Rsu1-dependent control of PTEN expression is regulated via ATF2 and cJun

Abstract

The Rsu1 protein contributes to cell adhesion and migration via its association with the adaptor complex of Integrin linked kinase (ILK), PINCH, and Parvin (IPP), which binds to the cytoplasmic domain of β1 integrins joining integrins to the actin cytoskeleton. Rsu1 binding to PINCH in the IPP complex is required for EGF-induced adhesion, spreading and migration in MCF10A mammary epithelial cells. In addition, Rsu1 expression inhibits Jun kinase but is necessary for the activation of MKK4 and p38 Map kinase signaling essential for migration in MCF10A cells. The data reported here examines the links between MKK4-p38-ATF2 signaling and AKT regulation in MCF10A cells. Ectopic Rsu1 inhibited AKT1 phosphorylation while Rsu1 depletion induced AKT activation and AKT1 phosphorylation of MKK4 on serine 80, blocking MKK4 activity. Rsu1 depletion also reduced the RNA for lipid phosphatase PTEN thus implicating PTEN in modulating levels of activated AKT in these conditions. ChIP analysis of the PTEN promoter revealed that Rsu1 depletion prevented binding of ATF2 to a positive regulatory site in the PTEN promoter and the enhanced binding of cJun to a negatively regulatory PTEN promoter site. These results demonstrate a mechanism by which Rsu1 adhesion signaling alters the balance between MKK4-p38-ATF2 and cJun activation thus altering PTEN expression in MCF10A cells.

Keywords: Adhesion; MKK4: ATF2: PTEN; Migration; Rsu1.

Fig. 1

The Rsu1 is necessary for activation of p38 Mapk and MKK4 but not MKK3/6 in response to EGF. (a, b) Cell lysates were isolated from control siRNA and Rsu1 siRNA transfected MCF10A cells following EGF treatment (100 ng/ml) for the indicated time. Immunoblot analysis was performed with equal amounts of protein samples as indicated in Materials and Methods and the levels of phospho-p38 (T180/Y182), phospho-MKK4 (S257 and T261), phospho-MKK3/6 (S189/S207), phospho-JNK (T183/Y185), were detected. Total p38, JNK, MKK4 and MKK3/6 were measured and α-tubulin and Rsu1 antibodies were used for loading controls and depletion levels, respectively. c MCF10A cells stably expressing control vector (pBabe), Rsu1-myc (Rsu1) or Rsu1-N92D-myc (Rsu1-N92D) were transfected with negative control siRNA or endogenous Rsu1 specific siRNA and treated with EGF (100 ng/ml) for 10 min as described in Materials and Methods. The lysates were subjected by western blot and analyzed with phosphorylation specific antibodies indicated. d Lysates from MCF10A cells depleted of MKK4, MKK3 or MKK6 were examined for levels of phospho-p38, phospho-JNK and phospho-Erk. Quantitation is expressed as fold stimulation compared to T = 0 for control siRNA transfected cells set as 1

Fig. 2

Rsu1-dependent changes in AKT signaling pathway. a MCF10A, T47D and MDA-MB-231 cells were infected with adenoviral vector encoding HA-tagged Rsu1 or empty vector. At 72 h post infection cell lysates were prepared and analyzed by western blot with phosphorylation specific antibodies, phospho-AKT (T308) and phospho-AKT (S473). Antibodies directed against total AKT, ILK, Rac1 and Rsu1were used to determine expression levels of those proteins. Quantitation shows the level of phosphorylated AKT normalized to total AKT and the level set in the control lane = 1. b MCF10A lysates were isolated from control siRNA and Rsu1 siRNA transfected MCF10A cells after EGF treatment for the indicated times. Immunoblot analysis was performed with equal amounts of protein samples and separated by SDS-PAGE for the levels of phospho-MKK4 (S80), phospho-AKT (p S473), total MKK4, AKT and PTEN. α-tubulin and Rsu1 antibodies were used for internal and depletion controls. c MCF10A RNA from control siRNA and Rsu1 siRNA transfected cells was used to determination level of PTEN RNA by quantitative real-time PCR. Quantitation is expressed as fold stimulation or relative amount compared to T = 0 for control siRNA transfected cells set as 1

Fig. 3

PTEN gene regulation by MKK4-p38 Mapk signaling and Rsu1-dependent expression of PTEN in MCF10A cells. a Determination of PTEN RNA levels was measured by quantitative real-time PCR following transfection with siRNAs for the following: Rsu1, p38α, MKK3, MKK4 and MKK6. The 18S RNA was used for normalization. Bar graphs show the fold change of PTEN expression quantified as the ratios to the control siRNA level. The data show means ± SD, n = 3. b RNA levels of PTEN were measured by quantitative real-time PCR following SB 203580 treatment of MCF10A cells (0, 5 or 20 μM). The 18S RNA was used for normalization. Bar graphs show the fold change of PTEN expression quantified as the ratios to the control, the means ± SD, n = 3. c Immunoblot analysis was performed with equal amounts of protein samples for the levels of phospho-AKT (S473) and phospho-p38 (T180/Y182), total AKT, p38, PTEN expression and α-tubulin was for internal controls

Fig. 4

ATF2 and cJun bind on the ATF2 site of the PTEN promoter. a Schematic representation of putative two ATF2 binding sites on the PTEN promoter. b Chromatin immunoprecipitation (ChIP) assay was performed for occupancy of the binding factors in the two ATF2 sites in the PTEN promoter. Purified chromatin DNA from either control siRNA (C) or Rsu1-specific siRNA (R) MCF10A cells was immunoprecipitated with anti-ATF2 (Top) and anti-cJun (Bottom) antibodies for binding to the promoter of PTEN. The immunoprecipitated complexes were amplified by PCR using the primer sets (either site 1 or site 2) as described in Materials and Methods. The PCR products were analyzed on the agarose gel with duplicated samples (C; control siRNA and R; Rsu1 siRNA transfected MCF10A cells). Input is shown as positive control and immunoprecipitation with anti-normal rabbit IgG antibodies was used as negative control (Negative control). c ChIP experiment was performed with EGF stimulated MCF10A cells. Cells were maintained in media without EGF for 16 h then stimulated for 30 min. Real-time PCR analysis of chromatin bounded ATF2 (left) or cJun (right) were measured by quantitative PCR methods with either control siRNA (control) or Rsu1-specific siRNA (Rsu1) MCF10A cells with or without EGF stimulation. d Immunoblot analysis was performed using cell lysates from control siRNA (control) and Rsu1 specific siRNA (Rsu1) MCF10A cells for the levels of phospho-ATF2 (p-ATF2, T71), phospho-cJun (p-cJun, S63), total ATF2 and cJun. α-tubulin and Rsu1 antibodies were used for internal controls and depletion levels, respectively

Fig. 5

Schematic model of the regulation of PTEN promoter in MCF10A cells by Rsu1 and IPP complex. Rsu1 plays a role in the regulation of PTEN gene transcription via MKK4-p38-ATF2 and MKK4-JNK-cJun. p38 MAPK can be constitutively activated by detachment or by blocking Rsu1-PINCH1 interaction and this is mediated by MKK3/6 signaling. However, EGF-induced p38 MAPK phosphorylation is dependent on Rsu1 and occurs through MKK4 and possibly MEKK1. In MCF10A cells, PTEN gene transcription is regulated through the ATF2/AP1 by the binding of AFT2 and cJun