A Combined Approach Reveals a Regulatory Mechanism Coupling Src's Kinase Activity, Localization, and Phosphotransferase-Independent Functions

Abstract

Multiple layers of regulation modulate the activity and localization of protein kinases. However, many details of kinase regulation remain incompletely understood. Here, we apply saturation mutagenesis and a chemical genetic method for allosterically modulating kinase global conformation to Src kinase, providing insight into known regulatory mechanisms and revealing a previously undiscovered interaction between Src's SH4 and catalytic domains. Abrogation of this interaction increased phosphotransferase activity, promoted membrane association, and provoked phosphotransferase-independent alterations in cell morphology. Thus, Src's SH4 domain serves as an intramolecular regulator coupling catalytic activity, global conformation, and localization, as well as mediating a phosphotransferase-independent function. Sequence conservation suggests that the SH4 domain regulatory interaction exists in other Src-family kinases. Our combined approach's ability to reveal a regulatory mechanism in one of the best-studied kinases suggests that it could be applied broadly to provide insight into kinase structure, regulation, and function.

Keywords: Src; activity; allostery; kinase; regulation.

Figure 1.. Dissection of intramolecular kinase regulation using a multidisciplinary approach.

A. Structural features of Src’s catalytic domain (CD) (PDB: 3DQW), showing the C-spine (Catalytic, green), R-spine (regulatory, blue), helix αC (yellow), and the activation (A, pink)-loop. B. Linear schematic of Src kinase (Unq = Unique domain). C. Src kinase in the closed, autoinhibited (left, PDB: 2SRC) and an open (right, PDB: 1Y57) global conformation. The SH3 domain is shown in orange, the SH2 domain in green and the CD in purple. D. Deep mutational scanning for the simultaneous measurement of the activity of nearly all possible single mutants of a kinase. E. Cysteine Installation for Modulating Allostery and Targeted Inhibition of Kinases (CystIMATIK), a method for selectively modulating intramolecular regulatory domain interactions and global conformation of a drug-sensitized kinase with conformation-selective, ATP-competitive probes.

Figure 2.. Multiplex measurement of the activity of 3,506 single amino acid Src variants.

A. Schematic of a yeast growth-based deep mutational scan (DMS) of the Src catalytic domain. B. Individually-assessed growth curves for yeast expressing Src^myr WT, K298M, T341I, or a control vector (n=3). C. Src and phospho-tyrosine (pTyr) immunoblots of yeast expressing Src^myr variants for 24 hr. D. Scatterplot showing activity score correlations between two independent transformations of the Src^myr variant library (Pearson’s R = 0.91). E. Activity scores for variants classified as gain-of-function (n=403, green), WT-like (n=1288, orange), or loss-of-function (n=1681, blue). F. Position-averaged activity scores mapped onto the Src catalytic domain (PDB: 3DQW). Nonsense mutants were excluded from the average score. Sequence-activity map of Src catalytic domain. Black dots in the map indicate the WT amino acid, gray tiles indicate missing data. Bar graph indicates relative evolutionary conservation at each position as determined by Kullback-Leibler entropy. Secondary structure and functional motif annotations were obtained from the ProKinO database. G. Dot plot of individually assessed growth rates compared to activity scores for a panel of Src^myr variants (n=3; Pearson’s R = −.97). Growth rates for WT, K298M, and T341I from Figure 2B are shown for comparison. H. Src and phospho-tyrosine immunoblots of yeast expressing Src^myr variants. I. Correlation between yeast DMS-derived activity scores and the ratio of phospho-tyrosine/Src levels in HEK293Ts for a panel of Src^myr variants (n=3; Pearson’s R = 0.97; Figures S1E, S1F). Points represent individual measurements and the horizontal lines indicate the mean of all measurements. See also Figure S1, Tables S2 and S3.

Figure 3.. Large-scale mutagenesis data reveal Src’s regulatory interfaces.

A. Hierarchical clustering of Src residues with at least five gain-of-function mutations based on spatial coordinates of the atomic centroids of each sidechain (right panel) projected onto the Src structure (left panel, PDB: 2SRC). B. Structural detail (left panels) and activity scores (right panels) for every variant at each residue comprising clusters that overlap with the SH2-CD (cluster 4, top), SH3/Linker-CD (cluster 7, middle) and αF pocket (cluster 2, bottom) interfaces (PDB: 2SRC). In the left panels, CD residues that are not part of a cluster are shown in white, SH2-linker residues in tan, SH2 residues in green, and SH3 residues in orange. C. Phosphotransferase activity of purified Src^FL WT, T293D, E381T, or I444K (n=3-6). D. Schematic of the SH3 pulldown assay. To detect global conformation, Src is incubated with an immobilized SH3 domain ligand. Closed, SH3-engaged Src is unable to bind to the resin, whereas open, SH3-disengaged Src binds. After washing, retained Src is eluted and quantified by western blot or in-gel fluorescence. E. Percent retained Src in the SH3 pulldown assay with purified Src^FL WT, T293D, or D368K (n=3-6). F. Sequence alignment of Src-family kinase αF pocket residues. G. Representative micrographs (left) and percent bleb quantification (right) for SYFs expressing either Src^GFP WT or Src^GFP E381T. Scale bars = 10 μm. Each point represents a replicate transfection with multiple cells imaged and scored in a double-blind fashion. Horizontal lines indicate the mean of all replicates. See Table S5 for the total number of replicates and cells analyzed. *p <0.05. See also Figure S2.

