PKD autoinhibition in trans regulates activation loop autophosphorylation in cis

Significance Many eukaryotic protein kinases are activated by phosphorylation of their activation loop. Some protein kinases can autoactivate by phosphorylating their own activation loop. This reaction is widely believed to occur exclusively in trans (by a copy of the same protein) and has been observed in a wide array of kinases that undergo stimulus-induced dimerization. Here, we describe the inverse mechanism for activation of protein kinase D (PKD), an essential kinase involved in membrane trafficking. We show that inactive PKD is dimeric and that its activation depends on dissociation of its kinase domains followed by activation loop autophosphorylation in cis (by itself). Nature has, therefore, found two solutions to the same problem that are simply the inverse of one another.

Stoichiometric phosphorylation of PKD1 KD (1P) was achieved by incubating the washed, GST-PKD1 KD bound beads with 1mM ATP and 5 mM MgCl2 at 4°C overnight followed by 1 h at room temperature. After TEV cleavage, the phosphorylated species were separated on a MonoQ 5/50 GL column (Cytiva) and characterized by intact mass spectrometry. PKD1 ULD proteins were purified according to (3).

Analytical size exclusion chromatography
A Superdex 200 increase 3.2/300 size exclusion column (Cytiva), connected to an Äkta Pure with a Micro configuration (Cytiva) was equilibrated in buffer (20 mM HEPES pH 7.4, 150 mM KCl, 1 mM EDTA, 1 mM TCEP, 1 % (v/v) glycerol) at 4°C. 25 µL protein samples were clarified by centrifugation (5 min, 21 000 g) prior to injection at constant flow rate. Peak elution volumes are reported, while peak concentration was determined by integrating the peak area for 20 µL centered on the highest point.

Differential scanning fluorimetry (DSF)
Thermal stability measurements of PKD1 KD were performed in 20 mM Tris pH 7.5, 150 and, in the absence of a concentration-dependent change in melting temperature, the measurements were averaged and presented as replicates.

Sample preparation for mass spectrometry
The Coomassie-stained gel band was de-stained with a mixture of acetonitrile (Chromasolv®, Sigma-Aldrich) and 50 mM ammonium bicarbonate (Sigma-Aldrich). The proteins were reduced using 10 mM dithiothreitol (Roche) and alkylated with 50 mM iodoacetamide. Trypsin (Promega; Trypsin Gold, Mass Spectrometry Grade) was used for proteolytic cleavage. Digestion was carried out with trypsin at 37°C overnight. Formic acid was used to stop the digestion and the extracted peptides were desalted using C18 Stagetips (4).

Sample analysis
Peptides were analyzed on an UltiMate 3000 HPLC RSLC nanosystem (Thermo Fisher Scientific) coupled to a Q Exactive HF-X, equipped with a nano-spray ion source using coated emitter tips (PepSep, MSWil). Samples were loaded on a trap column (Thermo Fisher Scientific, PepMap C18, 5 mm × 300 μm ID, 5 μm particles, 100 Å pore size) at a flow rate of 25 μL min -1 using 0.1% TFA as mobile phase. After 10 min, the trap column was switched in-line with the analytical C18 column (Thermo Fisher Scientific, PepMap C18, 500 mm × 75 μm ID, 2 μm, 100 Å) and peptides were eluted applying a segmented linear gradient from 2% to 80% solvent B (80% acetonitrile, 0.1% formic acid; solvent A 0.1% formic acid) at a flow rate of 230 nL/min over 120 min. The mass spectrometer was operated in data-dependent mode, survey scans were obtained in a mass range of 350-1600 m/z with lock mass activated, at a resolution of 120,000 at 200 m/z and an AGC target value of 1E6. The 15 most intense ions were selected with an isolation width of 1.2 Thomson for a max. of 150 ms, fragmented in the HCD cell at stepped normalized collision energy at 26%, 28%, and 30%. The spectra were recorded at an AGC target value of 1E5 and a resolution of 60,000. Peptides with a charge of +1, +2, or >+7 were excluded from fragmentation, the peptide match feature was set to preferred, the exclude isotope feature was enabled, and selected precursors were dynamically excluded from repeated sampling for 20 seconds within a mass tolerance of 8 ppm.
Raw data were processed using the MaxQuant software package 1.6.17.0 (5) and searched against the Uniprot human reference proteome (January 2020, www.uniprot.org) as well as a database of most common contaminants. The search was performed with standard identification settings: full trypsin specificity allowing a maximum of two missed cleavages. Carbamidomethylation of cysteine residues was set as fixed, oxidation of methionine and acetylation of protein N-termini as variable modifications. All other settings were left at default. Results were filtered at a false discovery rate of 1% at protein and peptide spectrum match level. To identify crosslinked peptides, the spectra were searched using pLink software 2.3.9 (6) against the sequences of the top 8 non-contaminant proteins from the MQ search sorted by iBAQ.
Carbamidomethylation of cysteine was set as fixed, oxidation of methionine and acetylation of protein N-termini as variable modifications. The enzyme specificity was set to trypsin allowing 4 missed cleavage sites. Crosslinker settings were selected as EDC.
Search results were filtered for 1% FDR (false discovery rate) on the PSM level (peptidespectrum matches) and a maximum precursor mass deviation of 5 ppm. To remove low quality PSMs, additionally an e-Value cutoff of < 0.001 was applied. Cross-link maps were generated in xiNET (7).

