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SKAP2 modular organization differently recognizes SRC kinases depending on their activation status and localization

Open AccessPublished:November 21, 2022DOI:https://doi.org/10.1016/j.mcpro.2022.100451

      Highlights

      • Characterization of the SKAP2 modular organization recognizing SRC kinases;
      • Each module is affected differently by the activation and/or the subcellular localization of SRC kinases;
      • There are, however, subtle differences in SKAP2 binding between SRC kinases, which have not been explored further in this article;
      • Some mutations in HCK kinase induce its degradation probably by the proteasome in which Lys7 and Tyr390 play a role.

      Abstract

      Dimerization of SRC Kinase Adaptor Phosphoprotein 2 (SKAP2) induces an increase of binding for most SRC kinases suggesting a fine-tuning with trans-phosphorylation for kinase activation. This work addresses the molecular basis of SKAP2-mediated SRC kinase regulation through the lens of their interaction capacities. By combining a luciferase complementation assay and extensive site-directed mutagenesis, we demonstrated that SKAP2 interacts with SRC kinases through a modular organization depending both on their phosphorylation-dependent activation and subcellular localization. SKAP2 contains three interacting modules consisting in the dimerization (DIM) domain, the SRC homology 3 (SH3) domain, and the second inter-domain located between the pleckstrin homology (PH) and the SH3 domains. Functionally, the DIM domain is necessary and sufficient to bind to most activated and myristyl SRC kinases. In contrast, the three modules are necessary to bind SRC kinases at their steady state. The PH and SH3 domains of SKAP2 as well as tyrosines located in the interdomains modulate these interactions. Analysis of mutants of the SRC kinase family member HCK supports this model and shows the role of two residues, Y390 and K7, on its degradation following activation. In this article, we show that a modular architecture of SKAP2 drives its interaction with SRC kinases, with the binding capacity of each module depending on both their localization and phosphorylation state activation. This work opens new perspectives on the molecular mechanisms of SRC kinases activation, which could have significant therapeutic impact.

      Graphical abstract

      Keywords

      List of abbreviations:

      DIM (domain domain of dimerization), DIMPH (dimerization and Pleckstrin homology domains), SKAP1 (SRC Kinase Adaptor Phosphoprotein 1), SKAP2 (SRC Kinase Adaptor Phosphoprotein 2), BLK (B Lymphocyte Kinase), FGR (Feline Gardner-Rasheed sarcoma viral homolog), FRK (Fyn Related src family tyrosine Kinase), FYN (FGR-YES1-related Novel protein), HCK (Hematopoietic Cell Kinase), LCK (Lymphocyte-Specific Kinase), LYN (LCK/Yes-related Novel protein tyrosine kinase), PH domain (Pleckstrin Homology domain), SRMS (Src-Related kinase lacking C-terminal regulatory tyrosine and N-terminal Myristylation Sites), SH2 domain (Src Homology 2 domain), SH3 (domain Src Homology 3 domain), NLR (Normalized Luciferase Ratio)

      Introduction

      The SRC kinase family is composed of the SrcA subfamily with FGR, FYN, SRC, and YES, the SrcB subfamily with BLK, HCK, LCK, and LYN, and finally FRK (
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      Figure thumbnail gr1
      Figure 1Study of the interaction between SKAP2 deletion mutants and either N1-fused or C1-fused HCK. (A) Schematic illustrating inactive (bottom) and active (top) forms of HCK. (B) Schematic of SKAP2 deletion mutants. (C) Normalized NLR of the eight SKAP2 deletion mutants with N1-fused (blue) and C1-fused (red) HCK in a logarithmic scale. Normalized NLR is the ratio of the NLR between a N2-SKAP2 deletion mutant and one N1- or C1- SRC kinase to the corresponding NLR between wild type N2-SKAP2 and the same Gluc1 SRC kinase.
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      In this work, we wished to better characterize the underlying mechanism of this regulation. We show that the interaction of SKAP2 with SRC kinases depends on three modules, each of whose binding capacity is differently modulated by the activation and/or the subcellular localization of SRC kinases. Interestingly, the modular architecture of another member of the same family, SKAP1, is different. We studied SRC kinase mutations using the SRC kinase family member HCK, which is the only one with a resolved complete 3D structure amongst the nine studied (
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      Experimental Procedures

      Plasmids, mutagenesis, BP and LR cloning, and luciferase complementation assay

      Previously used plasmids and a more detailed methodology will be found elsewhere (
      • Bureau J.F.
      • Cassonnet P.
      • Grange L.
      • Dessapt J.
      • Jones L.
      • Demeret C.
      • Sakuntabhai A.
      • Jacob Y.
      The SRC-family tyrosine kinase HCK shapes the landscape of SKAP2 interactome.
      ). The ORF of the new SKAP2 deletion mutants flanked by two gateway sites were amplified by PCR, cloned into pCR-II TOPO vector using TOPO TA cloning kit (Thermo Fisher Scientific, Waltham) and transferred into pDONR207 vector. Mutagenesis of SKAP2, and HCK ORFs were performed using QuickChange Lightening Site-Directed Mutagenesis kit (Agilent Technologies, Les Ulis) according to the manufacturer’s protocol. Supplementary Table S1 shows the new primers used for all these purposes. After each mutagenesis and PCR amplification, the insert was completely sequenced at GATC services using LightRun Barcodes (Eurofins Genomics, Ebersberg, Germany). Sequence alignments were performed using DNA Strider (
      • Marck C.
      DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers.
      ). The ORF of SKAP2 mutants were transferred into pSPICA-N2 vector and those of SRC kinases and HCK mutants into pSPICA-N1 and pSPICA-C1 ones. The pSPICA vectors are mammalian expressing vectors designed for luciferase complementation assay. They expressed Gaussia princeps Luciferase fragment 1 (amino acid residues from 18 to 109) or 2 (amino acid residues from 110 to 185) at the N or C-terminal part of the fusion protein. The luciferase complementation assay was performed as described in human HEK293T cells (
      • Cassonnet P.
      • Rolloy C.
      • Neveu G.
      • Vidalain P.O.
      • Chantier T.
      • Pellet J.
      • Jones L.
      • Muller M.
      • Demeret C.
      • Gaud G.
      • Vuillier F.
      • Lotteau V.
      • Tangy F.
      • Favre M.
      • Jacob Y.
      Benchmarking a luciferase complementation assay for detecting protein complexes.
      ). Briefly the first day, 30 000 HEK293T cells are cultured in 100 μl of DMEM supplemented with 10% Fetal Bovine Serum and antibiotics per well of microplate 96 wells (Greiner, Kremsmünster). One day later, cells were transfected with 100 ng GPCA plasmid pair using polyethylenimine (PEI) method. The third day, culture medium was discarded, cells were washed once with 150 μl of Phosphate Buffer Saline without calcium and magnesium (PBS) and incubated for 25 min in 40 μl of lysis buffer. Luminescence monitoring was performed after addition of native Coelenterazine on a Centro XS3 LB 960 microplate luminometer (Berthold Technologies, Thoiry). Transfections using PEI were performed at least in triplicate. Protein-protein interactions were monitored by measuring interaction-mediated normalized luminescence ratio (NLR) (
      • Cassonnet P.
      • Rolloy C.
      • Neveu G.
      • Vidalain P.O.
      • Chantier T.
      • Pellet J.
      • Jones L.
      • Muller M.
      • Demeret C.
      • Gaud G.
      • Vuillier F.
      • Lotteau V.
      • Tangy F.
      • Favre M.
      • Jacob Y.
      Benchmarking a luciferase complementation assay for detecting protein complexes.
      ). Briefly, NLR is the ratio between the interaction signal and a control one, the sum of signals of each component of the interaction with the complementary empty hemi-luciferase expressing vector. A negative control, FAM102B, was used in each experiment as positive controls to verify the status of the SKAP2 mutants. For each experiment, a threshold was defined as NLR mean + SEM of sample FAM102B. These data are not presented in this article.

