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Published in final edited form as: Nat Cell Biol. 2009 Mar 29;11(5):545–556. doi: 10.1038/ncb1861

a, HeLa cells were co-transfected with YFP-SSH1L and vector, constitutively-active PKD1 (PKD1.CA) or kinase-inactive PKD1 (PKD1.KD). SSH1L was immunoprecipitated with a α-GFP/YFP antibody and analyzed for phosphorylation by PKD1 using the α-pMotif PKD-substrate antibody. Blots were re-probed for SSH1L expression and control blots indicate PKD1 transgene expression (α-PKD1) or equal loading (α-actin). b, HeLa cells were transfected with RNAi control or PKD-RNAi. The next day cells were transfected with HA-SSH1L. After 48h, cells were stimulated with H₂O₂ (10 mM, 10 min) as indicated. SSH1L was immunoprecipitated (α-HA) and samples were analyzed using the α-pMotif PKD-substrate antibody. Blots were re-probed for SSH1L expression and control blots were probed with α-actin (loading control) or α-PKD1 (to show effective knockdown; numbers show knockdown of PKD1 relative to control). c, The PKD1 consensus motif indicates preference for serines with an arginine at -3 and leucine at -5 relative to the phosphorylated serine. Both serines, Ser937 and Ser978, in the serine-rich region of SSH1L fulfill the criteria of ideal phosphorylation consensus sequences. Also shown is the phosphorylation sequence of the known PKD1 substrate Hsp27. d, Purified GST-fusion proteins of SSH1L encompassing the putative PKD1 phosphorylation sites S937 and S978 as well as fusion proteins of SSH1L.S937A, SSH1L.S978A and SSH1L.S937A/S978A mutants were subjected a PKD1 kinase assay. Substrate phosphorylation was detected with the α-pMotif PKD-substrate antibody. Purified GST alone or GST-Hsp27 served as negative or positive controls. Control blots show substrate loading (α-GST) and PKD1 (α-PKD1). e, Purified GST (control) and GST-SSH1L were subjected to an in vitro kinase reaction with purified, active PKD1. Western blots of resolved proteins were probed with α-pS978-SSH1L. Control blots show substrate loading (α-GST) and PKD1 (α-PKD1). f, HeLa cells were co-transfected with Myc-tagged SSH1L or SSH1L.S978A and vector or constitutively-active PKD1 (PKD1.CA). SSH1L was immunoprecipitated (α-myc) and analyzed for phosphorylation at S978 (α-pS978-SSH1L). Blots were re-probed for SSH1L expression (α-SSH1L) and control blots were probed for PKD1 (α-PKD1). Uncropped images of Figs. 2a, 2d and 2f are shown in Supplementary information Fig. S6.

a, HeLa cells were co-transfected with YFP-SSH1L and vector, constitutively-active PKD1 (PKD1.CA) or kinase-inactive PKD1 (PKD1.KD). SSH1L was immunoprecipitated with a α-GFP/YFP antibody and analyzed for phosphorylation by PKD1 using the α-pMotif PKD-substrate antibody. Blots were re-probed for SSH1L expression and control blots indicate PKD1 transgene expression (α-PKD1) or equal loading (α-actin). b, HeLa cells were transfected with RNAi control or PKD-RNAi. The next day cells were transfected with HA-SSH1L. After 48h, cells were stimulated with H₂O₂ (10 mM, 10 min) as indicated. SSH1L was immunoprecipitated (α-HA) and samples were analyzed using the α-pMotif PKD-substrate antibody. Blots were re-probed for SSH1L expression and control blots were probed with α-actin (loading control) or α-PKD1 (to show effective knockdown; numbers show knockdown of PKD1 relative to control). c, The PKD1 consensus motif indicates preference for serines with an arginine at -3 and leucine at -5 relative to the phosphorylated serine. Both serines, Ser937 and Ser978, in the serine-rich region of SSH1L fulfill the criteria of ideal phosphorylation consensus sequences. Also shown is the phosphorylation sequence of the known PKD1 substrate Hsp27. d, Purified GST-fusion proteins of SSH1L encompassing the putative PKD1 phosphorylation sites S937 and S978 as well as fusion proteins of SSH1L.S937A, SSH1L.S978A and SSH1L.S937A/S978A mutants were subjected a PKD1 kinase assay. Substrate phosphorylation was detected with the α-pMotif PKD-substrate antibody. Purified GST alone or GST-Hsp27 served as negative or positive controls. Control blots show substrate loading (α-GST) and PKD1 (α-PKD1). e, Purified GST (control) and GST-SSH1L were subjected to an in vitro kinase reaction with purified, active PKD1. Western blots of resolved proteins were probed with α-pS978-SSH1L. Control blots show substrate loading (α-GST) and PKD1 (α-PKD1). f, HeLa cells were co-transfected with Myc-tagged SSH1L or SSH1L.S978A and vector or constitutively-active PKD1 (PKD1.CA). SSH1L was immunoprecipitated (α-myc) and analyzed for phosphorylation at S978 (α-pS978-SSH1L). Blots were re-probed for SSH1L expression (α-SSH1L) and control blots were probed for PKD1 (α-PKD1). Uncropped images of Figs. 2a, 2d and 2f are shown in Supplementary information Fig. S6.