Colocalisation of PKD1 and αvβ3 integrin at intracellular vesicles following inhibition of receptor recycling. NIH 3T3 fibroblasts were transfected with αvβ3 integrin in combination with GFP-PKD1. Cells were serum-starved and challenged with 10 ng/ml PDGF-BB in the absence (A) and presence (B, C) of 60 μM primaquine (PQ) for 5 min (B) or 10 min (C). Following fixation, cells were permeabilised in 0.2% (v/v) Triton X-100 and stained for β3 integrin by indirect immunofluorescence using a Texas red-conjugated secondary antibody (red) and GFP-PKD1 was visualised directly (green). Colocalisation of the two fluorophores is shown in yellow in the merged panels. The areas encompassed by the white boxes in (B) and (C) are enlarged four-fold in (B′) and (C′), respectively. Images are presented as a single confocal optical slice centred approximately 0.5 μm above the plane of the substratum. Bar, 10 μm.

Catalytically inactive PKD1 associates with αvβ3 in the absence of primaquine. (A, B) NIH 3T3 fibroblasts were transfected with αvβ3 integrin in combination with GFP-PKD1 or GFP-PKD^D733A. Cells were serum-starved, challenged with 10 ng/ml PDGF-BB for 10 min in the absence and presence of 0.6 mM primaquine (PQ), lysed and immunoprecipitated (IP) using magnetic beads coupled to monoclonal antibodies against human β3 integrin, PKD1, GFP or control antibody (RG-16). Immobilised material was analysed by Western blotting. (C) Cells were transfected with PKD1 or PKD^D733A and fractionated on 10–40% (v/v) iodixanol gradients. The linearity of the gradient was confirmed by refractometry (•). The sedimentation profile of PKD1 (▪) and β3 integrin (▴; red line) was determined by Western blotting followed by densitometric scanning. These profiles represent the mean values from a pool of four experiments. The sedimentation positions of intracellular compartments were determined by Western blotting with antibodies against the following markers: calreticulin (ER), Rab11 and Rab11 (endosomes) and biotinylated cell surface proteins (plasma membrane).

PDGF-stimulated recycling of αvβ3 integrin requires association with active PKD1. (A) NIH 3T3 fibroblasts were transfected with mu6pro, mu6pro-CON or mu6pro-PKD1 using the Amaxa ‘Nucleofector'. Cells were lysed 24 and 48 h following transfection and the lysates were probed for the presence of PKD1, PKC-ζ and vinculin (as a loading control) by Western blotting. (B, C) Cells were transfected with αvβ3 integrin or αvβ3 with mutated β3 subunits, in combination with the mu6pro RNAi vectors or PKD1 constructs as indicated. ‘Short-loop' recycling of αvβ3 in the presence and absence of 10 ng/ml PDGF-BB was determined as described previously (Roberts et al, 2001). Values are mean±s.e.m., n>5. To evaluate the PDGF-dependent component of recycling, the data from (B) are presented following subtraction of the appropriate basal values (C).

Incorporation of αvβ3 integrin into newly forming focal adhesions requires association with active PKD1. (A, B) NIH 3T3 fibroblasts were transfected with mu6pro-CON or mu6pro-PKD1 using the Amaxa Nucleofector and allowed to grow to confluence over 48 h. Following wounding, the cells were allowed to migrate in DMEM supplemented with 1% (v/v) FCS and 10 ng/ml PDGF-BB. Photographs of the wounds were taken at various times post-wounding and the degree of wound closure was determined by delineation of the wound envelope using the ‘NIH image' software (values are mean±s.e.m., n=10 fields). (C) Confluent monolayers of NIH 3T3 fibroblasts expressing αvβ3 integrin in combination with mu6pro-CON or mu6pro-PKD1 were wounded as for (A) and the cells were allowed to migrate for 4 h. Surface αvβ3 was visualised by indirect immunofluorescence (red) and cells were counterstained with phalloidin (green). Surface-only αvβ3 staining was obtained by addition of the primary antibody prior to detergent permeabilisation. Bar, 10 μm. (D) NIH 3T3 fibroblasts were transfected with αvβ3 integrin or αvβ3 with mutated β3 subunits, in combination with the PKD1-RNAi vector (mu6pro-PKD1) or appropriate control vectors (mu6pro, mu6pro-CON), PKD1 or catalytically inactive PKD as indicated. Following trypsinisation, cells were allowed to adhere to fibronectin-coated coverslips for 45 min or 24 h. β3 integrin was visualised by indirect immunofluorescence (red). Bar, 10 μm.