Proteomics data deposition
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (8) with the dataset identifier PXD031997.

Kinase assays
Assays probing the fraction of autophosphorylated PKD KD at the reaction plateau at different concentrations were performed at 30°C overnight. After termination of the reaction, aliquots corresponding to 0.5 pmol of PKD KD were subjected to SDS-PAGE to separate the phosphorylated protein from excess [γ-32P] ATP. The gels were stained with silver stain (Invitrogen), dried and exposed to a phosphor screen overnight. The fraction of phosphorylated PKD KD was calculated from the internal standard, assuming a single phosphate is incorporated per PKD KD molecule (confirmed by intact mass spectrometry).
To test the reversible nature of the plateau, a reaction containing PKD KD was left to reach completion over 2 h at 30°C. A fraction was then diluted to a final concentration of 50 nM and allowed to react for an additional 18 h under the same conditions. Aliquots corresponding to 0.5 pmol of each reaction were subjected to SDS-PAGE and analyzed as described above.
Aliquots corresponding to 0.5 pmol of active kinase were subjected to SDS-PAGE. The gels were washed three times in dH2O and processed as described above. The signal was normalized to PKD1 KD (0P) at 50 nM.
Autophosphorylation time course experiments were performed at 30°C with 50 nM PKD1 KD . Samples were taken from the reaction volume, terminated and aliquots corresponding to 0.5 pmol of PKD1 KD were subjected to SDS-PAGE and processed as described above.
Substrate phosphorylation time course assays with PKD1 ULD-5G/10G-KD were performed on ice with 50 nM protein and 100 µM biotinylated Syntide2 (biotin-PLARTSVAG (GenScript)). Samples were taken from the reaction volume, terminated, and spotted on a nitrocellulose membrane. The membrane was washed five times with 20 ml of 75 mM H3PO4 and exposed to a phosphor screen for 2 h together with a set of internal standards.
The reactions were started by adding 10 nM PKD1 KD and terminated after 2 min. Aliquots were spotted onto a nitrocellulose membrane and processed as described above.

Dynamic light scattering
Dynamic light scattering measurements were performed on 10 µM PKD KD in 100 mM Tris pH 7.5, 150 mM KCl, 1 mM TCEP, 1% Glycerol, 10 mM MgCl2 1 mM ATP at 20°C using a DynaPro NanoStar instrument (Wyatt Technology Corp.). Buffer and protein stocks were cleared from aggregate particles by ultracentrifugation at 186,000 x g for 1.5 h prior to the measurements.