      Western blot

      Pooled triplicate samples from luciferase complementation assay diluted in Laemmli x1 buffer were passed through Qiashredder column (Qiagen, Hiden, Germany) before eating at 95°C during 5 min. Samples were loaded on a gel with PageRuler Plus Prestained Protein Ladder (ThermoFisher, Waltham). After 2h migration on a 4-12% NuPAGE Trisminigel (ThermoFisher Scientific, Waltham) at 120V at room temperature in x1 MES buffer, proteins were transferred to a PVDF membrane at 5°C. After saturating the membrane with 1x PBS, Tween 0.25% 5% milk during 30 min at room temperature, an overnight hybridization at 4°C with a primary antibody in 1x PBS, tween 0.25%, BSA 1% buffer was performed followed by 1h hybridization at room temperature with a secondary antibody in the same buffer. Revelation was performed on G:Box system (Syngene, Cambridge, UK) after incubation with Pierce ECL2 Western Blotting substrate (ThermoFisher Scientific, Waltham)). Table S2 shows the different antibodies used.

      Experimental Design and Statistical Rationale

      Approximation of the mean and the variance of a ratio were performed using Taylor expansions with a null covariance between the numerator and the denominator. Two-sample z test and two-way ANOVA after log-normal transformation were used to compare these ratios. Differential interaction scatterplots and PPI-mutation plots were performed as described in (
      • Marck C.
      DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers.
      ). Three levels of significance were used for the P-value lower than 0.05 (*), 0.01 (**), and 0.001 (***). Bonferroni correction was used for multiple testing with an initial threshold p = 0.05. To compare different experiments, we used modified NLR for each SRC kinases as the ratio between NLR of the SKAP2 mutant and NLR of wild-type SKAP2 protein. Each mutant experiment was repeated at least three times. Statistical analyses were performed using Stata/IC version 10.1 (Stata Corporation, College Station, USA). Comparison of mean to theorical value (i.e. Mean ratio to 1 and its log-normal transformation to 0) and between two means was performed by using Student t test and unpaired Student t test, respectively. Comparison of mean to theorical value was also studied using binomial distribution after calculating the number of samples upper/lower to theoretical value. Comparison of two means was also performed by Kruskal-Wallis test. Parametric and non-parametric tests giving similar results, only results of the parametric are shown in Table1. Two-way ANOVA were performed after a log-normal transformation of the modified NLR and using analytic weights. For data modelling, the full model contains nine explanatory variables and the data on the N1- and C1-fused SRC kinases were analyzed separately. For generating the variable explaining which domains are necessary to bind to either N1-fused SRC kinases or C1-fused ones, the following protocol was used. Two-way ANOVA was performed on deletion SKAP2 mutant data using five variables, each explaining the effect of a SKAP2 region. Significant variables and all their possible region combinations were used in a second two-way ANOVA. Significant variables of the second analysis were used to generate the definitive variable describing which regions are necessary for defining the binding site. An explicative model was generated from of this full model by a step-by-step procedure that removed at each step the lowest non-significant variable until no non-significant variables were present.
      Table 1Statistical analysis of the interaction between SKAP2 mutants and SRC kinases
      MutantNb ExpUnivariate model
      (n)N1 effect+C1 effect+DC1-N1+
      DIMPH3 (
      • Nguyen G.T.
      • Xu S.
      • Adams W.
      • Leong J.M.
      • Bunnell S.C.
      • Mansour M.K.
      • Sykes D.B.
      • Mecsas J.
      Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation.
      )
      14.47 (-) ***NS3.48 (+) ***
      DomDIM23 (
      • Nguyen G.T.
      • Xu S.
      • Adams W.
      • Leong J.M.
      • Bunnell S.C.
      • Mansour M.K.
      • Sykes D.B.
      • Mecsas J.
      Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation.
      )
      49.25 (-) ***8.10 (-) ***3.72 (+) ***
      DLink1PH3 (
      • Nguyen G.T.
      • Xu S.
      • Adams W.
      • Leong J.M.
      • Bunnell S.C.
      • Mansour M.K.
      • Sykes D.B.
      • Mecsas J.
      Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation.
      )
      NS4.97 (-) ***1.94 (-) *
      DPH3 (
      • Nguyen G.T.
      • Xu S.
      • Adams W.
      • Leong J.M.
      • Bunnell S.C.
      • Mansour M.K.
      • Sykes D.B.
      • Mecsas J.
      Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation.
      )
      7.47 (-) ***9.11 (-) ***1.70 (-) *
      DPHLink23 (
      • Nguyen G.T.
      • Xu S.
      • Adams W.
      • Leong J.M.
      • Bunnell S.C.
      • Mansour M.K.
      • Sykes D.B.
      • Mecsas J.
      Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation.
      )
      10.20 (-) ***15.53 (-) ***NS
      DSH33 (
      • Nguyen G.T.
      • Xu S.
      • Adams W.
      • Leong J.M.
      • Bunnell S.C.
      • Mansour M.K.
      • Sykes D.B.
      • Mecsas J.
      Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation.
      )
      18.12 (-) ***NS3.62 (+) ***
      Link2SH33 (
      • Nguyen G.T.
      • Xu S.
      • Adams W.
      • Leong J.M.
      • Bunnell S.C.
      • Mansour M.K.
      • Sykes D.B.
      • Mecsas J.
      Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation.
      )
      10.20 (-) ***15.53 (-) ***NS
      SH33 (
      • Nguyen G.T.
      • Xu S.
      • Adams W.
      • Leong J.M.
      • Bunnell S.C.
      • Mansour M.K.
      • Sykes D.B.
      • Mecsas J.
      Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation.
      )
      150 (-) ***69.49 (-) ***2.09(+) *
      SKAP13 (
      • Nguyen G.T.
      • Xu S.
      • Adams W.
      • Leong J.M.
      • Bunnell S.C.
      • Mansour M.K.
      • Sykes D.B.
      • Mecsas J.
      Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation.
      )
      NS14.62 (-) ***5.73 (-) ***
      3YF3 (
      • Nguyen G.T.
      • Xu S.
      • Adams W.
      • Leong J.M.
      • Bunnell S.C.
      • Mansour M.K.
      • Sykes D.B.
      • Mecsas J.
      Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation.
      )
      6.80 (-) ***NS2.31 (+) *
      D129K4 (
      • Marck C.
      DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers.
      )
      4.49 (+) ***6.47 (+) ***2.71 (+) **
      D129K-3YF3 (
      • Nguyen G.T.
      • Xu S.
      • Adams W.
      • Leong J.M.
      • Bunnell S.C.
      • Mansour M.K.
      • Sykes D.B.
      • Mecsas J.
      Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation.
      )
      NS5.53 (+) ***3.15 (+) **
      DIM-3 (
      • Nguyen G.T.
      • Xu S.
      • Adams W.
      • Leong J.M.
      • Bunnell S.C.
      • Mansour M.K.
      • Sykes D.B.
      • Mecsas J.
      Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation.
      )
      16.27 (-) ***11.78 (-) ***NS
      D129K-DIM-3 (
      • Nguyen G.T.
      • Xu S.
      • Adams W.
      • Leong J.M.
      • Bunnell S.C.
      • Mansour M.K.
      • Sykes D.B.
      • Mecsas J.
      Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation.
      )
      2.37 (-) *3.78 (-) ***NS
      E15AR18A3 (
      • Nguyen G.T.
      • Xu S.
      • Adams W.
      • Leong J.M.
      • Bunnell S.C.
      • Mansour M.K.
      • Sykes D.B.
      • Mecsas J.
      Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation.
      )
      3.10 (+) **2.93 (+) **NS
      E15AR18A-DIM-3 (
      • Nguyen G.T.
      • Xu S.
      • Adams W.
      • Leong J.M.
      • Bunnell S.C.
      • Mansour M.K.
      • Sykes D.B.
      • Mecsas J.
      Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation.
      )
      8.32 (-) ***3.12 (-) **NS
      K161E3 (
      • Nguyen G.T.
      • Xu S.
      • Adams W.
      • Leong J.M.
      • Bunnell S.C.
      • Mansour M.K.
      • Sykes D.B.
      • Mecsas J.
      Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation.
      )
      2.82 (+) **NSNS
      R140M3 (
      • Nguyen G.T.
      • Xu S.
      • Adams W.
      • Leong J.M.
      • Bunnell S.C.
      • Mansour M.K.
      • Sykes D.B.
      • Mecsas J.
      Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation.
      )
      NS2.63 (-) **1.70 (-) *
      R140M-D129K3 (
      • Nguyen G.T.
      • Xu S.
      • Adams W.
      • Leong J.M.
      • Bunnell S.C.
      • Mansour M.K.
      • Sykes D.B.
      • Mecsas J.
      Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation.
      )
      NS2.70 (-) **NS
      W336K3 (
      • Nguyen G.T.
      • Xu S.
      • Adams W.
      • Leong J.M.
      • Bunnell S.C.
      • Mansour M.K.
      • Sykes D.B.
      • Mecsas J.
      Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation.
      )
      5.57 (+) ***4.68 (+) ***NS
      Y151FY197F3 (
      • Nguyen G.T.
      • Xu S.
      • Adams W.
      • Leong J.M.
      • Bunnell S.C.
      • Mansour M.K.
      • Sykes D.B.
      • Mecsas J.
      Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation.
      )
      2.31 (+) *NS2.33 (-) *
      + t value with its sense and its level of significance. Bold: significant after Bonferroni correction. Red:loss of interaction
      NS: Non significant