Association of kinases with integrins following addition of primaquine. NIH 3T3 fibroblasts were transfected with αvβ3 or α5β1 integrin alone (A, B), or αvβ3 in combination with GFP-PKD1 (C, D). Cells were serum-starved, challenged with 10 ng/ml PDGF-BB for 10 min in the absence and presence of 0.6 mM primaquine (PQ), and lysed in a buffer containing 0.5% (v/v) Triton X-100 and 0.25% (v/v) Igepal CA-630. Primaquine was added 5 min following PDGF addition and 5 min prior to lysis. Lysates were immunoprecipitated (IP) using magnetic beads coupled to monoclonal antibodies against human β3 or α5 integrin (A–C), PKD or GFP (D), or control antibody (RG-16; B–D). Immobilised material was analysed using the Kinexus 1.2 protein kinase screen (A) or Western blotting (B–D).

Association of PKD1 with the β3 integrin cytodomain. (A) Description and nomenclature of the GST-β3 integrin cytodomain fusion proteins employed for the present study. GST fusion proteins were expressed in E. coli strain BL-21 and purified as described previously (Woods et al, 2002). (B) Lysates from NIH 3T3 fibroblasts were incubated with magnetic beads conjugated to GST, or GST-β3^727–762. Immobilised material was analysed by Western blotting with anti-PKD1 (upper panel), and the loading of the GST fusion proteins was confirmed by Western blotting for GST (lower panel). (C–F) GST and GST-cytodomain fusion proteins were immobilised on the surface of microtitre wells. These were then incubated with either cell lysate (C–E) or biotinylated talin-FERM domain (residues 86–410; mouse sequence) (F). Bound PKD1 was detected by ELISA using antibodies recognising PKD1 (C–E) and binding of the biotinylated talin-FERM domain was detected with HRP-conjugated streptavidin (F).

Colocalisation of αvβ3 integrin with catalytically inactive GFP-PKD^D733A. NIH 3T3 fibroblasts (A–C) or primary cultured MEFs (D) were transfected with αvβ3 integrin in combination with GFP-PKD^D733A. Cells were serum-starved and challenged with 10 ng/ml PDGF-BB in the absence (A, C, D) and presence (B) of 60 μM primaquine (PQ). Cells were stained for β3 integrin (red) and GFP-PKD1 was visualised directly (green). Colocalisation of the two fluorophores is shown in yellow in the merged panels. The areas encompassed by the white boxes are enlarged four-fold in (A′) and (B′) and two-fold in (D′). Bar, 10 μm.

Association of αvβ3 with PKD1 requires the activity of Rab4. (A) NIH 3T3 fibroblasts were transfected with αvβ3 integrin in combination with Rab4, Rab4^S22N, Rab4^N121I or Rab11^N124I. Cells were serum-starved in the absence and presence of 100 nM wortmannin (WMN), challenged with 10 ng/ml PDGF-BB in the presence of 0.6 mM primaquine (PQ), lysed and immunoprecipitated (IP) using magnetic beads coupled to monoclonal antibodies against human β3 integrin. Immobilised material was analysed by Western blotting. (B) Cells were transfected with αvβ3 integrin in combination with GFP-PKD^D733A and Rab4^S22N, serum-starved and challenged with 10 ng/ml PDGF-BB. Cells were stained for β3 integrin (red) and GFP-PKD1 was visualised directly (green). The areas encompassed by the white box in (B) is enlarged four-fold in (B′). Bar, 10 μm. (C) Cells were transfected with GFP-PKD1 in combination with Rab4 or Rab4^S22N. Following serum starvation, the cells were challenged with 10 ng/ml PDGF in the absence or presence of 0.6 mM primaquine. The cellular content of GFP-PKD1 and phospho-Ser⁹¹⁶GFP-PKD1 was determined by Western blotting.