 15 µg of non-targeting siRNA plasmid (lane 1) or PKCε specific siRNA plasmid (lane 2) (Ambion, Austin, TX) for 48hr and whole cell lysates were prepared as described in Materials and Methods. The whole cell lysates (25 µg protein) were immunoblotted and indicated protein expression levels were detected with appropriate antibodies. β-actin was used as a control for gel loading variations. Protein quantification (normalized to β-actin) was done as described in Materials and Methods (right side). Expression levels of: a (i and ii), PKC isoforms (PKCε, PKCδ and PKCα), b (i and ii), pStat3Ser727, pStat3Tyr705, Stat3, and c (i and ii), Stat3 regulated genes (Bcl-xL, cdc25A and COX-2). d: Human cancer cell invasion. Cells were transfected with non-targeting siRNA plasmid or PKCε specific siRNA plasmid (Ambion, Austin, TX) and cell invasion was determined as described in Materials and Methods. (i): Photographs of invading cells. The migrant cells were stained with crystal violet and photographed the invading cells (40× magnification), (ii): Number of invading cells was estimated by colorimetric measurements at 560 nm according to assay instructions (Chemicon International, Temecula, CA). Each value in the graph is the mean ± S.E. from three separate wells.

Figure 4. PKCε integrates with MAPK cascade to phosphorylate Stat3Ser727

a: Tissue extracts of prostate cancer from TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice (50 µg protein) were used for reciprocal IP experiments with antibodies specific to PKCε, Stat3, Raf-1, MEK1/2, and ERK1/2. The immunoprecipitates were subjected to western blot analysis using the indicated antibodies. b, c and d: DU145 cells were transfected with non-targeting siRNA (Lanes 1 and 2) or PKCε specific siRNA (Lanes 3 and 4) (from Dharmacon Inc., Lafayette, CO), and whole cell lysates were prepared as described before (3). The lysates (25 µg protein) were immunoblotted and indicated protein expression levels were detected with the appropriate antibodies. β-actin was used as a control for gel loading variations. Protein quantification (normalized to β-actin) was performed as described in Materials and Methods. Each value is the mean ± S.E. of three independent experiments. e: EMSA. DU145 total cells were suspended in buffer A [10 mmol/L HEPES (pH 7.9), 1.5 mmol/L MagCl₂, 10 mmol/L KCl, 0.5 mmol/L DTT, 0.2 mmol/L PMSF]. After 15 min of incubation on ice, the cells were pelleted and resuspended in buffer B [20 mmol/L HEPES (pH 7.9), 20 mmol/L NaF, 1.5 mmol/L MgCl₂, 1 mmol/L Na₃VO₄, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT, 0.5 mmol/L PMSF, 420 mmol/L NaCl, 20% glycerol, 1 µg/mL leupeptin, 1 µg/mL aprotinin]. The samples were then centrifuged and the clear supernatant was used for EMSA as described in Materials and Methods. Lane 1, free probe only, Lane 2, nontargeting; lane 3, PKCε siRNA and Lane 4, mutant probe.

Figure 5. PKCε integrates with ERK1/2 to phosphorylate Stat3Ser727

DU145 cells were untransfected (lane 1), or transfected with nontargeting ERK1/2 siRNA (lane 2), or ERK1/2 specific siRNA (lane 3), or nontargeting PKCε siRNA (lane 4), or PKCε-specific siRNA (lane 5) (ERK1/2 specific siRNA from Santa Cruz Biotechnology, Santa Cruz, CA and PKCε-specific siRNA from Dharmacon Inc., Lafayette, CO), for 48hr and whole-cell lysates were prepared as described in Materials and Methods. a: The protein extracts (25 µg protein) were immunoblotted and indicated protein expression levels were detected with the appropriate antibodies. β-actin was used as a control for gel loading variations. b: The quantification of proteins (normalized to β-actin) was done as described in Materials and Methods. i: % of control not treated with PKCε-siRNA, ii: % of control not treated with ERK1/2-siRNA.