Structural insights into regulation of the PEAK3 pseudokinase scaffold by 14-3-3 - PubMed (original) (raw)
Structural insights into regulation of the PEAK3 pseudokinase scaffold by 14-3-3
Hayarpi Torosyan et al. Nat Commun. 2023.
Abstract
PEAK pseudokinases are molecular scaffolds which dimerize to regulate cell migration, morphology, and proliferation, as well as cancer progression. The mechanistic role dimerization plays in PEAK scaffolding remains unclear, as there are no structures of PEAKs in complex with their interactors. Here, we report the cryo-EM structure of dimeric PEAK3 in complex with an endogenous 14-3-3 heterodimer. Our structure reveals an asymmetric binding mode between PEAK3 and 14-3-3 stabilized by one pseudokinase domain and the SHED domain of the PEAK3 dimer. The binding interface contains a canonical phosphosite-dependent primary interaction and a unique secondary interaction not observed in previous structures of 14-3-3/client complexes. Additionally, we show that PKD regulates PEAK3/14-3-3 binding, which when prevented leads to PEAK3 nuclear enrichment and distinct protein-protein interactions. Altogether, our data demonstrate that PEAK3 dimerization forms an unusual secondary interface for 14-3-3 binding, facilitating 14-3-3 regulation of PEAK3 localization and interactome diversity.
© 2023. The Author(s).
Conflict of interest statement
N.J. is a founder of Rezo Therapeutics and a shareholder of Rezo Therapeutics, Sudo Therapeutics, and Type6 Therapeutics. N.J. is a SAB member of Sudo Therapeutics, Type6 Therapeutic and NIBR Oncology. The Jura laboratory has received sponsored research support from Genentech, Rezo Therapeutics and Type6 Therapeutics. The Krogan Laboratory has received research support from Vir Biotechnology, F. Hoffmann-La Roche, and Rezo Therapeutics. Nevan Krogan has previously held financially compensated consulting agreements with the Icahn School of Medicine at Mount Sinai, New York and Twist Bioscience Corp. He currently has financially compensated consulting agreements with Maze Therapeutics, Interline Therapeutics, Rezo Therapeutics, and GEn1E Lifesciences, Inc. He is on the Board of Directors of Rezo Therapeutics and is a shareholder in Tenaya Therapeutics, Maze Therapeutics, Rezo Therapeutics, and Interline Therapeutics. The other authors declare no competing interests.
Figures
Fig. 1. Structure of the PEAK3/14-3-3 complex.
a Representative Coomassie-stained SDS-PAGE gel analysis of the purified PEAK3/14-3-3 complex and b corresponding size exclusion chromatography profile resolved on a Superdex 200 10/300 Increase column. Pink bar on the chromatogram indicates collected fractions. Blue tick on the x-axis represents the elution volume of Bovine γ-globulin (158,000 Da) as a reference for the PEAK3/14-3-3 tetrameric complex (161,801 Da). SDS-PAGE and size exclusion chromatography results are representative of at least 3 independent experiments. c Cartoon schematic of PEAK3 domain structure depicting location of the 14-3-3 binding site. d Sequence logo (Weblogo) demonstrating conservation of the 14-3-3 binding site in PEAK3 across evolution. PEAK3 homolog sequences were collected by BLAST search in the Refseq database and clustered at 80% sequence identity. e Cryo-EM map of the PEAK3/14-3-3 complex colored according to local resolution determined by ResMap. f Structure of the PEAK3/14-3-3 complex and zoomed-in view of PEAK3 N-terminal 14-3-3 consensus binding sites superimposed with the cryo-EM map, demonstrating occupancy of both canonical substrate binding grooves in 14-3-3 by phosphorylated 14-3-3 consensus sequences of PEAK3. PEAK3 pseudokinase domains (PsK and PsK’) are in gray and the resolved regions of the N-terminal PEST linkers are shown in black. SHED domain helices αN1 are in yellow, αJ in pink, αK in blue, and αL in purple. 14-3-3ε is shown in green and 14-3-3β is shown in light green.