      Conservation of the DIM domain of SKAP1 and SKAP2

      The N-terminal fragment of human SKAP1 and SKAP2 protein structures were predicted from I-TASSER server (
      • Yang J.
      • Yan R.
      • Roy A.
      • Xu D.
      • Poisson J.
      • Zhang Y.
      The I-TASSER Suite: protein structure and function prediction.
      ) using two crystal structures of a N-terminal fragment of murine SKAP2 containing both the helical dimerization domain and the PH domain (ID PDB: 2OTX and 1U5E). Clustalw program (
      • Larkin M.A.
      • Blackshields G.
      • Brown N.P.
      • Chenna R.
      • McGettigan P.A.
      • McWilliam H.
      • Valentin F.
      • Wallace I.M.
      • Wilm A.
      • Lopez R.
      • Thompson J.D.
      • Gibson T.J.
      • Higgins D.G.
      Clustal W and Clustal X version 2.0.
      ) was used to align respectively 37 and 38 protein sequences of SKAP1 and SKAP2 from the Coelacanth to mammals. ConSurf server was used to visualize evolutionary conservation in these 2 macromolecules from their respective predict structure and aligned sequences using a maximum likelihood procedure. UCSF Chimera version 1.13.1 was used to visualize the conservation of these structures using a script from ConSurf server (
      • Ashkenazy H.
      • Abadi S.
      • Martz E.
      • Chay O.
      • Mayrose I.
      • Pupko T.
      • Ben-Tal N.
      ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules.
      ,
      • Pettersen E.F.
      • Goddard T.D.
      • Huang C.C.
      • Couch G.S.
      • Greenblatt D.M.
      • Meng E.C.
      • Ferrin T.E.
      UCSF Chimera--a visualization system for exploratory research and analysis.
      ).

      Results

      We finely analyzed the interactions between SKAP2 and nine SRC kinases. The aims were (i) to define SRC binding sites on SKAP2, (ii) to clarify how SKAP2/SRC interactions impact on the SRC activation, (iii) how sub-cellular localization of SRC kinases is involved in their binding to SKAP2. For that purpose, we used a luciferase complementation assay in HEK293T cells where the C-terminal hemi-gaussia2 luciferase (Gluc2) is fused at the N-terminus of SKAP2 (N2-SKAP2) and the N-terminal hemi-gaussia1 luciferase (Gluc1) is fused either to the N-terminus (N1) or C-terminus (C1) of the SRC kinases (
      • Bureau J.F.
      • Cassonnet P.
      • Grange L.
      • Dessapt J.
      • Jones L.
      • Demeret C.
      • Sakuntabhai A.
      • Jacob Y.
      The SRC-family tyrosine kinase HCK shapes the landscape of SKAP2 interactome.
      ). It is necessary to study the two orientations of hemi-gaussian luciferase fused to SRC kinases for evaluating the effect of their activation and/or subcellular localization on this interaction. One major assumption of this paper is that there is a general SKAP2 architecture, which defines its interaction with all the SRC kinases, and which can be studied by the mean value of their PPI signal. To assess the effect of SKAP2 deletions or mutations, the Normalized Luciferase Ratio (NLR) (
      • Cassonnet P.
      • Rolloy C.
      • Neveu G.
      • Vidalain P.O.
      • Chantier T.
      • Pellet J.
      • Jones L.
      • Muller M.
      • Demeret C.
      • Gaud G.
      • Vuillier F.
      • Lotteau V.
      • Tangy F.
      • Favre M.
      • Jacob Y.
      Benchmarking a luciferase complementation assay for detecting protein complexes.
      ) generated by their binding to SRC kinase has been divided by the binding of wild-type SKAP2 to the same SRC kinase. This generated a relative NLR enabling to standardize the interaction of mutated SKAP2 relative to the wild-type protein for the different SRC kinases. The interaction interface of SKAP2 with nine SRC kinases, BLK, FGR, FRK, FYN, HCK, LCK, LYN, SRMS, and YES, was studied with 21 SKAP2 mutants, eight of which are deletion mutants (Figure 1B) and the others contain non-synonymous mutations (Figure S1A). Study of deletion SKAP2 mutants interacting with C1-fused SRC kinases allowed to define which SKAP2 modules are important for this interaction. We analyzed the role of both sub-cellular localization and activation on these interactions by comparing C1-fused SRC kinase data with those of N1-fused SRC kinases. This role was also assessed by studying the interaction of wild-type SKAP2 with 24 HCK kinase mutants from 11 mutations (Figure S1B). A negative control, FAM102B, was used in each experiment as positive controls to verify the status of the SKAP2 mutants (not shown). We refined the role of the different modules by studying non-synonymous SKAP2 mutations.
      Figure 1C shows the initial study of the interaction between eight N2-fused SKAP2 deletion mutants (Figure 1B) with N1 or C1-fused HCK SRC kinase. The signal is greater or equal for most mutants when interacting with C1-fused HCK than with N1-fused HCK but varies considerably between mutants. This suggests that the localization of the hemi-luciferase fusion disturbs these interactions without suppressing them. We will first study the eight SKAP2 deletion mutants that interact with C1-fused SRC kinases.

      The DIM domain of SKAP2 is necessary and sufficient to bind C1-fused SRC kinases

      A previous study supports that the DIMPH SKAP2 mutant (Figure 1B), which contains the dimerization domain (DIM) and the Pleckstrin Homology domain (PH), is able to bind most C1-fused SRC kinases but not N1-fused ones (
      • Bureau J.F.
      • Cassonnet P.
      • Grange L.
      • Dessapt J.
      • Jones L.
      • Demeret C.
      • Sakuntabhai A.
      • Jacob Y.
      The SRC-family tyrosine kinase HCK shapes the landscape of SKAP2 interactome.
      ). To confirm and understand the structural basis of this result, the eight SKAP2 deletion mutants (Figure 1B), were studied with the nine C1-fused SRC kinases (Figure 2). A mutation is considered to destroy an interaction if the average of the relative NLRs among the nine SRC kinases is below 0.25. Only the two SKAP2 mutants lacking the DIM domain (Link2SH3 and SH3), did not bind to the C1-fused SRC kinases supporting, a necessary role of this domain. A similar binding pattern is observed in three SKAP2 mutants, DomDIM, DIMPH and ΔSH3, with the highest signals for YES and FRK, middle signals for HCK, LYN, FGR and the lowest signal for BLK, LCK, FYN and SRMS. This data support that the DIM domain is sufficient for the interaction but that other regions such as the SH3 domain also play a role. The study of the interaction between N2-SKAP2 and N1-fused SRC kinases has further defined these SKAP2 modules.
      Figure thumbnail gr2
      Figure 2Study of the interaction between SKAP2 deletion mutants and C1-fused SRC kinases. Graphs show normalized NLR of the eight SKAP2 deletion mutants with C1-fused SRC kinases in a logarithmic scale. Normalized NLR is the ratio of the NLR between a SKAP2 deletion mutant and one C1-fused SRC kinase to the corresponding NLR between wild type SKAP2 and the same C1-fused SRC kinase. For each graph, the normalized NRL mean ± SEM among SRC kinases is shown as its level of significance.

