PDCD10 interacted with MST4 in situ both overexpression and the endogenous proteins. (A) Cell lysates transfected with the indicated constructs were immunoprecipitated with the FLAG antibody to precipitate FLAG-PDCD10 and FLAG control protein. An MYC antibody was used to detect MST4 proteins in the immunoprecipitate. PDCD10 coimmunoprecipitated with MST4 proteins in 293T cells, whereas the control pCDEF-flag vector did not. (B) HeLa cell lysates in normal culture were immunoprecipitated with the rabbit-derived anti-MST4 antibody and the mouse-derived anti-PDCD10 antibody to precipitate corresponding proteins, respectively. Then the other antibodies were used to detect corresponding proteins in the immunoprecipitates. PDCD10 coimmunoprecipitated with MST4 proteins in HeLa cells, whereas the control mouse-derived IgG did not; on the other hand, MST4 coimmunoprecipitated with PDCD10 proteins in HeLa cells, whereas the control rabbit-derived IgG did not.

Expression levels of endogenous PDCD10 in human multicell lines by RT-PCR and immunoblotting.

PDCD10 modified normal and MST4-induced cellular growth. PC-3 and HeLa cells were transiently transfected with expression vectors PDCD10 and MST4. Cells proliferation was measured by MTT and ³H-TdR assays at indicated time points. (A) MTT assay for overexpression of PDCD10, MST4, and PDCD10 with cotransfected MST4 (B) ³H-TdR assay for overexpression of PDCD10, MST4, and PDCD10 with cotransfected MST4. (C) MTT assay for PDCD10 knockdown by double-stranded siRNAs. (D) MTT assay for PDCD10 knockdown by double-stranded siRNAs with overexpression of MST4. Error bars, SEM. Data are one representation of three separate experiments.

PDCD10-induced protection from anoikis. Cells transfected with pCDEF-PDCD10, pGC-PDCD10, with pCDEF vector plasmid (10 μg), and nonsilencing siRNA as mock were resuspended in RPMI 1640 and plated on the Poly-HEME plates. At 24 h after transfection, cells were harvested and stained with annexin V and propidium iodide (PI), and analyzed by FACS (n = 3). Apoptotic cells are expressed as a percentage of total cells. (A) Overexpression of PDCD10 inhibited anoikis compared with empty vectors (11.9, 24.8%, respectively). (B) An increase in apoptosis was observed after treatment with PDCD10 siRNA compared with nonsilencing (34.3, 25.6%, respectively).

RNAi of MST4 modified normal and PDCD10-induced cellular growth. HeLa cells were transiently transfected with expression vectors PDCD10 and MST4 siRNA. Cells proliferation was measured by MTT assays at indicated time points. (A) MTT assay for MST4 knockdown by double-stranded siRNAs in normal culture cells. (B) MTT assay for MST4 knockdown by double-stranded siRNAs with overexpression of PDCD10. Error bars, SEM. Data are one representation of three separate experiments.

A reporter gene assay of ERK dependent Elk1 activation was used to investigate the role of PDCD10 and MST4 in the ERK MAPK cascade by dual-luciferase reporter assays. Expression vectors and a reporter gene driven by Elk1-dependent promoter were transiently transfected into PC-3 cell lines, with the thymidine kinase promoter-Renilla luciferase reporter plasmid (pRL-TK) used as an internal control. Cells were lysed and the relative luciferase activity was measured (firefly luciferase for reporter and renilla luciferase activity for normalization of transfection efficiency). Reporter assays were performed after treatment with ERK specific inhibitor PD98059 (5 mM), p38 MAPK inhibitor SB20219 (10 mM), or JNK MAPK inhibitor JNK inhibitor II (0.1 μM), respectively. ELK1 represents the Gal4-ELK1 fusion expression vector. (A) The overexpression of PDCD10 and MST4 conferred a higher activation of the reporter gene. (B) Knockdown of PDCD10 showed inhibition of Elk1-dependent luciferase activity compared with nonsilencing vectors. Error bars, SEM; n = 3 per experiment.

RNAi of MST4 inhibited normal and PDCD10-induced ERK kinases activity. HeLa cells were transfected with RNAi of MST4, with nonsilencing as blank controls. Cell lysates were separated by SDS-PAGE and immunoblotted with anti-ERK and anti-phospho-ERK rabbit polyclonal antibodies, respectively. (A) Knockdown of MST4 significantly decreased ERK activity. (B) Overexpressed PDCD10 increased ERK activity, which was attenuated by effective siMST4, with nonsilencing as noneffective control.