Fig. 2. SHED-dependent dimerization of PEAK3.
a Overall structure of the PEAK3 dimer as part of the PEAK3/14-3-3 complex (14-3-3 is not shown). b XL-shaped helical bundle of one PEAK3 monomer, highlighting intramolecular interactions. c Intermolecular interactions between helices αN1 and αJ of each PEAK3 monomer within the SHED dimer. d,e Zoomed-in views of the interactions between the SHED domain and pseudokinase/N-lobe (d) and pseudokinase/C-lobe (e). f Structural alignment of PEAK family SHED domains (PDB ID: 6BHC for PEAK1, and PDB ID: 5VE6 for PEAK2) along αN1 and αJ helices, depicting movement of αJ and αL helices in PEAK3 relative to their position in PEAK1 and PEAK2. g Overlay of the PEAK1 (PDB ID: 6BHC), PEAK2 (PDB ID: 5VE6) and PEAK3 dimer structures aligned along one pseudokinase domain (shown at the bottom of each overlay). Dashed lines in red indicate interactions with distances ≤ 4 Å.
Fig. 3. Asymmetric binding mode of the PEAK3/14-3-3 complex.
a Cartoon depicting primary and secondary interactions within a 14-3-3/client complex. b PEAK3 binding to the amphipathic grooves of 14-3-3ε and 14-3-3β, highlighting key conserved interactions. c Known binding modes of 14-3-3/client complexes found in the Protein Data Bank. 14-3-3 monomers are shown in green and light green, 14-3-3 substrate monomers are shown in pink and light pink, and phosphorylated residues or phosphomimetics which engage the 14-3-3 binding grooves are shown as red spheres. See Methods for alignment details. d PEAK3/14-3-3 structure highlighting PEAK3 SHED domain (box e) and pseudokinase domain (box f-h) interactions with 14-3-3ε. e-h Zoomed-in view of PEAK3/14-3-3ε secondary interface, highlighting key interactions. Dashed lines in red indicate interactions with distances ≤ 4 Å.
Fig. 4. Unique features of the PEAK3 pseudokinase domain.
a Protein sequence alignment of PEAK1, PEAK2, and PEAK3 depicting secondary structure elements, sequence motifs corresponding to canonical catalytic motifs in active kinases, and key dimerization interface residues based on structures of PEAK1 (PDB ID: 6BHC), PEAK2 (PDB ID: 5VE6) and PEAK3 (PEAK3/14-3-3 complex). Conserved residues are shaded in gray, αC helix is shown in blue while catalytic and activation loops with their corresponding motifs are highlighted in green and red, respectively. Residues involved in PEAK3 dimerization and those shown to occlude the pseudoactive site and PEAK3/14-3-3 secondary interface residues are marked by the indicated symbols. b Comparison of PEAK3 pseudokinase domain (light gray) with PEAK1 (light pink, PDB ID: 6BHC) and PEAK2 (light blue, PDB ID: 5VE6) pseudokinase domains with key structural elements highlighted (catalytic loop [c-loop] in green, activation loop [a-loop] in red, αC helix in blue). c,d Zoomed-in view of the (c) DFG motif and (d) αC helix in PEAK3, overlayed on the structure of PKA (PDB ID: 1ATP), aligned along entire kinase domain. e Zoomed-in view of the packing between the αC helix and activation loop of PEAK3 and the 14-3-3ε monomer. f Zoomed-in view of the pseudoactive site of PEAK3 showing the occluded nucleotide-binding site, the orientation of the catalytic lysine and the substituted Gln 221 in the αC helix.
Fig. 5. PEAK3 homodimerization and both 14-3-3 binding interfaces are critical for PEAK3/14-3-3 complex formation.
a Diagram of PEAK3 mutants generated to probe PEAK3 binding to the primary site on 14-3-3. b Co-immunoprecipitation of endogenous 14-3-3 with FLAG-tagged WT PEAK3 and PEAK3 mutants (Δ14-3-3, S69A) transiently expressed in HEK293 cells. c Representative co-immunoprecipitation of endogenous 14-3-3 with PEAK3 variants carrying mutations in the secondary 14-3-3 binding interface (R147A, S225A, K293A, W298A, K293A/W298A) transiently expressed in HEK293 cells. d Quantification of co-immunoprecipitation data shown in panel (c) plotted as the mean with standard deviation from 3 independent experiments. Statistical significance was determined using One-way ANOVA Dunnett’s multiple comparisons test, *p < 0.05, **<0.01, ***p < 0.001, ****p < 0.0001. e Co-immunoprecipitation of endogenous 14-3-3 with homodimerization-deficient PEAK3 mutants (L146E, A436E, C453E) transiently expressed in HEK293 cells. f Co-immunoprecipitation of FLAG-tagged and HA-tagged WT PEAK3 and PEAK3 S69A transiently expressed in HEK293 cells. In panels b–f, protein levels were detected with the indicated antibodies by Western Blot. All co-immunoprecipitation data are representative of at least 3 independent experiments. Source data are provided as a Source Data file.