Protein digestion and MS/MS data collection
Protein samples were rapidly thawed and injected onto an integrated fluidics system containing a HDx-3 PAL liquid handling robot and climate-controlled (2°C) chromatography system (LEAP Technologies), a Dionex Ultimate 3000 UHPLC system, as well as an Impact HD QTOF Mass spectrometer (Bruker). The full details of the automated LC system are described in (9). The protein was run over one immobilized pepsin column MS/MS datasets were analyzed using PEAKS7 (PEAKS), and peptide identification was carried out by using a false discovery-based approach, with a threshold set to 0.1% using a database of known contaminants found in Sf9 cells (10). The search parameters were set with a precursor tolerance of 20 ppm, fragment mass error 0.02 Da, charge states from 1-8, leading to a selection criterion of peptides that had a -10logP score of 26.2 and 24.8 for PKD1 KD or PKD1 KD S738/742E, respectively.

Mass Analysis of Peptide Centroids and Measurement of Deuterium Incorporation
HD-Examiner Software (Sierra Analytics) was used to calculate the level of deuterium incorporation into each peptide. All peptides were manually inspected for correct charge state, correct retention time, and appropriate selection of isotopic distribution.  Table 2 according to published guidelines (11). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (8) with the dataset identifier PXD033139.

Protein extraction of cells and Western blotting
Whole cell extracts were obtained by solubilizing cells in lysis buffer (20 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM ethylene glycol tetra acetic acid (EGTA), plus Complete protease inhibitors and PhosSTOP (Roche Diagnostics, Basel, Switzerland)). Whole cell lysates were clarified by centrifugation for 15 min at 16,000 g and 4°C. Equal amounts of protein were loaded on 10% polyacrylamide gels or were run on NuPage Novex 4-12% Bis-Tris gels (Life Technologies) and blotted onto nitrocellulose membranes using the iBlot device (Life Technologies). Membranes were blocked for 30 min with 0.5% (v/v) blocking reagent (Roche Diagnostics) in PBS containing 0.05% (v/v) Tween-20. Membranes were incubated with primary antibodies overnight at 4°C, followed by 1 hr incubation with HRP-conjugated or IRDye-conjugated secondary antibodies at room temperature.
Proteins were visualized using an enhanced chemiluminescence detection system (Thermo Fisher Scientific, Waltham, MA, USA) or the Licor Odyssey system. Special care was taken not to overexpose in order to guarantee accurate quantifications.
Densitometry was performed using Image Studio Lite 4.0 (Li-COR Biosciences, Bad Homburg, Germany). For each protein, the integrated density of the signal was measured and corrected for background signals.

Immunofluorescence staining and confocal microscopy
Cells expressing PKD1-EGFP were grown on glass coverslips coated with 2,5 mg/ml collagen R (Serva, Heidelberg, Germany) and fixed for 15 min with 4% (v/v) paraformaldehyde. After washes in PBS, cells were incubated for 5 min with 1 M glycine in PBS and permeabilized for 2 min with 0.1% (v/v) Triton X-100 in PBS. Blocking was performed with 5% (v/v) bovine serum (PAN) in PBS for 30 min. Fixed cells were incubated with primary antibodies diluted in blocking buffer for 2 hr at room temperature. Following three washing steps with PBS, cells were incubated with Alexa-Fluor-546-labeled secondary antibodies in blocking buffer for 1 hr at room temperature.
Nuclei were counterstained with DAPI and mounted in ProLong Gold Antifade Reagent (Thermo Fisher Scientific). All samples were analyzed at room temperature using a confocal laser scanning microscope (LSM 710, Carl Zeiss) equipped with a Plan Apochromat 63x/1.40 DIC M27 (Carl Zeiss, Jena, Germany) oil-immersion objective. GFP was excited with the 488 nm line of an Argon laser, its emission was detected from 496 to 553 nm. Alexa546 was excited with a 561 nm DPSS laser, its emission was detected from 566 to 622 nm. Image acquisition for G-PKDrep ratiometric imaging was done as follows: z-stacks of 0.5 mm intervals were acquired throughout the cell and maximum intensity projections were calculated. GFP and Alexa546 channels were hereby acquired with the same pinhole setting that was adjusted to 1 AU in the Alexa 546 channel. Laser powers were adjusted to prevent fluorophore saturation and identical photomultiplier tube and laser settings were maintained throughout the whole experiment. Image processing and analysis was performed with Zen black 2.1 software. Regions of interest of identical Golgi areas of reporter expressing cells were selected in the GFP channel, mean pixel intensity values of the selected areas in both channels were measured and the Alexa546 to GFP ratio was calculated.