Palladin interacts with SH3 domains of SPIN90 and Src and is required for Src-induced cytoskeletal remodeling

Abstract

Palladin and SPIN90 are widely expressed proteins, which participate in modulation of actin cytoskeleton by binding to a variety of scaffold and signaling molecules. Cytoskeletal reorganization can be induced by activation of signaling pathways, including the PDGF receptor and Src tyrosine kinase pathways. In this study we have analyzed the interplay between palladin, SPIN90 and Src and characterized the role of palladin and SPIN90 in PDGF and Src-induced cytoskeletal remodeling. We show that the SH3 domains of SPIN90 and Src directly bind palladin's poly-proline sequence and the interaction controls intracellular targeting of SPIN90. In PDGF-treated cells, palladin and SPIN90 co-localize in actin-rich membrane ruffles and lamellipodia. The effect of PDGF on the cytoskeleton is at least partly mediated by the Src kinase since PP2, a selective Src kinase family inhibitor, blocked PDGF-induced changes. Furthermore, expression of active Src kinase resulted in coordinated translocation of both palladin and SPIN90 to membrane protrusions. Knock-down of endogenous SPIN90 did not inhibit Src-induced cytoskeletal rearrangement, whereas knock-down of palladin resulted in cytoskeletal disorganization and inhibition of remodeling. Further studies showed that palladin is tyrosine phosphorylated in cells expressing active Src indicating bidirectional interplay between palladin and Src. These results may have implications in understanding the invasive and metastatic phenotype of neoplastic cells induced by Src.

Figure 1. Palladin interacts with SPIN90 and Src

(A) Yeast-two hybrid analysis. Yeast cells were cotransformed with various palladin fragments in bait vector and either full length SPIN90 or Src SH3-SH2 in prey vector. Interaction was monitored by scoring growth on plates lacking histidine and by a filter based β-galactosidase assay. + = positive reaction, − = negative reaction. A scheme of the domains palladin 3Ig isoform is shown on top. The poly-proline (PP) regions are necessary for the interaction with SPIN90 and Src. Ig = immunoglobulin domain. (B) Affinity precipitation assay. In vitro translated (IVT) ³⁵S-labeled palladin 8–772 was allowed to bind GST-SPIN90 and GST-Src constructs coupled to glutathione-Sepharose beads. The panel is an autoradiograph showing the material eluted from beads. The SH3-domain of SPIN90 and SH3-SH2 domain of Src is sufficient in mediating the interaction. GST-FL-SPIN90 and IVT palladin have a similar molecular weight, which explains the slightly differential mobility of IVT protein in the gel. (C). Interaction between palladin poly-proline sequences, SPIN90 and Src. HEK293 cells were transfected with a HA-tagged full length SPIN90 or with wild type Src construct. The cleared lysates were allowed to bind GST (lane 2) or GST-palladin constructs containing the indicated residues on glutathione-Sepharose beads. Bound proteins were resolved in a 10% SDS-PAGE gel and detected either with a SPIN90 or a Src antibody. SPIN90 binding does not show marked preference between the poly-proline sequences, whereas Src binds preferably to the sequence contained within the 101–228 construct. Lane 1 shows immunoblotting of the cell lysates and bottom panel shows Fast Green staining of the membrane to demonstrate the equal loading of GST constructs. (D) Coimmunoprecipitation. COS-7 cells were co-transfected with HA-tagged palladin and GFP-tagged SPIN90. The cleared lysate was immunoprecipitated with an anti-HA antibody and the precipitate was blotted with anti-HA and anti-GFP antibody. The GFP-tagged protein is detected in the precipitate thus verifying the interaction.

