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First published online 29 January 2008
doi: 10.1242/jcs.013557

Journal of Cell Science 121, 514-521 (2008)
Published by The Company of Biologists 2008
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Functional interactions between phosphatase POPX2 and mDia modulate RhoA pathways

Yi Xie1, E-Jean Tan2, Shimei Wee2, Edward Manser2, Louis Lim2,3 and Cheng-Gee Koh1,*

1 School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Republic of Singapore
2 GSK/IMCB Group, Institute of Molecular and Cell Biology, Proteos, 61 Biopolis Drive, Singapore 138673, Republic of Singapore
3 Department of Molecular Neuroscience, Institute of Neurology, University College London, 1 Wakefield Street, London, WC1N 1PJ, UK


Figure 1
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  		Fig. 1. POPX interacts with mDia1 in the presence of RhoAV14. (A) A construct of flag-tagged full-length mDia1 was co-transfected with GFP-POPX2 and other cDNA constructs (HA-RhoAV14 or GST-SRC) into COS7 cells. Flag-immunoprecipitated proteins from cell lysates were analyzed by SDS-PAGE and western blotting using anti-GFP antibodies to detect co-precipitated POPX2. Lane 1, mDia-FL + POPX2; 2, mDia-FL + POPX2 + GST-SRC; 3, mDia-FL + POPX2 + HA-RhoV14; 4, mDia-FL + POPX2 + HA-RhoV14 + GST-SRC. (B) Schematic of the various mDia constructs. (C) GST-POPX2 was transfected with different Flag-tagged mDia1 constructs into COS7 cells. Cell lysates were incubated with glutathione beads. The bound protein was eluted and analyzed by SDS-PAGE and western blotting using anti-Flag antibodies to detect co-purification of mDia molecules. Lane 1, POPX2 + mDia-FL; 2, POPX2 + mDia-DN; 3, POPX2 + mDia-{Delta}N1; 4, POPX2 + mDia-{Delta}DAD; 5, POPX2 + mDia-GBD.

 

Figure 2
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  		Fig. 2. mDia1 co-immunoprecipitates with POPX2. (A) Protein lysates from COS7 cells were incubated with Protein G Sepharose beads covalently coupled with random IgG or affinity-purified anti-POPX2 antibodies. The eluates were analyzed by SDS-PAGE and western blotting using anti-mDia1 antibodies. Lane 1, COS7 total-protein lysate; 2, eluate from co-IP with random IgG; 3, eluate from co-IP with anti-POPX2 antibodies. (B) Dot-blot using 10 µg of MBP as control protein, 1 µg 6xHis-tagged mDia-DN and 10 µg 6xHis-tagged mDia-DN. The nitrocellulose membrane was dotted with MBP and 6xHis-tagged mDia-DN and incubated with bacterial-produced GST-POPX2. Far-western analysis was done using anti-GST antibodies to detect direct binding. (C) COS7 cells transfected with mDia-DN or mDia-{Delta}C were co-transfected with POPX2 or RhoV14 to determine the binding by co-IP and western blotting. Lane 1, HA-POPX2 + Flag-mDia-DN; 2, HA-POPX2 + Flag-mDia-{Delta}C; 3, HA-RhoV14 + Flag-mDia-DN; 4, HA-RhoV14 + Flag-mDia-{Delta}C.

 

Figure 3
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  		Fig. 3. POPX2 inhibits RhoAV14-induced and active-mDia-induced SRF-mediated transcription. The different plasmid constructs were transfected into COS7 cells as described in the Materials and Methods section. (A) POPX2 inhibits RhoA-induced SRF activity. The transfections were as labelled. (B) POPX2 inhibits active-mDia-induced SRF activity. The transfections were as labelled. (C) POPX2 does not inhibit CDC42-induced SRF activity. The transfections were as labelled.

 

Figure 4
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  		Fig. 4. POPX2 blocks the nuclear translocation of MAL. (A) HA-tagged MAL was co-transfected with GST-POPX2 or GST control vector into NIH3T3 cells. The cells were fixed and immunostained with antibodies to the tags to detect the location of MAL after growing in media containing 0.5% serum. The top panels show that HA-MAL was localized in the cytoplasm in the presence of GST-POPX2. The bottom panels show the nuclear location of HA-MAL in the absence of POPX2. DAPI stained the nuclei. (B) Various GST-tagged POPX cDNA constructs were co-transfected with HA-MAL and immunostained as in A. The transfected cells were scored for MAL nuclear localization. Lane 1: HA-MAL + GST control vector; 2, HA-MAL + GST-POPX1; 3, HA-MAL + GST-POPX2; 4, HA-MAL + GST-POPX2m; 5, HA-MAL + GST-POPX2N; 6, HA-MAL + GST-PPc; 7, HA-MAL + GST-PPm. Each transfection was repeated three times and approximately 100 transfected cells were scored per transfection. Scale bar: 25 µm.

 

Figure 5
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  		Fig. 5. POPX2 blocks the effect of dominant-negative mDia. (A) Flag-mDia-DN was transfected either with or without POPX2 into HeLa cells. The cells were fixed and immunostained with anti-Flag antibodies and rhodamine-coupled phalloidin. Cells transfected with just mDia-DN alone showed loss of stress fibres. Cells co-transfected with mDia-DN and POPX2 still contained stress fibres. (B) Transfection with different cDNAs and detection of stress fibres were performed as in A. The phenotype was scored by analyzing at least 100 cells per transfection. Each transfection was repeated three times. Lane 1, GFP; 2, mDia-DN; 3, POPX2; 4, POPX2m; 5, mDia-DN + POPX2; 6, mDia-DN + POPX2m; 7, mDia-DN + KID. Scale bars: 10 µm.

 

Figure 6
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  		Fig. 6. POPX2 knock-down causes rearrangement of the actin cytoskeleton and loss of stress fibres. (A) HeLa cells were microinjected with POPX2 siRNA and immunostained with rhodamine-coupled phalloidin. (B) HeLa cells were transfected with combinations of pXJ constructs of GFP or KID and POPX2 siRNAs. The cells were immunostained as in A. Three transfections were performed per construct. More than 100 cells were scored for each transfection. Lane 1, GFP; 2, GFP + control siRNA; 3, GFP + POPX2; 4, GFP + POPX2-siRNA1; 5, GFP + POPX2-siRNA2; 6, KID + POPX2-siRNA1. Scale bars: 10 µm.

 

Figure 7
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  		Fig. 7. A scheme representing a possible mechanism by which POPX2 negatively regulates transcription from the SRE. An increase in G-actin level results in inhibition of transcription from the SRE. Two different types of stress fibres are induced; one by ROK (SFR) and the other by mDia (SFM). SFR is formed from the bundling of existing actin filaments (F-actin) with myosin. SFM formation is catalyzed by the actin-nucleation and -polymerization activities of mDia. Downregulation of PAK activity by POPX2 results in stabilization and accumulation of SFR, which leads to less free F-actin in the cell. The increased G-actin:F-actin ratio will then inhibit SRE transcription. mDia has a positive effect on actin polymerization and enhances SRE transcription. Our current study shows that POPX2 blocks mDia enhancement of SRE response by its interaction with the mDia protein.

 

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© The Company of Biologists Ltd 2008