PDCD10/MST4 signaling pathway and CCM protein function. The CCM1/2 complex may modulate integrin-mediated cell adhesion via association with ICAP1 in the cytoplasm, as well as organize a p38 MAPK signaling complex (Zawistowski et al., 2005). However, it has been reported that MST4 activated MEK-1/ERK via a Ras/Raf independent pathway (Lin et al., 2001). These data, and ours, confirmed that PDCD10 (CCM3) interacted with MST4 and modulated the ERK MAPK cascade, but not P38 and JNK, resulting in growth induction and cellular transformation.

Colocalization of PDCD10 and MST4 in HeLa cells. (A) Confocal microscopy images of cells cotransfected with pEGFP-C3-PDCD10 (green) and pDsred-N1-MST4 (red) plasmids, with coexpression in yellow. Cell nucleus morphology was observed by DAPI staining (blue). a, b, and c represented three different cells. Cell b was transfected with pDsred-N1-MST4 plasmid and cell c with pEGFP-C3-PDCD10; however, cell a was cotransfected with both plasmids. It showed that either pEGFP-C3-PDCD10 or pDsred-N1-MST4 localized to the cytoplasm with some concentrated spots around the nucleus, and the distribution did not changed when cotransfected PDCD10 and MST4. (B) The same layer with 2× magnification of A, which showed that the distribution of MST4 entirely overlapped with PDCD10. (C) The HeLa cells costained by anti-PDCD10 antibody and FITC-conjugated goat anti-mouse IgG, anti-MST4 antibody, and TRITC-conjugated bovine anti-rabbit IgG in turn, for the endogenous immunofluorescence microscopy of PDCD10 and MST4. It exhibited that either PDCD10 or MST4 localized around the nucleus as highly concentrated spots, and the distribution of MST4 overlapped with PDCD10. Experiments were performed in triplicate, with similar results.

Transfection of siPDCD10-1 or siPDCD10-2 inhibited PDCD10 expression at both mRNA and protein levels. (A) RT-PCR PDCD10 mRNA expression data. PC-3 cells, which express high levels of PDCD10, were transfected with nonsilencing siRNA or the indicated siRNAs against PDCD10. At 24 h after transfection, total RNAs were extracted and RT-PCR experiments were performed as described (see Materials and Methods). Both siPDCD10-1 and siPDCD10-2 showed much stronger inhibitory effects for PDCD10 mRNA expression than nonsilencing siRNA. (B) Effects of siRNAs against PDCD10 on the expression of GFP PDCD10 fusion protein. PC-3 and HeLa cells were cotransfected with pEGFP-PDCD10 plasmid and siPDCD10-1 (c) and siPDCD10-2 (d), respectively. Plasmids encoding pEGFP-PDCD10 were also transfected into PC-3 and HeLa cells cotransfected with nonsilencing siRNA (a) as negative control, as well as siRNA against pEGFP (b) as positive control. At 24 h after transfection, cells were observed by fluorescence microscopy. siPDCD10-1 and -2–transfected cells show far fainter fluorescence than cells transfected with nonsilencing siRNA. Then cells were harvested and immediately analyzed by FACS Calibur (1 × 10⁴ cells) and CELLQuest software (BD Bioscience). Mean GFP-PDCD10 fluorescence intensity was quantificationally shown in C. (D) Immunoblot assessment on the effects of siPDCD10 on endogenous PDCD10 protein expression. HeLa cells were transfected with nonsilencing siRNA or siPDCD10. At 24 h after transfection, a significant decrease in the expression of endogenous PDCD10 was observed in siPDCD10–transfected cells, whereas no decrease was noted in nonsilencing siRNA transfected cells. β-actin was used as the internal control.

PDCD10 modified normal and MST4-induced cellular growth in colony forming assays. (A) Left two panels, PC-3 cells transfected with pCDEF-PDCD10, pCDEF-MST4, pCDEF-PDCD10, and pCDEF-MST4 or vector control in monolayer culture and selected with G418. Quantitative analyses of colony numbers are shown in the right panel (n = 3). (B) Representative sample of inhibition of colony formation in monolayer culture by siPDCD10. Right panel, PC-3 cells transfected with pGC-PDCD10, or nonsilencing control in monolayer culture. Quantitative analyses of colony numbers are shown in the right panel.