Fig. 6. 14-3-3 influences cellular localization and scaffolding range of PEAK3.
a Immunofluorescence-based imaging of FLAG-tagged PEAK3 transiently expressed in COS-7 cells. Representative confocal microscopy images show cells transfected with an empty vector (EV) control or PEAK3 variants: WT, S69A, R147A, S225A, K293A, W298A, or K293A/W298A. PEAK3 was detected with an anti-FLAG antibody (green), and cells were further stained with DAPI (blue, nucleus) and iFluor-647 conjugated phalloidin (red, actin). Scale bars: 10 µm. b Quantification of relative nuclear enrichment of PEAK3 under conditions of impaired 14-3-3 binding. The ratio of fluorescence intensity in the green channel after background subtraction measured in the nucleus to the fluorescence intensity of the non-nuclear portion of the cell is plotted for each PEAK3 variant; see Methods for details. Data are plotted as the mean with standard deviation, combining all cells from at least 3 independent experiments (n = 57, 65, 60, 69, 71, 53, and 48 total cells for WT, S69A, R147A, S225A, K293A, W298A, and K293A/W298A, respectively). Statistical significance was determined using One-way ANOVA Dunnett’s multiple comparisons test, **p < 0.01, ***p < 0.001, ****p < 0.0001. c Spectrum of protein-protein interactions engaged by the WT or S69A PEAK3 analyzed by immunoprecipitation/mass spectrometry approach. Edges represent significant specific interactions of PEAK3 WT or S69A as determined by SAINTexpress (BFDR < 0.05) compared to negative control (non-transfected cells). Node colors represent the log2 fold change enrichment determined by MSstats between WT and S69A PEAK3 (bold node border represents significant adjusted p value < 0.05). CORUM complexes are indicated by yellow circles. Source data are provided as a Source Data file.
Fig. 7. PKD regulates the PEAK3/14-3-3 interaction.
a Consensus sequence of PKD substrates aligned with PEAK3’s 14-3-3 consensus site. b Co-immunoprecipitation of endogenous 14-3-3 with FLAG-tagged WT PEAK3 transiently expressed in HEK293 cells treated with 0.1% DMSO or increasing concentrations of the PKD inhibitor CRT0066101 dihydrochloride. Quantification of co-immunoprecipitation data plotted as the mean with standard deviation from 3 independent experiments. Statistical significance was determined using One-way ANOVA Dunnett’s multiple comparisons test, ***p < 0.001, ****p < 0.0001. Protein levels were detected with anti-FLAG and anti-pan 14-3-3 antibodies by Western Blot. c ADP-Glo PKD2 in vitro kinase assay conducted with PEAK3-derived PKD consensus site peptides (WT and S69A) and a PKD2 substrate peptide (CREBtide). Data are plotted as the mean with standard deviation, combining 3 independent experiments. Statistical significance was determined using One-way ANOVA Dunnett’s multiple comparisons test, ****p < 0.0001. d Immunofluorescence-based imaging of FLAG-tagged PEAK3 transiently expressed in COS-7 cells. Representative confocal microscopy images show cells transfected with an empty vector (EV) control or indicated FLAG-tagged PEAK3 constructs, and treated with 0.1% DMSO or 10 µM PKD inhibitor CRT0066101. PEAK3 was detected with an anti-FLAG antibody (green), and cells were further stained with DAPI (blue, nucleus) and iFluor-647 conjugated phalloidin (red, actin). Scale bars: 10 µm. e Quantification of relative nuclear enrichment of PEAK3 under conditions of impaired 14-3-3 binding. The ratio of fluorescence intensity in the green channel after background subtraction measured in the nucleus to the fluorescence intensity of the non-nuclear portion of the cell is plotted for each PEAK3 variant; see Methods for details. Data are plotted as the mean with standard deviation, combining all cells from at least 3 independent experiments (n = 58, 64, and 57 total cells for WT, WT + CRT0066101, and S69A, respectively). Statistical significance was determined using One-way ANOVA Dunnett’s multiple comparisons test, ****p < 0.0001. Source data are provided as a Source Data file.