Novel signaling axis for ROS generation during K-Ras-induced cellular transformation

Abstract

Reactive oxygen species (ROS) are well known to be involved in oncogene-mediated cellular transformation. However, the regulatory mechanisms underlying ROS generation in oncogene-transformed cells are unclear. In the present study, we found that oncogenic K-Ras induces ROS generation through activation of NADPH oxidase 1 (NOX1), which is a critical regulator for the K-Ras-induced cellular transformation. NOX1 was activated by K-Ras-dependent translocation of p47(phox), a subunit of NOX1 to plasma membrane. Of note, PKCδ, when it was activated by PDPK1, directly bound to the SH3-N domain of p47(phox) and catalyzed the phosphorylation on Ser348 and Ser473 residues of p47(phox) C-terminal in a K-Ras-dependent manner, finally leading to its membrane translocation. Notably, oncogenic K-Ras activated all MAPKs (JNK, ERK and p38); however, only p38 was involved in p47(phox)-NOX1-dependent ROS generation and consequent transformation. Importantly, K-Ras-induced activation of p38 led to an activation of PDPK1, which then signals through PKCδ, p47(phox) and NOX1. In agreement with the mechanism, inhibition of p38, PDPK1, PKCδ, p47(phox) or NOX1 effectively blocked K-Ras-induced ROS generation, anchorage-independent colony formation and tumor formation. Taken together, our findings demonstrated that oncogenic K-Ras activates the signaling cascade p38/PDPK1/PKCδ/p47(phox)/NOX1 for ROS generation and consequent malignant cellular transformation.

Figure 1

K-Ras^V12 expression causes ROS generation and consequent malignant transformation in normal fibroblasts. (a) Levels of ROS as assessed by dichloro-dihydro-fluorescein diacetate (DCFDA). ROS is gradually increased in Rat2 fibroblasts after transduction with K-Ras^V12, compared with control vector MFG-transduced cells. (b) Levels of ROS as assessed by DCFDA fluorescence. Ectopic expression of antioxidant enzymes catalase or GPX blocks K-Ras-induced ROS generation. (c) Anchorage-independent colony formation in soft agar. Ectopic expression of antioxidant enzymes catalase or GPX attenuates K-Ras-induced colony-forming ability in soft agar. (d) Tumor formation in xenograft mice. Ectopic expression of antioxidant enzymes catalase or GPX in Rat2 fibroblasts suppresses K-Ras-induced tumor formation. Error bars represent mean±S.D. of triplicate samples. *P<0.001

Figure 2

NOX1 and p47^phox are required for K-Ras-induced ROS generation and subsequent malignant transformation. (a) Levels of ROS as assessed by DCFDA fluorescence in Rat2 cells or K-Ras^V12-transduced cells after transfection with siRNA targeting NOX1, NOX2, NOX3, NOX4, or scrambled siRNA. (b) Soft agar colony formation of K-Ras-transduced cells after transfection with siRNA targeting NOX1, NOX2, NOX3, NOX4, or scrambled siRNA and control vector MFG-transduced cells. (c) Levels of ROS as assessed by DCFDA fluorescence in K-Ras^V12-transduced cells after transfection with siRNA targeting p47^phox, Noxo1, or scrambled siRNA. (d) Soft agar colony formation of K-Ras^V12-transduced cells after transfection with siRNA targeting p47^phox, Noxo1, or scrambled siRNA and control vector MFG-transduced cells. (e) Western blot analysis for p47^phox after immunoprecipitation with anti-phospho-serine, -phospho-threonine or -phospho-tyrosine antibody or particulate after separation of cytosol and particulate. Error bars represent mean±S.D. of triplicate samples. *P<0.001

