Volume 19, Issue 12, Pages 2586-2597 (June 2017) PKN1 Directs Polarized RAB21 Vesicle Trafficking via RPH3A and Is Important for Neutrophil Adhesion and Ischemia-Reperfusion Injury  Qianying Yuan, Chunguang Ren, Wenwen Xu, Björn Petri, Jiasheng Zhang, Yong Zhang, Paul Kubes, Dianqing Wu, Wenwen Tang  Cell Reports  Volume 19, Issue 12, Pages 2586-2597 (June 2017) DOI: 10.1016/j.celrep.2017.05.080 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 19, 2586-2597DOI: (10.1016/j.celrep.2017.05.080) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 PKN1 Deficiency Attenuates Neutrophil Adhesion to Endothelial Cells (A) Validation of the lack of PKN1 proteins in neutrophils isolated from Pkn1m/m mice by western blot analysis. PKN2 and tubulin were detected as internal controls. (B–D) PKN1 deficiency impairs neutrophil infiltration into inflamed mouse peritonea (B), adhesion to endothelial cells under shear flow (C), and binding to ICAM1 (D). Data are presented as means ± SEM (∗p < 0.05; Student’s t test; n > 5). (E–G) Effect of PKN1 deficiency on neutrophil-endothelial cell interaction in inflamed cremaster muscle venues. Adhesion (E), transmigration of neutrophils (F), and rolling flux (G) were determined after stimulation of the cremaster muscle with TNF-α (0.5 μg) for 4 hr. All values are means of n = 5 animals (with five to seven vessels per animal) ± SEM (∗p < 0.05; ∗∗p < 0.01; Student’s t test). Cell Reports 2017 19, 2586-2597DOI: (10.1016/j.celrep.2017.05.080) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 PKN1 Deficiency Impairs PIP5K1C90 Polarization in Neutrophils (A and B) WT (A) or PKN1-defient (B) mouse neutrophils transfected with HA-PIP5K1C90 were placed on Fb-coated coverslips for 30 min. They were then fixed and stained by anti-HA and anti-EEA1 antibodies followed by secondary antibodies conjugated with Alexa Fluor 633 (colored in red) and Alexa 488 (colored in green). Cell contours are outlined, and scale bars are 2.5 μm for all of the figures. (C) Quantification of the effect of PKN1 deficiency on PIP5K1C90 polarization. The quantification was done from three independent observations, and more than 20 cells were examined in each observation. Data are presented as means ± SD (∗∗p < 0.01; Student’s t test). Cell Reports 2017 19, 2586-2597DOI: (10.1016/j.celrep.2017.05.080) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 RAB21 Is Polarized and Involved in PIP5K1C90 Polarization in Neutrophils (A–C) RAB21DN mutant inhibits PIP5K1C90 polarization in mouse neutrophils. Mouse neutrophils were cotransfected with HA-PIP5K1C90 and GFP (A) or RAB21DN-GFP (B) and sorted by FACS. GFP-positive cells were stimulated by Fb for 30 min and stained by anti-HA and anti-EEA1 antibodies, followed by secondary antibodies conjugated with Alexa Fluor 633 (colored in red) and Alexa 488 (colored in green). Two representative cells are shown in (A) and (B). (C) shows the quantification of the percentage of neutrophils showing PIP5K1C90 polarization from three independent experiments. More than 20 cells were examined in each experiment. Data are presented as means ± SD (∗p < 0.01; Student’s t test). Figure S2A shows localization of PIP5K1C90 and EEA1 staining in representative cells placed on PK-coated surfaces as a control. (D–F) Polarization of RAB21 in mouse neutrophils upon Fb stimulation. Mouse neutrophils were placed on coverslips coated with PK (D) or Fb (E) for 30 min and stained by anti-RAB21 and Alexa-488-conjugated secondary antibody (green) in presence of Alexa Fluor 647-phalloidin (red). (F) shows the quantification of the percentage of neutrophils showing RAB21 polarization from three independent experiments. More than 20 cells were examined in each experiment. Data are presented as means ± SD (∗p < 0.01; Student’s t test). (G) Polarization of RAB21 and PIP5K1C90 in neutrophils. Mouse neutrophils transfected with HA-PIP5K1C90 were placed on Fb-coated coverslips for 30 min and stained by anti-HA and anti-RAB21 antibody followed by secondary antibodies conjugated with Alexa Fluor 633 (colored in red) and Alexa 488 (colored in green). Figure S2C shows localization of PIP5K1C90 and RAB21 staining in cells placed on PK-coated surfaces as a control. (H) PKN1 deficiency impairs RAB21 polarization. Mouse neutrophils were placed on Fb-coated coverslips for 30 min and fixed and stained by anti-RAB21 and anti-EEA1 antibodies, followed by secondary antibodies conjugated with Alexa Fluor 633 (colored in red) and Alexa 488 (colored in green). The quantification for percentage of neutrophils showing RAB21 polarization was done from three independent observations, and more than 20 cells were examined in each observation. Data are presented as means ± SD (∗p < 0.01; Student’s t test). Cell Reports 2017 19, 2586-2597DOI: (10.1016/j.celrep.2017.05.080) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 RPH3A Binds to RAB21 RAB21 binds to RPH3A via RPH3A’s C2 domain in a Ca2+ and GTP-dependent