Fig 2: PKD1 interacts with KAT7 in vitro and in vivo.a KAT7 were pulled down by Flag-PKD1. Cellular extracts from HEK293T cells overexpressing FLAG-PKD1 were immunoprecipitated with anti-FLAG antibody and Protein G-Sepharose beads. After extensively washing the beads, immobilized proteins were eluted in SDS–PAGE sample buffer. The eluent was resolved by SDS–PAGE and silver stained. The protein bands were retrieved and analyzed by mass spectrometry. b–e PKD1 interacts with KAT7 in vivo. b HEK293T cells were co-transfected with HA-PKD1 and Flag-KAT7 plasmids. IP assay and subsequent western blotting were performed by using the indicated antibodies in the panel. c HEK293T cells were transfected with Flag-KAT7 and HeLa cells were transfected with Flag-PKD1, IP assay was carried out by using anti-FLAG antibody and followed by western blotting with anti-KAT7 or anti-PKD1 antibody, respectively. d, e Whole-cell lysates from HEK293T, HeLa, or H1299 cells were immunoprecipitated with anti-KAT7 or anti-PKD1 antibodies followed by immunoblotting with the indicated antibodies. f PKD1 interacts with KAT7 in vitro. GST pull-down assay were performed with bacterial expressed GST-KAT7 protein and HEK293T-expressed FLAG-PKD1. g The schematic representation of the key domains within KAT7 was depicted, and GST pull-down assay was performed by incubating GST-KAT7 or its various deletion mutants with HEK293T-expressed HA-PKD1.

Fig 3: The PKD1 kinase domain autophosphorylates on its activation loop in vitro. A, size-exclusion chromatography (S200 10/300) of the purified kinase domain of PKD1 (PKD1CAT). Inset, Coomassie-stained SDS-PAGE loaded with a molecular weight marker ranging from 15 to 250 kDa and the pooled and concentrated fractions of the first run (solid lines). For this, 500 µl of 3 mg/ml (solid line) were injected onto the column. Injecting only 0.3 mg/ml (dotted line) shifts the elution volume. Dashed line, expected elution volumes for a monomeric (37-kDa) or dimeric (75-kDa) kinase domain derived from calibration of the column with globular molecular weight standards. B, fluorescence anisotropy of Atto488-labeled PKD1CAT (80 nm) as a function of increased, unlabeled PKD1CAT. The averaged change in fluorescence anisotropy (black squares) and the S.D. were derived from 31–60 technical replicates. To derive a Kd, the individual data points were fitted with a single binding mode (magenta line). C, static light scattering (SEC-MALS) of PKD1CAT. The peak fractions of the first run (625 µg, dark magenta) were pooled, and the same volume was reinjected onto the system (48 µg, light magenta). The table summarizes the polydispersity (Mn/Mw), the experimentally determined molecular weight (Mexp.), and the oligomeric state (Mexp./Mtheor.) for two peaks of the first run (1 and 2) and one peak of the second run (3). The manually selected peak boundaries are indicated on the chromatogram. D, intact MS of purified PKD kinase domain (black, PKD1CAT) and the same protein incubated at a concentration of 10 µm with 1 mm ATP and 2 mm MgCl2 at room temperature overnight (magenta, PKD1CAT ATP). E, radiometric kinase assay using PKD1CAT, [?-32P]ATP, and a conventional CAMK peptide substrate (syntide 2, black) or a peptide resembling the activation loop of PKD1 (act. loop (S742), magenta). The increase in radioactivity was plotted against the substrate concentration. Fitting the data points for syntide 2 to a Michaelis–Menten equation, a Km of 56 ± 9 µm was derived. The experiment was repeated three times with similar results. F, table indicates the consensus substrate sequence of PKD, displaying a leucine (L) at the -5 position and an arginine (R) at the -3 position with respect to the phosphorylatable Ser (or Thr) residue. X, any amino acid. The synthetic peptide syntide 2, a CAMK substrate, conforms to the PKD consensus motif. Both phosphorylation sites in the activation loop (Ser738 and Ser742), however, do not resemble a PKD consensus substrate site. Syntide 2 and the peptide covering amino acids 736–750 of the activation loop of PKD1 (act. loop (S742)) were used in the radiometric kinase assay. G, activation loop phosphorylation investigated by immunoblot using the anti-Ser738/742 antibody. 1 µm recombinant PKD1CAT or 1 µm recombinant GST-PKD1CAT was incubated with 1 mm ATP and 2 mm MgCl2 at room temperature, and a sample was taken at the indicated time points (0, 15, 30, and 60 min and overnight (o/n)), mixed with 2× SDS-loading dye, and immediately boiled at 95 °C. 10 ng of each sample were then subjected to SDS-PAGE and blotted for immunoblot analysis. H, size-exclusion profile of PKD1CAT (solid line) and stoichiometrically phosphorylated PKD1CAT (P-PKD1CAT, dashed line). The black dashed lines indicate the elution volume expected for a monomeric or dimeric kinase domain and are derived from the calibration of the column with globular protein standards.

Fig 4: Expression of pPKD1 (Ser 916) in T98G cells treated with ZKK-3 following 24 h of incubation. (A) Expression of pPKD1 (Ser 916) kinase in T98G cells treated with ZKK-3 and different oxygen conditions was determined using western blot analysis. (B) Impact of ZKK-3 and different oxygen conditions on the expression of pPKD1 (Ser 916) kinase. (C) Impact of ZKK-3 and different oxygen conditions on the expression of pPKD1 (Ser 916) kinase calculated as pPKD1(Ser 916)/total PKD1 ratio. Normoxia at 0 µM ZKK-3 represents a reference level of pPKD1 (Ser 916) expression. *P<0.05 and **P<0.01 vs. control under corresponding oxygen conditions; #P<0.05 and ##P<0.01 as indicated. pPKD1, phosphorylated protein kinase D1; HBO, hyperbaric oxygen; ZKK-3, N,N'-dimethyl-S-(2,3,4,5,6-pentabromobenzyl)-isothiouronium bromide.

Fig 5: PKD can monophosphorylate cTnI in rat adult cardiomyocytes. Cardiomyocytes infected with adenovirus carrying PKDWT/EGFP (+) or control EGFP (-) were stimulated with ET-1 (100 nmol/liter for 5, 10, or 20 min), PDBu (200 nmol/liter, 20 min), or isoproterenol (Iso, 10 nmol/liter, 10 min). A, representative immunoblots (IB) of p744/748 PKD and total PKD (top panel; the vertical line shows where an empty lane has been removed) and relative levels of p744/748 PKD normalized to total PKD (bottom panel). B, representative Phos-tag gels of total TnI and phosphoserine 23/24 cTnI (top panel) and proportion of cTnI unphosphorylated, monophosphorylated, and bisphosphorylated cTnI after the various treatments. Symbols show the data from individual experiments; vertical bars show the mean ± S.D. (n = 3–5).