Src-mediated tyrosine phosphorylation of Protein Kinase D2 at focal adhesions regulates cell adhesion

Dependent on their cellular localization, Protein Kinase D (PKD) enzymes regulate different processes including Golgi transport, cell signaling and response to oxidative stress. The localization of PKD within cells is mediated by interaction with different lipid or protein binding partners. With the example of PKD2, we here show that phosphorylation events can also contribute to localization of subcellular pools of this kinase. Specifically, in the present study, we show that tyrosine phosphorylation of PKD2 at residue Y87 defines its localization to the focal adhesions and leads to activation. This phosphorylation occurs downstream of RhoA signaling and is mediated via Src. Moreover, mutation of this residue blocks PKD2’s interaction with Focal Adhesion Kinase (FAK). The presence and regulation of PKD2 at focal adhesions identifies a novel function for this kinase as a modulator of cell adhesion and migration.

A B C Supplemental Figure S1, relates to Figure 1: A: NMuMG cell express PKD2 and PKD3, but not PKD1. RT-PCR analysis using specific primer sets for mouse PKD1 (CTGGACATGTGGTCTGTTGG and GCTCACTGAGGGCTTTCATC), PKD2 (GCAGATATCCAGGAGGGTGA and GCACAGCGTTTGTGACAGTT) and PKD3 (CATCAGCAGCGTTTACCAGA and CACACAGCTTCACCTGAGGA). All experiments have been performed at least three times with similar results. B: PKD3 can not be immunoprecipitated with the anti-pY95/87 antibody. HeLa cells (3.5 x 10 6 cells per 10 cm plate) were subjected to immunoprecipitation analysis using the anti-pY95/87 antibody, and then probed for PKD3. Control Western blots of lysates were evaluated for PKD3 expression (input). C: Y87-phosphorylated PKD2 co-localizes with Paxillin. MDA-MB-231 and NMuMG cells in ibidi channel μ-slides were analyzed by immunofluorescence to determine the localization of endogenous pY87-PKD2 and Paxillin (secondary antibodies used: Alexa Fluor 488, Alexa Fluor 568). Scale bars indicate 10 μm.
Supplemental Figure S2, relates to Figure 1 Supplemental Figure S2, relates to Figure 1: PKD2 can be detected at the focal adhesions. A: Hek293T cells (0.5 x 10 6 cells/well of a 6 well plate) were co-transfected with GFP-Paxillin and vector or FLAG-PKD2. PKD2 was immunoprecipitated (anti-FLAG) and samples were analyzed for coimmunoprecipitated Paxillin (anti-GFP), and then then re-probed for PKD2. Control Western blots of lysates were evaluated for GFP-Paxillin expression (input) using anti-GFP antibodies. B: HeLa cells (6000 cells/channel, ibidi channel μ-Slide) were analyzed for the localization of endogenous PKD2 by immunofluorescence analysis (anti-PKD2 and Alexa Flour 488). F-actin was visualized with Alexa Flour 633-Phalloidin. Images shown represent single Z-stack slices (apical to basal, step size 0.5 µm). Scale bars indicate 10 μm.

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Supplemental Figure S3, relates to Figure 2: Loss of PKD2 is associated with reduced cell spreading. A: HeLa cells (0.2 x 10 6 cells/well, 6-well plate) were infected with lentivirus expressing non-target (scr) shRNA, or two independent shRNA sequences targeting PKD2. After 48 hours, cells (6000/channel) were reseeded in ibidi channel slides. Cells were subjected to immunofluorescence analysis to visualize F-actin which was stained with phalloidin. The scale bar indicates 10 μm. B: Shows quantitation for cell area, determined using Image J software in cells expressing scr-shRNA (n=50), PKD2-shRNA#1 (n= 50) and PKD2-shRNA#2 (n=50). The asterisk indicates statistical significance (p < 0.05) as compared to cells expressing scr-shRNA.

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Supplemental Figure S4, relates to Figure 3 Supplemental Figure S4, relates to Figure 3: Detection of active Src at focal adhesions of HeLa cells growing in channel slides. HeLa cells in ibidi channel μslides were transfected with RFP-LifeAct for visualization of F-actin. Cells were fixed and the localization of endogenous pY418-Src was determined by immunofluorescence analysis (secondary antibody used: Alexa Fluor 488). Scale bars indicate 10 μm.
Supplemental Figure S5, relates to Figure 5: Active RhoA, but not active Rac1 or Cdc42 induce PKD2 phosphorylation at Y87. HeLa cells (3 x 10 5 cells per well, 6 well plate) were co-transfected with FLAG-tagged PKD2 and GST-tagged versions of indicated RhoGTPases or control. PKD2 was immunoprecipitated (anti-FLAG) and samples were analyzed for PKD2 phosphorylation at Y87 (anti-pY87), and then then re-probed with anti-FLAG (PKD2). Control Western blots of lysates were evaluated for GST expression (input) using anti-GST antibodies.
Supplemental Figure S5, relates to Figure 5 Supplemental Figure S6, relates to Figure 6: Prevention of Y87-phosphorylation decreases cell adhesion and cell migration. A, B: HeLa cells were transfected with GFPtagged versions of PKD2 or PKD2.Y87F (A shows cells before reseeding) and then re-plated on fibronectin-coated (10 μg/ml) glass coverslips at a density of 0.02 x 10 6 cells/coverslip. Fluorescent images were acquired at the indicated times to determine the number of cells attached. B shows representative areas. Scale bar indicates 25 μm. A quantitation for the percentage of cells attached at 4 and 12 hours is shown in Figure 6B of the main manuscript. C: Western blots corresponding to Figure 6E. Indicated samples were analyzed for expression of GFP-tagged wildtype or mutant PKD2 using anti-GFP antibodies. Staining of the same blots for β-actin (anti-β-actin antibody) served as a loading control.
Supplemental Figure S6, relates to Figure 6 A B C Supplemental Figure