Figure 3

PKCδ phosphorylates p47^phox for K-Ras-induced ROS generation. (a) Kinase assay for PKC-α, -β, -δ in K-Ras^V12-transduced cells and control vector MFG-transduced cells with MARKS as substrate. (b) Levels of ROS as assessed by DCFDA fluorescence in control vector MFG-transduced cells or cells transfected with DN-PKCα, DN-PKCβ, DN-PKCδ, or control pcDNA and subsequently transduced with K-Ras^V12. (c) Soft agar colony formation in control vector MFG-transduced cells or cells transfected with dominant-negative mutant PKCα, PKCβ, PKCδ, or control vector pcDNA and subsequently transduced with K-Ras^V12. (d) Tumor growth curves of xenografts derived from parental Rat2 cells, K-Ras^V12-transduced cells, or K-Ras^V12-transduced cells that are transfected with DN-PKCδ. (e) Western blot for p47^phox after immunoprecipitation with anti-phospho-serine antibody in control vector MFG-transduced cells or cells that are transfected with DN-PKCδ and subsequently are transduced with K-Ras^V12. Also, western blot for p47^phox in particulate after separation of cytosol and particulate in cell lysates. (f) Western blot for PKC-α, -β, -δ after GST pull-down of p47^phox in K-Ras^V12-transduced cells and control vector MFG-transduced cells. (g) Kinase assay of PKCα, PKCβ, and PKCδ with GST-p47^phox fusion protein as substrate in K-Ras^V12-transduced and control vector MFG-transduced cells. Error bars represent mean±S.D. of triplicate samples. *P<0.001

Figure 4

PKCδ binds to the SH3-N domain and phosphorylates Ser348 and Ser379 residues in p47^phox for K-Ras^V12-induced ROS generation and consequent malignant transformation. (a) Constructs of GST-fusion proteins for the full length, PX domain, SH3-N domain, SH3-C domain, and PP domain in p47^phox. (b) Western blot for PKCδ and GST after GST pull-down of GST-fusion proteins in cells that are transfected with each construct of the GST-fusion protein and are subsequently transduced with K-Ras^V12. (c) Western blot for HA and Flag in cells that are transfected with HA-tagged WT p47^phox or Flag-tagged-PX, -SH3-N, -SH3-C, -PP domains in cells that are transfected with each construct of the GST-fusion protein and are subsequently transduced with K-Ras^V12. Also, western blot for PKCδ after immunoprecipitation with anti-HA or -Flag antibody in cells that are transfected with each construct of the GST-fusion protein and are subsequently transduced with K-Ras^V12. (d) Kinase assay of PKCδ with GST-fusion protein p47^phox wild type or mutant form, as substrate, that harbors a point mutation on Ser345, -348, -359, -370, -379 to Ala. (e) Western blot for PKCδ after immunoprecipitation with anti-HA antibody in cells that are transfected with HA-tagged WT p47^phox or HA-tagged p47^phox that harbors a point mutation on Ser345, -348, -359, -370, -379, -348/379, -345/359 to Ala. Also, western blot for HA to detect HA-tagged WT p47^phox or HA-tagged p47^phox that harbors a point mutation on Ser345, -348, -359, -370, -379, -348/379, -345/359 to Ala, in particulate after separation of cytosol and particulate in cell lysates. (f) Levels of ROS as assessed by DCFDA fluorescence in cells that are transfected with WT p47^phox or mutant p47^phox harboring a point mutation on Ser345, -348, -359, -370, -379, -348/379, -345/359 to Ala, and are subsequently transduced with K-Ras^V12 or control vector MFG. (g) Soft agar colony formation in control cells or cells that are transfected with WT p47^phox or mutant p47^phox harboring a point mutation on Ser345, -348, -359, -370, -379, -348/379, -345/359 to Ala, and are subsequently transduced with K-Ras^V12. WT, wild type; Error bars represent mean±S.D. of triplicate samples. *P<0.001