      SKAP2 modular organization for the binding to N1-fused SRC kinases depends on three modules

      When the Gluc1 fragment is fused to the N terminus of SRC kinases, five SKAP2 deletion mutants, DomDIM2, DIMPH, ΔSH3, SH3 and LINK2SH3, completely lost their binding capacity for SRC kinases and only ΔLINK1PH mutant showed no decrease in signaling compared to wild type SKAP2 (Figure 3). Data on ΔLINK1PH mutant support an important role for other SKAP2 domains, i.e. the DIM and SH3 domains and the linker 2. As previous results have shown that the DIM domain alone did not bind to N1-fused SRC kinases, these results suggest that the three modules DIM, SH3, and linker2 are necessary. Note that the two non-myristate SRC kinases, SRMS and FRK, do not have any particular profile compared to myristate ones, which supports that the localization defect induced by the N1 fusion is not prohibitive for the study of these interactions. Furthermore, differential interaction scatterplots, comparing the mean value of luminescence (NLR) induced by the interaction of each SKAP2 mutant to that induced by the interaction of wild type SKAP2, confirmed the major effect of the Gluc1 orientation on DomDIM2, DIMPH, and ΔSH3 SKAP2 mutants (Figure S3). These results support that the SKAP2 modular organization for binding to SRC kinases has its properties depending on the localization of the hemi-luciferase fusion.
      Figure thumbnail gr3
      Figure 3Study of the interaction between SKAP2 deletion mutants and either N1-fused or C1-fused SRC kinases. Graphs show normalized NLR of the eight SKAP2 deletion mutants with N1-fused (blue) and C1-fused (red) SRC kinases in a logarithmic scale. Normalized NLR is the ratio of the NLR between a SKAP2 deletion mutant and one SRC kinase either N1-fused or C1-fused to the corresponding NLR between wild type SKAP2 and the same SRC kinase fused at the same position. For each graph, the normalized NRL mean ± SEM among SRC kinases is shown as its level of significance.

      Role of SKAP2 dimerization

      To exclude an effect of dimerization specifically on the binding of activated C1-fused SRC kinases to DIM domain, a dimerization-invalidated mutant of SKAP2, DIM-, bearing the V24D, F27G, and V28E mutations (Figure S1A) (
      • Swanson K.D.
      • Tang Y.
      • Ceccarelli D.F.
      • Poy F.
      • Sliwa J.P.
      • Neel B.G.
      • Eck M.J.
      The Skap-hom dimerization and PH domains comprise a 3'-phosphoinositide-gated molecular switch.
      ) was studied (Figure 4 and Figure S4). A similar significant decrease of interaction signal of both N1-fused and C1-fused SRC kinases to SKAP2 DIM- mutant was detected without suppressing their binding capacity. This result strongly suggests that dimerization influences binding but in a different way to that of the DomDIM2 SKAP2 mutant, which has lost its ability to bind only to N1-fused SRC kinases.
      Figure thumbnail gr4
      Figure 4Effect of SKAP2 point mutations on interaction with SRC kinases. Each graph shows normalized NLR of each of the 12 SKAP2 mutants with N1-fused (blue) and C1-fused (red) SRC kinases in a logarithmic scale. Normalized NLR is the ratio of the NLR between a SKAP2 deletion mutant and one SRC kinase either N1-fused or C1-fused to the corresponding NLR between wild type SKAP2 and the same SRC kinase fused at the same position. For each graph, the normalized NRL mean ± SEM among SRC kinases is shown as its level of significance.

      Limited role of conserved tyrosines

      Classical views suggest that the binding of SRC kinases to SKAP2 occurs primarily through the SH2 domain of SRC kinases and one of the two phosphorylated tyrosine Y237 or Y261 motifs of SKAP2 (
      • Kouroku Y.
      • Soyama A.
      • Fujita E.
      • Urase K.
      • Tsukahara T.
      • Momoi T.
      RA70 is a src kinase-associated protein expressed ubiquitously.
      ,
      • Marie-Cardine A.
      • Verhagen A.M.
      • Eckerskorn C.
      • Schraven B.
      SKAP-HOM, a novel adaptor protein homologous to the FYN-associated protein SKAP55.
      ,
      • Curtis D.J.
      • Jane S.M.
      • Hilton D.J.
      • Dougherty L.
      • Bodine D.M.
      • Begley C.G.
      Adaptor protein SKAP55R is associated with myeloid differentiation and growth arrest.
      ). Our results of the SKAP2 deletion mutants do not support this hypothesis. To directly test it, a SKAP2 3YF mutant bearing three non-synonymous mutations, Y75F, Y237F, and Y261F, was studied (Figure S1A). Only a slight decrease of the signal for N1-fused SRC kinases was detected (Figures 4 and S4). This result confirms a limited role of these three tyrosines.
      Inactivation of the classical SH3 binding site has a completely different effect than the SH3 deletion
      The SH3 domain of SKAP2 interacts with proline-rich helix of its partners for which its tryptophan 336 is required (
      • Liu J.
      • Kang H.
      • Raab M.
      • da Silva A.J.
      • Kraeft S.K.
      • Rudd C.E.
      FYB (FYN binding protein) serves as a binding partner for lymphoid protein and FYN kinase substrate SKAP55 and a SKAP55-related protein in T cells.
      ). Interestingly, the study of W336K SKAP2 mutant (Figure S1A) shows a significant increase of signal for both N1-fused and C1-fused SRC kinases (Figure 4). The SH3 deletion has an opposite effect, significantly decreasing the signal for N1-fused SRC kinases (Figure 3) and inducing a specific pattern for C1-fused ones (Figure 2).

      Role of the PH domain by studying nonsynonymous mutations

      The PH domain of SKAP2 is known to bind to the phosphatidyl inositol 3, 4, 5 tri-phosphate (PI[3,4,5]P3). It also interacts with the DIM domain of the same molecule, which prevents the binding to PI[3,4,5]P3 (
      • Marck C.
      DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers.
      ). Three mutations in the PH domain known to affect the interaction with the DIM domain, D129K (
      • Alenghat F.J.
      • Baca Q.J.
      • Rubin N.T.
      • Pao L.I.
      • Matozaki T.
      • Lowell C.A.
      • Golan D.E.
      • Neel B.G.
      • Swanson K.D.
      Macrophages require Skap2 and Sirpalpha for integrin-stimulated cytoskeletal rearrangement.
      ,
      • Bourette R.P.
      • Therier J.
      • Mouchiroud G.
      Macrophage colony-stimulating factor receptor induces tyrosine phosphorylation of SKAP55R adaptor and its association with actin.
      ,
      • Swanson K.D.
      • Tang Y.
      • Ceccarelli D.F.
      • Poy F.
      • Sliwa J.P.
      • Neel B.G.
      • Eck M.J.
      The Skap-hom dimerization and PH domains comprise a 3'-phosphoinositide-gated molecular switch.
      ), R140M (
      • Swanson K.D.
      • Tang Y.
      • Ceccarelli D.F.
      • Poy F.
      • Sliwa J.P.
      • Neel B.G.
      • Eck M.J.
      The Skap-hom dimerization and PH domains comprise a 3'-phosphoinositide-gated molecular switch.
      ), and K161E (
      • Witte A.
      • Meineke B.
      • Sticht J.
      • Philipsen L.
      • Kuropka B.
      • Muller A.J.
      • Freund C.
      • Schraven B.
      • Kliche S.
      D120 and K152 within the PH Domain of T Cell Adapter SKAP55 Regulate Plasma Membrane Targeting of SKAP55 and LFA-1 Affinity Modulation in Human T Lymphocytes.
      ) and four new mutations located in the DIM domain (E15A, R18A), or in the PH domain (Y151F and Y197F) (Figure S1A) were studied alone or in combination. The R140M and K161E mutations are known to affect the binding of PI[3,4,5]P3 and D129K to prevent the intra-molecular interaction with DIM domain. Crystallographic data on the murine Skap2 support that E15A and R18A mutations located in the DIM domain prevent its interaction with the PH domain. Literature curation supports the view that Y151F and Y197F mutations affect the binding to PI[3,4,5]P3 and possibly to the DIM domain (
      • Swanson K.D.
      • Tang Y.
      • Ceccarelli D.F.
      • Poy F.
      • Sliwa J.P.
      • Neel B.G.
      • Eck M.J.
      The Skap-hom dimerization and PH domains comprise a 3'-phosphoinositide-gated molecular switch.
      ). As shown in Figures 4 and S3, these mutations have a limited effect on the interaction with SRC kinases, except D129K, which surprisingly increased signaling primarily with C1-fused SRC kinases. No of them has lost their binding capacity for SRC kinases and only some of them have a significant decrease.
      In conclusion, the data presented in the last four paragraphs show that non-synonymous mutations alone or in combination have a weaker effect on SRC kinase binding than deletion of the corresponding binding domain or region, consistent with a modular organization. It is noteworthy that also in this case, the two non-myristate SRC kinases, SRMS and FRK, do not have a particular profile compared to the myristate kinases, confirming that the data with the N1 fusion provide useful information for studying the molecular architecture of SKAP2.