Figure 1. Palladin interacts with SPIN90 and Src

(A) Yeast-two hybrid analysis. Yeast cells were cotransformed with various palladin fragments in bait vector and either full length SPIN90 or Src SH3-SH2 in prey vector. Interaction was monitored by scoring growth on plates lacking histidine and by a filter based β-galactosidase assay. + = positive reaction, − = negative reaction. A scheme of the domains palladin 3Ig isoform is shown on top. The poly-proline (PP) regions are necessary for the interaction with SPIN90 and Src. Ig = immunoglobulin domain. (B) Affinity precipitation assay. In vitro translated (IVT) ³⁵S-labeled palladin 8–772 was allowed to bind GST-SPIN90 and GST-Src constructs coupled to glutathione-Sepharose beads. The panel is an autoradiograph showing the material eluted from beads. The SH3-domain of SPIN90 and SH3-SH2 domain of Src is sufficient in mediating the interaction. GST-FL-SPIN90 and IVT palladin have a similar molecular weight, which explains the slightly differential mobility of IVT protein in the gel. (C). Interaction between palladin poly-proline sequences, SPIN90 and Src. HEK293 cells were transfected with a HA-tagged full length SPIN90 or with wild type Src construct. The cleared lysates were allowed to bind GST (lane 2) or GST-palladin constructs containing the indicated residues on glutathione-Sepharose beads. Bound proteins were resolved in a 10% SDS-PAGE gel and detected either with a SPIN90 or a Src antibody. SPIN90 binding does not show marked preference between the poly-proline sequences, whereas Src binds preferably to the sequence contained within the 101–228 construct. Lane 1 shows immunoblotting of the cell lysates and bottom panel shows Fast Green staining of the membrane to demonstrate the equal loading of GST constructs. (D) Coimmunoprecipitation. COS-7 cells were co-transfected with HA-tagged palladin and GFP-tagged SPIN90. The cleared lysate was immunoprecipitated with an anti-HA antibody and the precipitate was blotted with anti-HA and anti-GFP antibody. The GFP-tagged protein is detected in the precipitate thus verifying the interaction.

Figure 2. Palladin is tyrosine phosphorylated in cells expressing active Src kinase

HEK293 cells were co-transfected with HA-tagged palladin together with a constitutively active (CA) or a dominant-negative (DN) Src construct. Palladin was immunoprecipitated with anti-HA antibody and tyrosine phosphorylation analyzed with anti phosphotyrosine (pY) antibody. In cells expressing active Src, palladin is phosphorylated on tyrosine, whereas in cells expressing Src DN no phosphorylation is detected. As a negative control an HA-tagged amino-terminal (amino-acids 1–309/Y145F) ezrin construct was used.

Figure 3. Subcellular targeting of SPIN90 by palladin

(A–F).COS-7 cells were transfected with GFP-8-772 palladin and HA-tagged full-length (FL) SPIN90 or the SH3 domain of SPIN90 (SPIN90-SH3). SPIN90 was detected with anti-HA antibody. In cotransfected cells, both SPIN90 and SPIN90-SH3 are targeted to palladin-containing actin bundle. (G–K). In a mitochondrial targeting assay, COS-7 cells were cotransfected with palladin 8–228 or 228–387 in a vector containing an outer mitochondrial membrane (MOM) BiPro targeting sequence and with HA-SPIN90-SH3. MOM constructs were detected with a polyclonal palladin antibody and SPIN90-SH3 with HA mAb. When cotransfected with palladin 8-228-MOM construct, SPIN90-SH3 is recruited to the mitochondrial membrane (G,H). Instead, SPIN90-SH3 transfected with the palladin 222–387 MOM is distributed diffusely in the cytoplasm (J,K).

Figure 3. Subcellular targeting of SPIN90 by palladin

(A–F).COS-7 cells were transfected with GFP-8-772 palladin and HA-tagged full-length (FL) SPIN90 or the SH3 domain of SPIN90 (SPIN90-SH3). SPIN90 was detected with anti-HA antibody. In cotransfected cells, both SPIN90 and SPIN90-SH3 are targeted to palladin-containing actin bundle. (G–K). In a mitochondrial targeting assay, COS-7 cells were cotransfected with palladin 8–228 or 228–387 in a vector containing an outer mitochondrial membrane (MOM) BiPro targeting sequence and with HA-SPIN90-SH3. MOM constructs were detected with a polyclonal palladin antibody and SPIN90-SH3 with HA mAb. When cotransfected with palladin 8-228-MOM construct, SPIN90-SH3 is recruited to the mitochondrial membrane (G,H). Instead, SPIN90-SH3 transfected with the palladin 222–387 MOM is distributed diffusely in the cytoplasm (J,K).

Figure 4. Localization of SPIN90 and palladin in PDGF treated U251 cells

(A) U251 cells transfected with GFP-SPIN90 construct were serum-starved for 16h and stimulated with PDGF for 10 minutes. Cells were fixed and stained for endogenous palladin. Both palladin and SPIN90 are relocated to the PDGF-induced membrane ruffles (top row) and actin containing wave-like structures (second row). Treatment with the Src kinase family inhibitor PP2 prior to PDGF prevents the redistribution and both proteins remain in their normal subcellular positions (third and fourth row). (B) In the same experiment, the co-localization of cortactin, a known Src kinase substrate, and SPIN90 was compared. Cortactin is known to re-locate to the membrane after PDGF treatment and, as expected, cortactin co-localized with SPIN90 in the membrane ruffles in PDGF-treated cells. This re-location was Src-dependent as it was abolished by PP2.

