Research ArticleBiochemistry

The Src family kinase Fgr is a transforming oncoprotein that functions independently of SH3-SH2 domain regulation

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Science Signaling  23 Oct 2018:
Vol. 11, Issue 553, eaat5916
DOI: 10.1126/scisignal.aat5916
  • Fig. 1 Src family kinase domain organization and crystal structure of the inactive conformation.

    The x-ray crystal structure of inactive, near–full-length Src is modeled at the top as a ribbon diagram (left) and space-filling model (right). The linear domain organization of full-length, wild-type Src is shown below the models, along with the near–full-length version with the modified tail sequence (Src-YEEI) used in this study. The N-terminal unique domains of all Src family members are modified by myristoylation (Myr) and palmitoylation (not shown) in most cases. The unique region is followed by the SH3 and SH2 domains, which are joined to the bi-lobed kinase domain through the SH2-kinase linker. The activation loop (A-loop), an important dynamic element involved in kinase domain regulation, has a single tyrosine residue (Tyr416). Autophosphorylation of Tyr416 stabilizes the active form of the kinase. The kinase domain is followed by the C-terminal tail, with a single tyrosine residue, Tyr527; phosphorylation of this site by Csk induces intramolecular SH2 engagement important for negative regulation.

  • Fig. 2 Expression of wild-type Fgr induces oncogenic transformation of Rat-2 fibroblasts.

    (A) Micrographs of representative 14-day monolayer cultures of Rat-2 fibroblasts after infection with recombinant retroviruses carrying wild-type (WT) and tail mutants (Tyr527 to Phe; YF) of Fgr or Hck and selection with G418. Transformed foci appear as clumps of refractile cells growing on top of the contact-inhibited cell monolayer. Scale bar, 300 μm. (B) Colony-forming assays with the cells described in (A). Each cell population, as well as control cells transduced with an empty vector, was plated in semisolid medium and incubated for 2 weeks. Cultures were stained with Wright-Giemsa, and colonies were counted from scanned images of each plate using ImageJ software. Colony counts from each plate as a function of cellular input are shown, with the mean value represented by the horizontal bar (n = 3). This entire experiment was repeated twice using independently derived retroviral supernatants and produced comparable results. (C) Protein extracts from each of the Rat-2 cell populations shown in (A) were separated by SDS–polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose, and probed with phosphospecific antibodies to the activation loop phosphotyrosine (pY416), the tail phosphotyrosine (pY527), and each kinase protein. Immunoreactive proteins were visualized using secondary antibodies conjugated to infrared dyes and imaged using the LI-COR Odyssey system and software. Representative blots of three independent determinations are shown.

  • Fig. 3 Activation of recombinant Src family kinases by SH3- and SH2-binding peptides.

    (A and B) Near–full-length Fgr, Src, and Hck kinase activities were assayed in the presence of peptides that bind to the SH3 domain alone (VSL12), to the SH2 domain alone (pYEEI), or to both SH2 and SH3 (pFAK). The amino acid sequence of each peptide ligand is shown (A), in which amino acids involved in regulatory domain engagement are underlined. Each recombinant kinase was added to the assay at a concentration that yielded 20 to 25% of maximum activity, and the input of each peptide was varied over the range of peptide to kinase molar ratios shown (B). Each data point was measured in triplicate and is shown as the mean value ± SD; the error bars are smaller than the size of the data points.

  • Fig. 4 SPR analysis of peptide ligand binding to recombinant Fgr and Hck SH2 and SH3 domains.

    Biotinylated VSL12, pFAK, and pYEEI peptide ligands (sequences shown in Fig. 3) were immobilized on a streptavidin biosensor chip. Recombinant SH3 and SH2 domain proteins were then injected in triplicate over the range of concentrations shown until equilibrium was reached, followed by a 2-min dissociation phase. Reference-corrected sensorgrams were fit by a 1:1 Langmuir binding model, and equilibrium Kd values (M) were calculated from the resulting kinetic rate constants (koff/kon). The three replicate sensorgrams obtained at each domain protein concentration are shown (color code at top right), and a single representative fitted curve for each set is shown as a dark gray line. RU, resonance units.

  • Fig. 5 HX MS shows that near–full-length Fgr adopts an assembled conformation similar to inactive Src.

    Deuterium uptake by the Src and Fgr SH3-SH2 domains alone was compared to uptake in the near–full-length kinases by HX MS. Difference maps for both Fgr and Src are shown, where the level of deuteration in each SH3-SH2 peptide was subtracted from that in the near–full-length kinase (SH3-SH2-K) at each of the time points indicated, and the difference was colored according to the scale shown. Protection from deuterium exchange (increasingly deeper blue color) was observed in the SH3 and SH2 domains from both Src and Fgr when the kinase domain was also present. The sequences of 10 homologous peptides derived from the Src and Fgr SH3-SH2 region are indicated on the maps, from the N terminus (top) to the C terminus (bottom). The corresponding uptake plots for each peptide are shown in fig. S4. Conn, SH3-SH2 connector.