      Role of SRC kinase activation on the interaction with SKAP2

      Furthermore, the study of the SKAP2 paralog, SKAP1, supports an even more complex binding of SRC kinases (Figure 3). In contrast to previous results with deletion SKAP2 mutants, the PPI signal of the N1-fused SRC kinases with SKAP1 was higher than that of the C1-fused SRC kinases, while similar SRC kinases binding was expected from the high homology between SKAP1 and SKAP2. To reconcile these data, we took account that SRC kinases adopt different conformations affecting their kinase activity (
      • Roskoski Jr., R.
      Src protein-tyrosine kinase structure, mechanism, and small molecule inhibitors.
      ,
      • Yang S.
      • Blachowicz L.
      • Makowski L.
      • Roux B.
      Multidomain assembled states of Hck tyrosine kinase in solution.
      ) and that PPI signal between SKAP2 and SRC kinases was higher for activated than inactivated HCK kinase in our initial work (
      • Bureau J.F.
      • Cassonnet P.
      • Grange L.
      • Dessapt J.
      • Jones L.
      • Demeret C.
      • Sakuntabhai A.
      • Jacob Y.
      The SRC-family tyrosine kinase HCK shapes the landscape of SKAP2 interactome.
      ). We now suppose (i) that the orientation of the fused hemi-luciferase differently affects the conformations and activation of SRC kinases, (ii) that SKAP2 binding sites for SRC kinases exhibit different binding properties depending on their conformations, (iii) the latter is not the case for SKAP1. These hypotheses have three consequences: (
      • Ingley E.
      Src family kinases: regulation of their activities, levels and identification of new pathways.
      ) the DIM domain of SKAP1 binds less efficiently C1-fused SRC kinases than that of SKAP2; (
      • Bradshaw J.M.
      The Src, Syk, and Tec family kinases: distinct types of molecular switches.
      ) the DIM domain of SKAP2 has a greater increase of its binding capacity for activated HCK mutants than the wild-type SKAP2 and the DIM domain of SKAP1, independently of the orientation for the hemi-luciferase; (
      • Engen J.R.
      • Wales T.E.
      • Hochrein J.M.
      • Meyn 3rd, M.A.
      • Banu Ozkan S.
      • Bahar I.
      • Smithgall T.E.
      Structure and dynamic regulation of Src-family kinases.
      ) different properties of the SKAP2 binding site can be defined by studying N1-fused and C1-fused SRC kinases with SKAP2 deletion mutants. Results of the two first paragraphs are already consistent with the last hypothesis.

      Functional differences between the DIM domain of SKAP1 and SKAP2

      A phylogeny analysis of each SKAP1 and SKAP2 protein allows to define the amino acid conservation in both structures using CONSURF website (
      • Ashkenazy H.
      • Abadi S.
      • Martz E.
      • Chay O.
      • Mayrose I.
      • Pupko T.
      • Ben-Tal N.
      ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules.
      ). A stronger conservation of the DIM domain of SKAP2 than that of SKAP1 was detected (Figure S2). This loss of SKAP1 conservation during evolution suggests that the binding capacity to SRC kinases by its DIM domain has changed over time compared to the DIM domain of SKAP2. This gives a molecular support that the DIM domain of SKAP1 has a lower binding capacity for C1-fused SRC kinases than that of SKAP2. A differential interaction scatterplot of data from DomDIM1 SKAP1 mutant (y-axis) versus those from DomDIM2 SKAP2 mutant (x-axis) supports also the first consequence of the hypothesis (Figure 5). Each point is the data of a N1-fused (blue) or C1-fused (red) SRC kinase interacting with either the DIM domain of SKAP2 or that of SKAP1. The data of N1-fused SRC kinases were aligned along the diagonal supporting that their capacity of binding is similar for both SKAP1 and SKAP2 proteins. In contrast, most data of C1-fused SRC kinases are in the lower right-hand quadrant in agreement with a higher binding capacity for DomDIM2 SKAP2 mutant than for DomDIM1 SKAP1 mutant.
      Figure thumbnail gr5
      Figure 5Comparison of binding properties from DIM domains of SKAP1 and SKAP2 proteins. Upper panel: Differential interaction scatterplot of DIM domain of SKAP1 protein (DomDIM1; y-axis) versus DIM domain of SKAP2 protein (DomDIM2; x-axis). Interactions not affected by the mutation are aligned on the diagonal. For DomDIM1, PPI disruptive mutations are located on the lower right-hand quadrant in contrast to mutations stabilizing interaction that are located on the upper left-hand one. Interactions for N1-fused SRC kinase are in blue circle and those for C1-fused SRC kinase are in red square. Error bar: Standard Error to the Mean (SEM). A robust linear regression, which takes account of outliers, was performed on data using mmregress Stata module. Linear regression equation is log10(DomDIM1) = 1.065* log10(DomDIM2) + 0.162 for N1-fused SRC kinases. Lower panels: PPI-mutation plot of respectively, SKAP2 protein, DomDIM1, and DomDIM2 domains with N1-fused (blue) and C1-fused (red) HCK mutants and the corresponding schematics of HCK organization. In PPI-mutation plot, Y-axis represents NLR of each HCK mutant in a logarithmic scale and X-axis, the position of the mutation. NLR are normalized according to that of HCK-N1. Arrows showed the position of the HCK mutations; wild type and replacement amino acid identities at mutated position involved in domain or motif inactivation are indicated in red and blue respectively.
      Study of HCK mutants agrees with the second consequence (Figure 5). First, note that HCK mutations known to mimic the activated kinase such as W93K, R150A, P228A and Y501F increase the binding signal to wild-type SKAP2, DomDIM1 or DomDIM2 independently of the N1 or C1 fusion. The only exception is N1-W93K for which an explanation will be given later. This supports that both configurations of the hemi-luciferase fusion in SRC kinases allow to study the molecular architecture of SKAP2 independently of their effects on kinases, since both detected an increase of binding capacity for activated HCK mutants. Second, the PPI signal strength of the HCK mutants known to mimic the activated kinase and C1-fused wild-type HCK was stronger for the DIM domain of SKAP2 than for either the DIM domain of SKAP1 or wild-type SKAP2 itself. These results support that the DIM domain of SKAP2 has a greater increase of its binding capacity for activated HCK mutants and C1-fused wild-type HCK than the DIM domain of SKAP1 and SKAP2 itself. These results are consistent with the first two consequences described above.