Transfection of siMST4-1 or siMST4-2 inhibited MST4 expression at both mRNA and protein levels. (A) RT-PCR MST4 mRNA expression data. HeLa cells, which express MST4, were transfected with nonsilencing siRNA or the indicated siRNAs against MST4. At 24 h after transfection, total RNAs were extracted, and RT-PCR experiments were performed as described (see Materials and Methods). Both siMST4-1 and siMST4-2 showed strong inhibitory effects for MST4 mRNA expression, whereas MST4 mRNA expression was not inhibited by nonsilencing siRNA. (B) Effects of siRNAs against MST4 on the expression of GFP-MST4 fusion protein. HeLa cells were cotransfected with pEGFP-MST4 plasmid and siMST4-1 (c), siMST4-2 (d), respectively. Plasmids encoding pEGFP-MST4 were also transfected into HeLa cells cotransfected with nonsilencing siRNA (a) as negative control, as well as siRNA against pEGFP (b) as positive control. At 24 h after transfection, cells were observed by fluorescence microscopy. Both siMST4-1 and siMST4-2 transfected cells show far fainter fluorescence than cells transfected with nonsilencing siRNA. Then cells were harvested and immediately analyzed by FACS Calibur (1 × 10⁴ cells) and CELLQuest software (BD Bioscience). Mean GFP-MST4 fluorescence intensity was quantificationally shown in C. (D) Immunoblot assessment on the effects of siMST4 on endogenous MST4 protein expression. HeLa cells were transfected with nonsilencing siRNA or siMST4. At 24 h after transfection, a significant decrease in the expression of endogenous MST4 was observed in both siMST4-1 or siMST4-2–transfected cells, whereas no decrease was noted in nonsilencing siRNA transfected cells. β-actin was used as the internal control.

The effect of MST4 RNAi on the protection from anoikis induced by PDCD10. Cells transfected with overexpression vectors of PDCD10 and siRNA against MST4, with pCDEF vector plasmid (10 μg) and nonsilencing siRNA as mock were resuspended and plated on the Poly-HEME plates. (A) At 24 h after transfection, cells were harvested and stained with annexin V and PI, and analyzed by FACS (n = 3). (B) Apoptotic cells are expressed as a percentage of total cells. An increase of apoptosis was observed after treatment with siMST4-1 compared with nonsilencing siRNA (37.6%, 24.7%, respectively), as well as overexpression of MST4 inhibited anoikis compared with the empty vectors (16.8%, 25.5%, respectively). After cotransfection of siMST4-1 and pCDEF-PDCD10, an increase of apoptosis was also observed compared with cotransfection of nonsilencing and pCDEF-PDCD10 (36%, 15.2%, respectively).

Interaction between PDCD10 and MST4 modulated ERK activity, but not JNK and P38 as measured by Western blot. HeLa cells were transfected with expression vectors, with pCDEF and nonsilencing siRNA as blank controls. Cell lysates were separated by SDS-PAGE and immunoblotted with anti-ERK, anti-JNK and anti-P38, as well as anti-phospho-ERKs, anti-phospho-JNK and anti-phospho-P38 rabbit polyclonal antibodies, respectively. And the positive control were MEK1 for ERK-MAPK, MEKK for JNK-MAPK, and MEK3 for P38-MAPK. (A) showed that overexpressed PDCD10 or MST4 increased ERK activity at similar levels. Co-overexpressed PDCD10 accelerated MST4-induced ERK activation significantly. In contrast, knockdown of PDCD10 significantly decreased ERK activity. (B and C) Either overexpressed PDCD10 or siPDCD10 did not influence JNK and P38 activity, as well as MST4.

The MST4 kinase activity assays in vitro. The kinase assays were performed according to the protocol of the HTScan Mst4 kinase assay kit. The reaction system included 10 Units recombinant Mst4 kinase and 200 μM cold ATP with substrate, Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Biotinylated Peptide. The HeLa cells overexpressing and down-regulating PDCD10 were lysed and immunoprecipitated with anti-PDCD10 mAb and protein G beads. After incubating with the reaction system for 30 min at 25°C, the patterns were performed with directed ELISA and detected the absorbance at 450 nm.