  • Fig. 6 Substitution of the Fgr activation loop with the conserved TAR motif suppresses kinase and transforming activity in Rat-2 cells.

    (A) Left: Alignment of the activation loop sequences from the eight mammalian Src family members. Fgr is unique among the Src family in that the Thr-Ala-Arg (TAR) sequence adjacent to the activation loop tyrosine (YP) is substituted with Asn-Pro-Cys (NPC). Right: Src activation loop structure in the inactive conformation with the side chains of the TAR motif shown. The activation loop tyrosine (Y416) makes a hydrogen bond with the catalytic aspartate (D386) to stabilize the inactive structure (PDB: 2SRC). (B) Rat-2 cells expressing wild-type Fgr (WT), the tail mutant (YF), wild-type with the TAR substitution in the activation loop, and vector control cells were plated in triplicate in semisolid medium and incubated for 2 weeks. Cultures were stained with Wright-Giemsa, and colonies were counted from scanned images of each plate using ImageJ software. Average colony counts, ±SD, from each plate as a function of cellular input are shown from a representative of two experiments. *P < 0.05 by Student’s t test. (C) Protein extracts from the Rat-2 cell populations shown in (B) were immunoblotted for activation loop phosphorylation (pY416) and Fgr protein abundance. Data are the average ratio of pY416 to Fgr protein signals from three independent determinations ± SD; **P < 0.05 by Student’s t test.

  • Fig. 7 Wild-type Fgr is active in TF-1 myeloid cells and reduces the GM-CSF requirement for proliferation.

    TF-1 myeloid cells were infected with recombinant retroviruses carrying the wild-type human Fgr coding sequence or an empty vector as a negative control, followed by selection with puromycin. (A) Cultures of TF-1/Fgr cells were expanded in the presence of GM-CSF. Cells were washed free of GM-CSF, split into two aliquots, and then incubated overnight in the presence or absence of GM-CSF (1000 pg/ml) or the pan-Src family kinase inhibitor A-419259 as indicated at the top. Fgr was immunoprecipitated from cell lysates and probed with phosphospecific antibodies to the activation loop phosphotyrosine (pY416), the tail phosphotyrosine (pY527), and total Fgr protein. Immunoreactive proteins were visualized using secondary antibodies conjugated to infrared dyes and imaged using the LI-COR Odyssey system and software. Images of representative blots are shown. Bar graphs below the images show the average intensity ratios for the phosphotyrosine signals divided by the Fgr protein abundance from three independent determinations. Ratios were normalized to the values for the +GM-CSF/no inhibitor conditions and are shown as mean values ± SE. *P < 0.05 compared to dimethyl sulfoxide control, all others not significant (by Student’s t test). (B) TF-1 cells expressing Fgr or vector (TF-1/vector and TF-1/Fgr, respectively) and vector control cells were plated in soft-agar colony-forming assays (1000 or 2500 cells per 35-mm plate) in the absence or presence of a suboptimal concentration of GM-CSF (100 pg/ml is 10% of the normal concentration used in routine cell culture). Colonies were visualized with Wright-Giemsa stain and quantified using ImageJ after 2 weeks. Each bar represents the average number of colonies observed ± SE (n = 3). *P < 0.05 by Student’s t test. (C) Fgr and Src gene expression data from 163 AML bone marrow samples were downloaded from TCGA database. Data are shown as the number of kinase complementary DNA fragments per kilobase of transcript per million mapped reads (FPKM). The dotted lines indicate the mean transcript values.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/553/eaat5916/DC1

    Fig. S1. Analysis of intact Fgr-YEEI and phosphopeptides by MS.

    Fig. S2. In vitro kinase assay for recombinant near–full-length Fgr, Hck, and Src activity.

    Fig. S3. SPR analysis of Fgr-YEEI interaction with peptide ligands for the SH3 and SH2 domains.

    Fig. S4. Deuterium uptake by peptic peptides derived from near–full-length Fgr and Src compared to their isolated SH3-SH2 domains.

    Fig. S5. Fractional deuterium uptake by Src-YEEI and Fgr-YEEI.

  • This PDF file includes:

    • Fig. S1. Analysis of intact Fgr-YEEI and phosphopeptides by MS.
    • Fig. S2. In vitro kinase assay for recombinant near–full-length Fgr, Hck, and Src activity.
    • Fig. S3. SPR analysis of Fgr-YEEI interaction with peptide ligands for the SH3 and SH2 domains.
    • Fig. S4. Deuterium uptake by peptic peptides derived from near–full-length Fgr and Src compared to their isolated SH3-SH2 domains.
    • Fig. S5. Fractional deuterium uptake by Src-YEEI and Fgr-YEEI.

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