      Statistical validations

      Previous hypotheses were confirmed using univariate analyses combining at least three experiments. For each SKAP2 mutant, three effects were tested on N1-fused SRC kinases, and on C1-fused ones comparing to theorical mean, and on the difference between the signal of the C1-fused SRC kinases and that of N1-fused ones. Results were considered statistically significant only after Bonferroni’s correction (Table 1).
      Mutants with a loss of interaction were the same as in the previous analysis except for N1-fused LINK2SH3, which was just above the threshold (0.272 versus 0.25). Comparison of the deletion mutants shows that (i) for ΔSH3, DIMPH, the signal significantly decreases only for N1-fused SRC kinases; (ii) for only one mutant, ΔLINK1PH SKAP2, the signal significantly decreases specifically for C1-fused SRC kinases. For all other deletion mutants, the signal significantly decreases for both N1-fused and C1-fused SRC kinases. For DomDIM and ΔSH3 mutants, the signal for C1-fused SRC kinases was significantly higher than those for N1-fused ones. These results strongly support that the DIM domain is sufficient to interact with C1-fused SRC kinases even if other domains play a role. For example, the SH3 domain explains the similar binding pattern of C1-fused SRC kinases among the three SKAP2 mutants, DomDIM, DIMPH, and ΔSH3. For the binding of N1-fused SRC kinases, both DIM and SH3 domains are necessary. Finally, the second linker between the PH and the SH3 domains probably affects SRC kinase binding independently of the fusion position since the signal decreases more for the ΔPHLINK2 mutant than for the ΔPH and ΔLINK1PH mutants. The fact that the signal of SKAP1 decreases specifically for C1-fused SRC kinases agrees with previous hypotheses. Other statistical analyses for single point mutants show that for W336K and D129K SKAP2 mutants, the signal significantly increases while for DIM- SKAP2 mutant, it significantly decreases independently of the orientation of the fused Gluc1. For 3YF SKAP2 mutant, only the signal of N1-fused SRC kinases decreases. For D129K-3YF mutant, the signal significantly increases only for C1-fused SRC kinases. In contrast, it significantly decreases for DIM--D129K mutant. In conclusion, these results give a statistical support to our previous analyses.
      Second, a statistical modeling was performed taking account both literature curation and our results. Nine variables were generated, describing function possibly affected by mutations and a status for each variable was assigned for each mutant. The two variables describing how SKAP2 protein interacts with N1-fused or C1-fused SRC kinases were generated according to the following protocol: first, which of the five domains or regions significantly decreases the signal; second, which associations between the regions highlighted in the first part of this analysis significantly decrease the signal. The study of C1-fused SRC kinases data shows that the binding capacity of SKAP2 depends mainly on the presence of the DIM domain but is modulated by the presence of the SH3 domain and of the Link2 interdomain. The analysis of the N1-fused SRC kinases data shows that the binding site of SKAP2 protein depends on the presence of three regions, DIM, Link2, and SH3 altogether. A two-way ANOVA analysis was carried out after lognormal transformation of the dependent variable on these nine variables with either N1-fused or C1-fused SRC kinase data. This transformation was chosen since data are linearized after log-log transformation in differential interaction scatterplots (see Figures S3, S4). An explicative model was generated from a full model by a step-by-step procedure that removed at each step the lowest non-significant variable until no non-significant variables were present (Table 2).
      Table 2Statistical models of the interaction between SKAP2 mutants and SRC kinases
      N1-LocC1-loc
      VariablePartial SS+dfMSF (sense)P*Partial SS+dfMSF (sense)P*
      Explanatory Model595.445119.09255.61.09 10-142353.62844.20112.623.40 10-112
      loss of link between DIM-PHNS5.0015.0012.73(+)0.0004
      gain of PH binding site3.1313.136.71 (+)0.00985.1315.1313.07(+)0.0003
      SKAP1NS5.9215.9215.09(-)0.0001
      loss of PH binding siteNS4.5914.5911.70(-)0.0007
      DIM-10.17110.1721.82 (-)3.74 10-615.37115.3739.16(-)3.85 10-10
      W336K7.7917.7916.72 (+)4.96 10-511.37111.3728.96(+)5.41 10-8
      3YF5.3315.3311.44 (-)0.0008NS
      SKAP2 module 2431.441431.44926.01 (-)1.57 10-121NDNDNDNDND
      SKAP2 module 1NDNDNDNDND147.17273.58187.48(-)1.59 10-63
      Residual264.975700.47222.545670.39
      * NS: non significant (deleted variable during the procedure of minimization)
      + ND: not done
      Only 3 variables have similar effect on both N1-fused and C1-fused SRC kinase, the gain of function associated to D129K and W336K mutations, which increase the signal of interaction, the DIM- mutation, which has an opposite effect. The 3YF mutation, Y75F, Y237F, Y261F, specifically decreases the signal of interaction with N1-fused SRC kinases. Mutation located in the PI[3,4,5]P3-binding pocket of the PH domain and SKAP1 protein specifically decrease the signal of interaction with C1-fused SRC kinases, and the loss of association between the DIM and the PH domain, which has an opposite effect. In conclusion, this analysis strongly supports a modular organization of SKAP2 for binding to SRC kinases being composed of three modules, the DIM and SH3 domains and the interdomain between the PH and the SH3 domains, which each module having its SRC kinase binding capacity modulated differently by their activation and/or their sub-cellular localization. Also, note that the binding capacity of SKAP1 to SRC kinases decreases with C1-fused SRC kinases, in agreement with the loss of binding of its DIM domain (Figure 3).