Figure 5

K-Ras^V12-induced activation of p38 is required for ROS generation and consequent malignant transformation. (a) Western blot for activation status of ERK, p38, and JNK MAPK in K-Ras^V12-transduced cells and control vector MFG-transduced cells. (b and c) Levels of ROS as assessed by DCFDA fluorescence in cells that are transfected with DN-ERK, DN-p38, or DN-JNK (b) or treatment with SB203580, inhibitor specific to p38 MAPK (c), and are subsequently transduced with K-Ras^V12. (d) Soft agar colony formation in control cells or cells that are transfected with DN-ERK, DN-p38, or DN-JNK, and are subsequently transduced with K-Ras^V12. (e) Tumor growth curves of xenografts derived from parental Rat2 cells, K-Ras^V12-transduced cells, or K-Ras^V12-transduced cells that are transfected with DN-p38. (f) Western blot for the phosphorylation status on Thr505 of PKCδ in Rat2 cells that are transfected with DN-p38 or control pcDNA, and are subsequently transduced with K-Ras^V12 or control vector MFG. (g) Western blot for the analysis of interaction between PKCδ and p47^phox after GST-p47^phox pull-down and immunoprecipitation with anti-PKCδ in cells that are transfected with DN-ERK, DN-p38, or DN-JNK, and are subsequently transduced with K-Ras^V12. (h) Western blot for phosphorylation status on serine residues of p47^phox after immunoprecipitation with anti-phospho-serine antibody, or membrane translocation of p47^phox in cells that are transfected with DN ERK, DN-p38, or DN-JNK, and are subsequently transduced with K-Ras^V12. (i) Western blot for the analysis of interaction between PKCδ and p47^phox after co-immunoprecipitation with anti-p47^phox or anti-PKCδ antibody in cells that are transfected with WT p38 or control pcDNA, and are subsequently transduced with K-Ras^V12 or control vector MFG. (j) Levels of ROS as assessed by DCFDA fluorescence in cells that are transfected with WT p38 or control pcDNA, and are subsequently transduced with K-Ras^V12 or control vector MFG. WT, wild type; DN, dominant-negative mutant; Error bars represent mean±S.D. of triplicate samples. *P<0.001

Figure 6

PDPK1 interacts with and phosphorylates PKCδ for K-Ras-induced ROS generation and consequent malignant transformation. (a) Western blot analysis for phosphorylation status and kinase assay of PDPK1 using GST-PKCδ as substrate at 0, 24, 48, 72 h after transduction with K-Ras^V12. (b) Kinase assay of PDPK1 using GST-PKCδ or GST-PKCδ (T505A) as substrate in cells that are transduced with K-Ras^V12 or control vector MFG. (c) Levels of ROS as assessed by DCFDA fluorescence in cells that are transfected with siRNA targeting PDPK1 or control scrambled siRNA, and are subsequently transduced with K-Ras^V12 or control vector MFG. (d) Soft agar colony formation in cells that are transfected with siRNA targeting PDPK1 or control scrambled siRNA, and are subsequently transduced with K-Ras^V12 or control vector MFG. (e) Western blot analysis for phosphorylation status and kinase assay of PKCδ using GST-p47^phox as substrate, and membrane translocation of p47^phox in cells that are transfected with siRNA targeting PDPK1 or control scrambled siRNA, and are subsequently transduced with K-Ras^V12 or control vector MFG. (f) Western blot analysis for phosphorylation status, interaction with PKCδ and kinase assay using GST-PKCδ as substrate, of PDPK1. (g) Western blot for PDPK1 after immunoprecipitation with anti-PKC-α, -β, -δ antibody as indicated in cells that are transduced with K-Ras^V12 or control vector MFG. Error bars represent mean±S.D. of triplicate samples. *P<0.001

Figure 7

K-Ras-induced ROS generation in human cancer cell lines, driving the same signaling pathway as in normal fibroblasts. (a) Levels of ROS as assessed by DCFDA fluorescence in HCT116 and SW480 after transfection with three different siRNA-targeting K-Ras. (b) Western blot for activation status of p38, PDPK1, and PKCδ after transfection with three different siRNA-targeting K-Ras. (c and d) Levels of ROS as assessed by DCFDA fluorescence after treatment with SB203580, inhibitor specific to p38 MAPK (c) or siRNA targeting PDPK1 or p47^phox (d) in HCT116 and SW480. Error bars represent mean±S.D. of triplicate samples. *P<0.001

Figure 8

Schematic model for K-Ras-induced signaling cascade that leads to ROS generation and consequent malignant cellular transformation. Note that PKCδ, PDPK1, and p47^phox form a protein complex, thereby facilitating the signal cascade for ROS generation