Figure 4.. Cysteine Installation for Modulating Allostery and Targeted Inhibition of Kinases (CystIMATIK).

A. Structure of the CD of Src (PDB: 2SRC) bound to AMP-PNP. Positions that are important for sensitivity to CystIMATIK probes are shown as sticks (T341 (red) and V284 (blue)). B. Sequence alignment of Src and Hck with various kinases and the C-terminal kinase domain of p90 ribosomal protein S6 kinase, CTD-RSK2. C. CystIMATIK probes 1-3. IC₅₀ values (n=3, mean ± s.e.m), determined in the presence of 1 mM ATP, for Src^3D WT, Src^3D V284C, Hck^3D WT, and Hck^3D V284C are shown below each probe. Hck is numbered according to analogous Src residue. D. Crystal structures of the Src^CD V284C-1 (left; PDB:5SWH), Src^CD V284C-2 (middle; PDB:5TEH) and Src^CD V284C-3 (right; PDB:5SYS) complexes. Top panels: 1-3 are shown as sticks colored by atom. Bottom panels: Helix αC (yellow) and the Phe (pink) of the DFG motif are shown. C284, K298, and E313 are depicted as sticks. E. Percent retained Src in the SH3 pulldown assay for Src^FLAG V284C-expressing HEK293s treated with CystIMATIK probe 1, 2, or 3 (n=3). F. SH2 domain accessibility assay using Csk. Src^3D V284C-CystIMATIK probe complexes are incubated with Csk and γ³²P-ATP, and radioactive phosphate transfer to Src^3D V284C is quantified. Closed, SH2-engaged Src cannot be phosphorylated by Csk whereas open, SH2-disengaged Src is efficiently phosphorylated by Csk. G. Quantification of Csk’s phosphorylation of the Src^3D V284C-2 and Src^3D V284C-3 complexes. H. Kinome profiling of CystIMATIK probes 1-3. Profiled kinases are represented by grey circles and interacting kinases by red circles. For interacting kinases, circle size scales with log2 SILAC ratio of DMSO control over 10 μM of each CystIMATIK probe (a mean log2 SILAC ratio >1 cut-off was applied; n=3). The heatmap shows all kinase targets for each CystIIMATIK probe. For 4E, 4G, points represent individual measurements and the horizontal lines indicate the mean of all measurements. See also Figure S3 and Table S7.

Figure 5.. Conformational changes at the ATP-binding site drive phosphotransferase-independent cellular blebbing.

A. Representative micrographs (left) and percent bleb quantification (right) for Src^GFP V284C-expressing SYFs treated for 15 min with DMSO, CystIMATIK probe 1, 2, 3, or pre-treated with 2 for 15 min, followed by 3. B. Representative micrographs from time course experiments performed with Src^GFP V284C-expressing SYFs treated with CystIMATIK probe 2 or 3. Insets show membrane blebs. C. Percentage of Src^GFP V284C-expressing SYFs treated with 1, 3, or 10 μM 3 for 15 min showing blebs. Values for DMSO and 10 μM conditions were used previously in Figure 5A. D. Representative micrographs (left) and percent bleb quantification (right) for Src^GFP V284C-expressing SYFs pretreated with the Rock inhibitor GSK429286A, and then treated with DMSO or 3 for 15 minutes. E. Representative micrographs (left) and percent bleb quantification (right) for Src^GFP G2A/V284C-expressing SYFs treated with DMSO or 3 for 15 minutes. F. Structures of probes 4, 5, and 6 and IC₅₀ values for Src^FL WT determined in the presence of 1 mM ATP (n=3, mean ± s.e.m). G. Quantification of Src immunoblots for co-sedimentation assays performed with apo Src^FLAG, Src^FLAG-4, Src^FLAG-5, or Src^FLAG-6, and lipsosomes composed of 1:1:1 phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine (PC:PS:PE) or 2:1 PC and PS (2PC:PS) (Figures S4H, S4I). In all micrographs, scale bars = 10 μm. For 5A-5E, each point represents a replicate treatment with multiple cells imaged and scored in a double-blind fashion. See Table S5 for the total number of replicates and cells analyzed. Horizontal lines indicate the mean of all replicates. ***p < 0.001. See also Figure S4.