      Analysis of HCK mutants

      Two non-exclusive hypotheses from literature curation might explain differences between N-terminal and C-terminal fused SRC kinases: a different sub-cellular localization and their level of activation. First, most SRC kinases such as HCK, are located to membranes by N-terminal myristylation and/or palmitoylation (
      • Patwardhan P.
      • Resh M.D.
      Myristoylation and membrane binding regulate c-Src stability and kinase activity.
      ,
      • Sato I.
      • Obata Y.
      • Kasahara K.
      • Nakayama Y.
      • Fukumoto Y.
      • Yamasaki T.
      • Yokoyama K.K.
      • Saito T.
      • Yamaguchi N.
      Differential trafficking of Src, Lyn, Yes and Fyn is specified by the state of palmitoylation in the SH4 domain.
      ). This process should be blocked by the N-terminal fusion of a hemi-luciferase. Second, a major process of activation is the dephosphorylation of Tyr501, which suppresses its intramolecular binding to the SH2 domain (
      • Engen J.R.
      • Wales T.E.
      • Hochrein J.M.
      • Meyn 3rd, M.A.
      • Banu Ozkan S.
      • Bahar I.
      • Smithgall T.E.
      Structure and dynamic regulation of Src-family kinases.
      ,
      • Roskoski Jr., R.
      Src protein-tyrosine kinase structure, mechanism, and small molecule inhibitors.
      ). C-terminal fusion of a hemi-luciferase might decrease the phosphorylation of Tyr501 by CSK kinase by masking a C-terminal binding site. To dissociate these two non-exclusive effects, we studied different HCK mutants. In our previous paper (
      • Bureau J.F.
      • Cassonnet P.
      • Grange L.
      • Dessapt J.
      • Jones L.
      • Demeret C.
      • Sakuntabhai A.
      • Jacob Y.
      The SRC-family tyrosine kinase HCK shapes the landscape of SKAP2 interactome.
      ), we have shown that the binding capacity of SKAP2 for SRC kinases specifically increases with their activation. We repeat this experiment by studying HCK mutants mimicking an activated kinase state (W93F, R150M, P228A, Y501F mutations alone or in combination). Two HCK mutations affecting its subcellular localization have also been studied only with C-terminal fusion of Gluc1: the G2A mutation suppressing any possibility of lipidation and the C3S mutation affecting palmitoylation. Interestingly, the kinase domain spontaneously adopts an active conformation in presence of its N-terminal region containing tryptophan 234, which induces rearrangement of two hydrophobic structures, the catalytic (CS) and the regulatory (RS) spines (
      • Sicheri F.
      • Moarefi I.
      • Kuriyan J.
      Crystal structure of the Src family tyrosine kinase Hck.
      ,
      • Gonfloni S.
      • Frischknecht F.
      • Way M.
      • Superti-Furga G.
      Leucine 255 of Src couples intramolecular interactions to inhibition of catalysis.
      ,
      • Kornev A.P.
      • Taylor S.S.
      Defining the conserved internal architecture of a protein kinase.
      ,
      • Kornev A.P.
      • Taylor S.S.
      Dynamics-Driven Allostery in Protein Kinases.
      ,
      • Meng Y.
      • Roux B.
      Computational study of the W260A activating mutant of Src tyrosine kinase.
      ,
      • LaFevre-Bernt M.
      • Sicheri F.
      • Pico A.
      • Porter M.
      • Kuriyan J.
      • Miller W.T.
      Intramolecular regulatory interactions in the Src family kinase Hck probed by mutagenesis of a conserved tryptophan residue.
      ). Twenty-two HCK mutants generated from 11 mutations (Figure S1B) and fused with hemi-luciferase1 at their N or C-terminus were tested for their binding to SKAP2 protein (Figure S5). HCK mutants bearing known mutations that activate kinase activity have a higher signal of interaction with SKAP2 than wild-type HCK independently of the Gluc1 orientation. N1-fused W93F HCK mutant is the only discordant result. These results confirmed that both fusion localizations detected the increase of PPI signal induced by SRC activation supporting their interest to define SKAP2 modular architecture.
      Four mutations, Y30F, W93F, W234A, and RS, induced differential signals of HCK kinase depending on the orientation of the fused luciferase fragment. RS mutation is a couple of mutations, I309A-T311A, located in the regulatory spine and known to inactivate the kinase activity (
      • Meharena H.S.
      • Chang P.
      • Keshwani M.M.
      • Oruganty K.
      • Nene A.K.
      • Kannan N.
      • Taylor S.S.
      • Kornev A.P.
      Deciphering the structural basis of eukaryotic protein kinase regulation.
      ,
      • Meharena H.S.
      • Fan X.
      • Ahuja L.G.
      • Keshwani M.M.
      • McClendon C.L.
      • Chen A.M.
      • Adams J.A.
      • Taylor S.S.
      Decoding the Interactions Regulating the Active State Mechanics of Eukaryotic Protein Kinases.
      ). Also, the signal completely disappeared for C1-fused G2A HCK mutant. However, these effects are not mainly due to a difference of binding capacity but to a difference of protein steady state level (Figure 6). N2-SKAP2 and N1- or C1-HCK mutants and wild-type were detected by a polyclonal antibody against Gaussia princeps luciferase. The level of HCK protein is followed by the decrease of the upper band (HCK) relative to the lower band (SKAP2) as well as by the appearance of small-sized degradation products. The level of HCK protein and the binding capacity were recovered by adding a second Y390F mutation to the four mutants of HCK C1-Y30F, N1-W93K, N1-W234A, C1-RS, and only protein level was rescued for C1-G2A (Figures 6A, 6B, and S4). Literature curation (
      • Mohapatra B.
      • Ahmad G.
      • Nadeau S.
      • Zutshi N.
      • An W.
      • Scheffe S.
      • Dong L.
      • Feng D.
      • Goetz B.
      • Arya P.
      • Bailey T.A.
      • Palermo N.
      • Borgstahl G.E.
      • Natarajan A.
      • Raja S.M.
      • Naramura M.
      • Band V.
      • Band H.
      Protein tyrosine kinase regulation by ubiquitination: critical roles of Cbl-family ubiquitin ligases.
      ,
      • Hornbeck P.V.
      • Zhang B.
      • Murray B.
      • Kornhauser J.M.
      • Latham V.
      • Skrzypek E.
      PhosphoSitePlus, 2014: mutations, PTMs and recalibrations.
      ) as well as the use of an expression vector support than the main effect on HCK steady state is due to degradation. Interestingly, small-sized degradation products are detected with C1-fused wild type and mutant HCK but not with N1-fused wild type and mutant HCK suggesting an N-terminal degradation. To support a role of ubiquitination, we studied K7R mutation in which degradation possibly does not occur. For all the mutants tested, C1-fused Y30F HCK, N1-fused W93K HCK, N1-fused W234A HCK, a full binding capacity was recovered when K7R mutation was present as well as protein stability for two of them (Figures 6B and S7). In conclusion, our present work did not confirm a role of W93K HCK mutation on SKAP2 binding as suggested in our original work but suggested that different HCK mutations have an effect on protein stability. The following model supported by literature suggests that ubiquitination of Lysine 7 after the binding of Cbl ubiquitin ligase by its SH2 domain on phosphorylated tyrosine 390 motif activates proteasome degradation (
      • Mohapatra B.
      • Ahmad G.
      • Nadeau S.
      • Zutshi N.
      • An W.
      • Scheffe S.
      • Dong L.
      • Feng D.
      • Goetz B.
      • Arya P.
      • Bailey T.A.
      • Palermo N.
      • Borgstahl G.E.
      • Natarajan A.
      • Raja S.M.
      • Naramura M.
      • Band V.
      • Band H.
      Protein tyrosine kinase regulation by ubiquitination: critical roles of Cbl-family ubiquitin ligases.
      ,
      • Hornbeck P.V.
      • Zhang B.
      • Murray B.
      • Kornhauser J.M.
      • Latham V.
      • Skrzypek E.
      PhosphoSitePlus, 2014: mutations, PTMs and recalibrations.
      ). Interestingly, the binding capacity to SKAP2 is poorly recovered for G2A HCK mutant suggesting that membranous localization of SRC kinases is necessary to bind SKAP2 at least for this HCK isoform. Note also that the steady state of N2 SKAP2 is slightly affected depending on HCK mutants.
      Figure thumbnail gr6
      Figure 6Role of protein stability on the signal of interaction between five HCK mutants and SKAP2. The upper panel shows normalized NLR of the samples loaded on the poly-acrylamide gel on the same order. The middle panel shows the western blot with a polyclonal antibody against Gaussia princeps luciferase detecting both SKAP2-N2 and HCK-N1 or HCK-C1 wild type and mutant proteins. Left lane: PageRuler Plus Prestained Protein Ladder with their corresponding molecular weight. Upper arrow: HCK-N1 and HCK-C1 wild type and mutant proteins. Lower arrow: SKAP2-N2. The lower panel shows the western blot on the same membrane using an antibody against GAPDH.