manner. Purified recombinant proteins were used in a GST-pull-down assay under the conditions described in the figure. Proteins were then detected by western blot analysis. C2, the C2 domain; Zn, the Zinc binding domain. Cell Reports 2017 19, 2586-2597DOI: (10.1016/j.celrep.2017.05.080) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 RPH3A Is Involved in PIP5K1C90 and RAB21 Polarization (A and B) RPH3A deficiency impairs polarization of PIP5K1C90 and RAB21 in mouse neutrophils. Mouse neutrophils transfected with HA-PIP5K1C90 were placed on Fb-coated coverslips for 30 min and stained by anti-EEA1 and anti-HA (A) or anti-RAB21 (B) antibodies, followed by secondary antibodies conjugated with Alexa 488 (colored in green) and Alexa Fluor 633 (colored in red). The percentage of neutrophils showing polarization was quantified from three independent observations, and more than 15 cells were examined in each observation. Data are presented as means ± SD (∗∗p < 0.01; Student’s t test). (C–E) RPH3A deficiency impairs neutrophil binding to ICAM1 (C), adhesion to endothelial cells under shear flow (D), and infiltration into inflamed mouse peritonea (E). Data are presented as means ± SEM (∗p < 0.05; ∗∗p < 0.01; Student’s t test; n > 4). (F–H) Effect of RPH3A deficiency on neutrophil-endothelial cell interaction in inflamed cremaster muscle venues. Adhesion (F), transmigration of neutrophils (G), and rolling flux (H) were determined after stimulation of the cremaster muscle with TNF-α (0.5 μg) for 4 hr. All values are means of n = 3 animals (with five to seven vessels per animal) ± SEM (∗∗p < 0.01; Student’s t test). Cell Reports 2017 19, 2586-2597DOI: (10.1016/j.celrep.2017.05.080) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 PKN1-Mediated Phosphorylation of RPH3A Is Required for Polarization (A) PKN1 phosphorylates RPH3A. Phosphorylation of recombinant RPH3A protein by recombinant PKN1 protein was performed in an in vitro kinase assay, and RPH3A phosphorylation was detected by western blot analysis using an anti-phospho-AKT substrate motif antibody. (B) RPH3A residues S234 and S271 are phosphorylated by PKN1. WT RPH3A and its mutants SA1 (S234A) and SA (S234 and 271A) were subjected to an in vitro kinase assay using [32P]ATP. Phosphorylated RPH3A ([32P]RPH3A) was detected by a phosphoimager, whereas the inputs were detected by western blot analysis. (C) RPH3A phosphorylation at Ser 234 depends on PKN1. WT and PKN1-null neutrophils were analyzed by western blot analysis using an anti-phospho-Ser234 RPH3A, anti-RPH3A, PKN1, or actin antibody. (D and E) WT RPH3A, but not its phosphorylation-defective mutant, can rescue RAB21 polarization in RPH3A-null cells. RPH3A-null neutrophils were transfected with RPH3A-HA (D) or RPH3A-SA-HA (E). The cells were then stained with an anti-HA antibody and anti-RAB21 antibody. Only anti-HA-staining-positive cells were examined. (F) Phosphorylated RPH3A shows enhanced binding to RAB21. Purified RPH3A protein was first incubated with PKN1 in the presence or absence of ATP in a kinase assay buffer for 30 min at 37°C. Phosphorylated RPH3A (pRPH3A) is evidenced by its upshift in the blot compared to unphosphorylated RPH3A (RPH3A). These RPH3A proteins were then subjected to a pull-down assay with GTPγS-bound RAB21. The proteins were detected by western blot analysis. Cell Reports 2017 19, 2586-2597DOI: (10.1016/j.celrep.2017.05.080) Copyright © 2017 The Author(s) Terms and Conditions

Figure 7 PKN1 Deficiency Attenuates Renal Ischemia-Reperfusion Injury (A) Schematic presentation of a model for RPH3A and its phosphorylation to regulate directional vesicle trafficking and PIP5K1C90 polarization. Integrin engagement triggers endocytosis of PIP5K1C90, which traffics through EEA1-positive endosomes and emerges in RAB21-positive recycling vesicles. GTP-bound RAB21 exits from the endosomes and recruits PKN1-phosphorylated RHP3A. This process is required for polarized RAB21 vesicle trafficking and PIP5K1C90 polarization. (B and C) Loss of PKN1 reduces renal neutrophil infiltration. Neutrophil infiltration into reperfused kidneys was evaluated by flow cytometry after dissociated kidney cells were stained with CD11b and Ly6G (B) and by immunostaining of renal tissue sections with anti-Ly6B (C). Representative flow cytometric charts are shown in Figure S5. Data in (B) are presented as means ± SEM (∗∗p < 0.01; Student’s t test; n = 5). (D) Blood creatinine contents from mice that were subjected to the renal ischemia-reperfusion procedure. Data are presented as means ± SEM (∗∗p < 0.01; Student’s t test; n = 5). (E) Histological examination of renal tissue injury. Renal histological sections from mice that were subjected to the renal ischemia-reperfusion procedure were stained with periodic acid-Schiff (PAS) dye, and the representative sections are shown. Cell Reports 2017 19, 2586-2597DOI: (10.1016/j.celrep.2017.05.080) Copyright © 2017 The Author(s) Terms and Conditions