Figure 5. SPIN90 and palladin co-localize in cells expressing active Src

U251 cells were co-transfected with a constitutively active (Src CA) or a dominant negative (Src DN) Src construct together with GFP-SPIN90 and stained for endogenous palladin with palladin Ab-3Ig antibody. Under basal conditions SPIN90 has a predominantly diffuse localization in the cytoplasm whereas palladin localizes to stress fiber dense bodies (Control). In the presence of Src DN, the localization of palladin and SPIN90 is similar to control cells. Expression of Src CA results in formation of numerous membrane projections and ruffles, which contain both palladin and SPIN90 (arrows).

Figure 6. Depletion of palladin results in disruption of actin cytoskeleton and loss of stress fibers

(A). U251 cells were transfected with a palladin specific siRNA oligonucleotide or with a non-targeting control siRNA. The specific siRNA markedly reduced the amount of palladin as compared to control. The alpha-tubulin blot serves as a loading control. (B). Transfected cells were stained with Ab-3Ig palladin antibody and with labeled phalloidin to visualize F-actin. The intensity of palladin staining is markedly reduced and the remaining staining pattern was diffuse compared to the control cells, which showed the typical stress fiber dense region staining. Depletion of palladin resulted in loss of stress fibers and disorganization of the cytoskeleton as visualized by phalloidin staining.

Figure 7. The role of palladin and SPIN90 in Src induced actin cytoskeleton remodeling

To study the role of palladin and SPIN90 in Src induced actin remodeling, U251 cells were co-transfected with the palladin and SPIN90 siRNA together with constitutively active Src (Src CA). Transfected cells were stained for SPIN90, Src, and cortactin. (A). In cells expressing active Src, all three proteins were located in cell extensions (arrows). However, cells in which palladin was depleted by siRNA exhibited hardly any membrane protrusions and the localization of SPIN90, Src and cortactin was mostly diffuse. (B). U251 cells were transfected with the GFP-conjugated SPIN90 siRNA construct or a control GFP-siRNA. Staining with a SPIN90 antibody shows a clear reduction in staining intensity of the cells transfected with specific siRNA as compared to the non-transfected cells or cells transfected with control GFP-siRNA. Arrows mark transfected cells. (C). Cells co-expressing Src CA and GFP-SPIN90 –siRNA or GFP-control-siRNA demonstrate equal and normal Src-induced actin remodeling. The cells have numerous membrane projections, which contain both palladin and Src.

Figure 7. The role of palladin and SPIN90 in Src induced actin cytoskeleton remodeling

To study the role of palladin and SPIN90 in Src induced actin remodeling, U251 cells were co-transfected with the palladin and SPIN90 siRNA together with constitutively active Src (Src CA). Transfected cells were stained for SPIN90, Src, and cortactin. (A). In cells expressing active Src, all three proteins were located in cell extensions (arrows). However, cells in which palladin was depleted by siRNA exhibited hardly any membrane protrusions and the localization of SPIN90, Src and cortactin was mostly diffuse. (B). U251 cells were transfected with the GFP-conjugated SPIN90 siRNA construct or a control GFP-siRNA. Staining with a SPIN90 antibody shows a clear reduction in staining intensity of the cells transfected with specific siRNA as compared to the non-transfected cells or cells transfected with control GFP-siRNA. Arrows mark transfected cells. (C). Cells co-expressing Src CA and GFP-SPIN90 –siRNA or GFP-control-siRNA demonstrate equal and normal Src-induced actin remodeling. The cells have numerous membrane projections, which contain both palladin and Src.

Figure 7. The role of palladin and SPIN90 in Src induced actin cytoskeleton remodeling

To study the role of palladin and SPIN90 in Src induced actin remodeling, U251 cells were co-transfected with the palladin and SPIN90 siRNA together with constitutively active Src (Src CA). Transfected cells were stained for SPIN90, Src, and cortactin. (A). In cells expressing active Src, all three proteins were located in cell extensions (arrows). However, cells in which palladin was depleted by siRNA exhibited hardly any membrane protrusions and the localization of SPIN90, Src and cortactin was mostly diffuse. (B). U251 cells were transfected with the GFP-conjugated SPIN90 siRNA construct or a control GFP-siRNA. Staining with a SPIN90 antibody shows a clear reduction in staining intensity of the cells transfected with specific siRNA as compared to the non-transfected cells or cells transfected with control GFP-siRNA. Arrows mark transfected cells. (C). Cells co-expressing Src CA and GFP-SPIN90 –siRNA or GFP-control-siRNA demonstrate equal and normal Src-induced actin remodeling. The cells have numerous membrane projections, which contain both palladin and Src.