      Discussion

      In a previous paper (
      • Bureau J.F.
      • Cassonnet P.
      • Grange L.
      • Dessapt J.
      • Jones L.
      • Demeret C.
      • Sakuntabhai A.
      • Jacob Y.
      The SRC-family tyrosine kinase HCK shapes the landscape of SKAP2 interactome.
      ), protein-protein interactions (PPIs) between SKAP2 and most of its partners were finely analyzed. However, for SRC kinases we were only able to town down the role of the SH2 domain of SRC kinases, at least for HCK and those of three SKAP2 phosphorylated tyrosines, Y75, Y237, Y261. Interestingly, DIMPH, a deletion mutant of SKAP2, has completely lost its capacity to bind N1-fused SRC kinases but has retained it for most C1-fused SRC kinases. The purpose of the present paper is to solve how SKAP2 and SRC kinases interact. One major difficulty to study SRC kinases comes from their activation, which seems to increase the binding to SKAP2 for the most part independently of the classical binding sites inside their SH2 and SH3 domains (Figure S5). In particular, the PPI signal of SKAP2 for wild-type HCK is lower than that for W93K R150M HCK double mutant, in which each classical binding site for both the SH3 and SH2 domain were inactivated. So, we decided to concentrate our efforts on SKAP2 binding sites. The interactions of 21 SKAP2 mutants, eight of which are deletion mutants with nine SRC kinases fused with hemi-gaussia luciferase at either their N- or C-terminus were studied. The localization of the fused hemi-luciferase on SRC kinases probably affects both their activation and sub-cellular localization. This was the main reason to study N1-fused SRC kinases: evaluating the effect of both activation and subcellular localization by comparing data obtained with N1-fused SRC kinases and those with C1-fused ones. Note also that the analysis of the data defining SKAP2 architecture was simpler for the N1-fused SRC kinases than the C1-fused ones. Furthermore, rare N-terminal fusions of FGR kinases have been reported during cancers (
      • Stransky N.
      • Cerami E.
      • Schalm S.
      • Kim J.L.
      • Lengauer C.
      The landscape of kinase fusions in cancer.
      ) supporting to directly evaluate the effects of these fusions by our approach. Lastly, we would like to emphasis that cytosolic SRC kinase family member localization has been evocated in many reviews (
      • Salaun C.
      • Greaves J.
      • Chamberlain L.H.
      The intracellular dynamic of protein palmitoylation.
      ) and has been confirmed experimentally (
      • Kasahara K.
      • Nakayama Y.
      • Kihara A.
      • Matsuda D.
      • Ikeda K.
      • Kuga T.
      • Fukumoto Y.
      • Igarashi Y.
      • Yamaguchi N.
      Rapid trafficking of c-Src, a non-palmitoylated Src-family kinase, between the plasma membrane and late endosomes/lysosomes.
      ,
      • Konitsiotis A.D.
      • Rossmannek L.
      • Stanoev A.
      • Schmick M.
      • Bastiaens P.I.H.
      Spatial cycles mediated by UNC119 solubilisation maintain Src family kinases plasma membrane localisation.
      ,
      • Mohn H.
      • Le Cabec V.
      • Fischer S.
      • Maridonneau-Parini I.
      The src-family protein-tyrosine kinase p59hck is located on the secretory granules in human neutrophils and translocates towards the phagosome during cell activation.
      ,
      • Robbins S.M.
      • Quintrell N.A.
      • Bishop J.M.
      Myristoylation and differential palmitoylation of the HCK protein-tyrosine kinases govern their attachment to membranes and association with caveolae.
      ). Further studies of HCK mutants and of SRMS and FRK, two non-myristyl SRC kinases confirmed the role of their activation and subcellular localization and supported that their effects on SKAP2 modular architecture might be deduced from the comparison of results on both localization of the protein fusion. Direct NLR between wild-type SKAP2 and the nine N1 or C1-fused wild-type SRC kinases gives a last insight to support this view (Figure S6). This signal greatly varies depending on the SRC kinases and the localization of the hemi-luciferase fusion. Interestingly, the fusion localization has a small effect for the two non-myristate SRC kinases, SRMS and FRK, but also for four other myristate SRC kinases, FYN, LCK, LYN, and YES1. In contrast, the signal was higher for C1-fused than N1-fused proteins for three SRC kinases, BLK, FGR et HCK. This pattern differing from the previous one defined from the study of the three deletion mutants, DomDIM, DIMPH, and ΔSH3, supports that luciferase complementation assay associated to mutagenesis is an efficient tool to study this interaction even if each fusion localization has its own limitation. The different patterns support the view that some important differences exist among SRC kinases even if a global SKAP2 modular architecture emerges from the present work. Study of SKAP2 deletion mutants shows that the DIM domain is necessary and sufficient to bind most C1-fused SRC kinases. However, this result does not explain how SKAP1 protein, a paralog of SKAP2, binds SRC kinases. We were thus supposing that the SKAP2 modular organization for binding to SRC kinases depends on their conformations and localizations, in contrast to SKAP1 one. This main difference is due to different properties of their DIM domains. Three different results support this hypothesis: First the strength of interaction of most SRC kinases for the DIM domain of SKAP2 is higher than for the DIM domain of SKAP1; Second the strength of interaction of activated HCK mutants is higher for the DIM domain of SKAP2 than for the DIM domain of SKAP1 and wild-type SKAP2 independently of the fusion localization; Third this SKAP2 modular organization containing the DIM and the SH3 domain, and the second linker is modulated depending on the activation status and the sub-cellular organization of SRC kinases. In addition to previous discussion, these three independent confirmations support that this protein-protein interaction study is insight full despite the positional effect of the fusion protein on SRC kinases.
      Our present results did not support a role for the SH3 domain of HCK in initiating the interaction with SKAP2 protein. In our initial study (
      • Bureau J.F.
      • Cassonnet P.
      • Grange L.
      • Dessapt J.
      • Jones L.
      • Demeret C.
      • Sakuntabhai A.
      • Jacob Y.
      The SRC-family tyrosine kinase HCK shapes the landscape of SKAP2 interactome.
      ), the protein level of HCK mutants was not evaluated. The present data with literature curation support that at least five mutations affect HCK protein stability depending on the orientation of hemi gaussia1 fusion (Figure 6, Figure S7). Previous articles have reported that SRC kinase activation induces proteasome degradation after the binding of Cbl ubiquitin ligase by its SH2 domain on phosphorylated tyrosine 390 binding motif (
      • Mohapatra B.
      • Ahmad G.
      • Nadeau S.
      • Zutshi N.
      • An W.
      • Scheffe S.
      • Dong L.
      • Feng D.
      • Goetz B.
      • Arya P.
      • Bailey T.A.
      • Palermo N.
      • Borgstahl G.E.
      • Natarajan A.
      • Raja S.M.
      • Naramura M.
      • Band V.
      • Band H.
      Protein tyrosine kinase regulation by ubiquitination: critical roles of Cbl-family ubiquitin ligases.
      ). Our data agree with this model and suggest a degradation where ubiquitination of lysine7 plays a central role.
      A statistical modeling supports that the SKAP2 modular architecture for SRC binding is composed of three modules, the DIM and the SH3 domain, and the interdomain between the PH and SH3 domains with some modulations depending on different factors. Our results confirmed and extended the limited role of the three SKAP2 tyrosines, Y75, Y237, and Y261 (Figure 4 and S3). However, we are not able to conclude if this limited effect affects directly or indirectly the binding to SRC kinases. Interestingly inactivation of SH3 binding capacity by W336K mutation, which increases the signal of interaction with SRC kinases, independently of the orientation of hemi gaussia1 fusion, has a completely different effect than the SH3 deletion (Table 1). This suggests a new hypothesis where SKAP2 would be maintained away from SRC kinases through interaction of its SH3 domain with other interactors, independently of its role for their SRC binding. The study of mutations inside the PI[3,4,5]P3-binding pocket of the PH domain strongly suggests that mutations of this site modulate SKAP2 interaction with SRC kinases by decreasing its localization close to SRC kinases (Table 2). Interestingly, D129K SKAP2 mutation possibly increases the strength of the interaction with SRC kinases by two different mechanisms: it makes the PI[3,4,5]P3-binding pocket accessible to PI[3,4,5]P3 and/or induces another function, possibly a new site for positively charge lipids (
      • Swanson K.D.
      • Tang Y.
      • Ceccarelli D.F.
      • Poy F.
      • Sliwa J.P.
      • Neel B.G.
      • Eck M.J.
      The Skap-hom dimerization and PH domains comprise a 3'-phosphoinositide-gated molecular switch.
      ,
      • Vonkova I.
      • Saliba A.E.
      • Deghou S.
      • Anand K.
      • Ceschia S.
      • Doerks T.
      • Galih A.
      • Kugler K.G.
      • Maeda K.
      • Rybin V.
      • van Noort V.
      • Ellenberg J.
      • Bork P.
      • Gavin A.C.
      Lipid Cooperativity as a General Membrane-Recruitment Principle for PH Domains.
      ,
      • Yamamoto E.
      • Domanski J.
      • Naughton F.B.
      • Best R.B.
      • Kalli A.C.
      • Stansfeld P.J.
      • Sansom M.S.P.
      Multiple lipid binding sites determine the affinity of PH domains for phosphoinositide-containing membranes.
      ). Our statistical modeling suggests that both mechanisms affect this binding (Table 2). Study of the DIM- SKAP2 mutant shows the positive role of dimerization in the PPI strength of both N1-fused and C1-fused SRC kinases even if the mechanism is less clear. One interesting hypothesis is a positive allosteric interaction modulating the SKAP2 modular architecture.
      A similar structure to the SKAP2 dimer consisting in a four-helixes bundle, has been already reported to have a central role in kinase activation through allosteric control (
      • Lavoie H.
      • Sahmi M.
      • Maisonneuve P.
      • Marullo S.A.
      • Thevakumaran N.
      • Jin T.
      • Kurinov I.
      • Sicheri F.
      • Therrien M.
      MEK drives BRAF activation through allosteric control of KSR proteins.
      ). Future work will determine whether this is a new example of a protein-protein interaction that allosterically modulates kinase activation and/or trafficking with interesting new therapeutic consequences such as in oncology (

      Kung, J. E., and Jura, N. (2016) Structural Basis for the Non-catalytic Functions of Protein Kinases. Structure (London, England : 1993) 24, 7-24

      ,
      • Paul M.D.
      • Hristova K.
      The RTK Interactome: Overview and Perspective on RTK Heterointeractions.
      ,
      • Wacker D.
      • Stevens R.C.
      • Roth B.L.
      How Ligands Illuminate GPCR Molecular Pharmacology.
      ).

      Conclusions

      In this article, we show that the modular architecture of SKAP2 for binding to SRC kinase family involves its DIM domain, the SH3 domain, and the second interdomain. The binding capacity of each module to SRC kinases differs depending on the localization and the activation of the kinases. Also, we propose that the stability of SRC kinase family member HCK is affected by proteasome-mediated degradation. These data are necessary to understand how SKAP2, which is associated multiple diseases from susceptibility to autoimmune disorders to some cancer prognostics, affects SRC kinase functions and what is its relationship with its own functions. Modulating specifically this interaction might have interesting therapeutic consequences.

      Availability of data and materials

      The datasets used and/or analyzed during the current study as well as mutants generated in the present study are available from the corresponding author on reasonable request.
      This article contains supplemental data.

      Ethics approval and consent to participate

      This research has received an OGM agreement from the Ministère de l’Education Nationale (Dossier DUO n°9216).

      Competing interests

      The authors declare no competing interests.

      Acknowledgement

      We thank Chen Kuang-Yu, Tim Krischuns and Guillaume Dugied for regularly providing HEK 293T cell culture, and the Dana-Farber Cancer Institute (Boston, MA, USA) for providing us with ORFs.

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