Figure 6.. Direct interaction of the SH4 and catalytic domain regulates Src activity and dictates cellular phenotype.

A. Proposed SH4 domain “fastener” model (left) and the Src constructs used for biochemical characterization (right). B. Quantification of Src immunoblots for co-sedimentation assays performed with Src^FLAG WT or Src^FLAG E381T, and liposomes composed of PC:PS:PE or 2PC:PS. C. Phosphotransferase activity of purified Src^FL, Src^3D or Src^ΔSH4 with either the WT or E381T sequence. (n=4-6). Values for Src^FL WT and E381T were used previously in Figure 3C. D. Autophosphorylation quantification of Src^FL or Src^ΔSH4 with either the WT or E381T sequence at various time points after ATP addition (n=3). E. Percent retained Src in the SH3 pulldown assay with purified Src^FL or Src^ΔSH4 with either the WT or E381T sequence (n=3-5). Values for Src^FL WT were used previously in Figure 3E. F. Model showing the global conformation of Src^FL K445C-5 or Src^FL K445C-6. G. Isotope-coded maleimide labeling of Src^FL K445C. An example mass spectrum of the light and heavy maleimide-labeled peptide containing K445C (αF peptide). The inset shows the location of the αF and control peptides. H. Peak intensity ratios of the maleimide-labeled αF and control peptides from the Src^FL K445C-5 or Src^FL K445C-6 complexes (n=4). Peak intensity ratios of the maleimide-labeled αF peptide from the Src^3D K445C-5 or Src^3D K445C-6 complexes (n=2) are also shown. I. SH4 pulldown assay schematic. Src variants are incubated with the immobilized SH4 domain (residues 1-18) of Src and the amount of retained Src is quantified after washing and elution. Src^CD variants with mutations outside (left) and within (right) the αF pocket are shown. J. Percent retained Src in the SH4 pulldown assay with purified Src^CD WT, E381T, I444K, T293D, or W285T (n=3). K. Percent retained Src in the SH4 pulldown assay with purified Src^CD WT or Src^FL WT (n=3). Points represent individual measurements and the horizontal lines indicate the mean of all measurements. See also Figure S5.

Figure 7.. Functional characterization of the SH4 domain/αF pocket interaction.

A. Sequence alignment of SH4 domain-lacking human non-receptor tyrosine kinases (SH4-) at αF pocket residues. Colors indicate classification of the analogous mutation from the Src DMS. B. Phosphotransferase activity of purified Fyn^FL WT or Fyn^3D WT (n=3). C. Percent retained Fyn in the SH3 pulldown assay with purified Fyn^FL WT or Fyn^3D WT (n=3). D Representative micrographs (top panel) and percent bleb quantification (bottom panel) for Fyn^myr-expressing SYFs treated with DMSO or CystIMATIK probe 3 for 15 min. E. DMS of Src’s SH4 domain. Dots indicate WT amino acid at that residue. F. Percent retained Src^TAMRA-3D in an SH4 pulldown assay with immobilized WT, P8N, or P8N/K5I SH4 domain (n=3). G. Proposed model of SH4 domain-mediated autoinhibition (myristoyl (green), unstructured SH4 and Unique (gray), SH3 (orange), SH2 (green), and CD (purple)). For 7D, each point represents a replicate treatment with multiple cells imaged and scored in a double-blind fashion. Horizontal lines indicate the mean of all replicates. See Table S5 for the total number of replicates and cells analyzed. *p <0.05; **p < 0.01, ***p < 0.001. See also Figure S6.

Scheme 1:

a) Diisopropylamine, n-BuLi, ethyl formate, THF, −78 °C; b) Ammonium hydroxide, 1,4-Dioxane, 60 °C; c) K₃PO₄, PdCl₂(dppf)₂. 4-Chlorophenylboronic acid, 1, 4-Dioxane/H₂O, 85 °C (microwave); d) K₃PO₄, PdCl₂(dppf)₂. 4-Phenoxyphenyl boronic acid, 1, 4-Dioxane/H₂O, 85 °C (microwave); e) 2-cyano-N-isopropylacetamide, DB U, THF, RT.

Scheme 2:

a) 2-Bromoethyl methyl ether, CS₂CO₃, DMF, 50 °C; b) Diisopropylamine, n-BuLi, ethyl formate, THF, −78 °C to RT; c) 3-(Trifluorome thyl)benzoic acid, EDCI, HOAt, DMF, RT; d) KF, MeOH, RT; e) PdCl₂(PPh₃)₂, CuI, Et₃N, DMF, 50 °C; f) 2-Cyano-N-methylacetamide, DBU, THF, RT.