PLCγ1 SH2C domain mediates phosphorylation of remote tyrosine. (A) One and five micromolar PLCγ1 SH2C-linker substrate (wild-type: lanes 1–3) or indicated mutants (lanes 4–39) were subjected to in vitro phosphorylation by 475 nM full-length FLAG-tagged Itk enzyme. Lane 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, and 39 are no Itk enzyme controls for the wild-type substrate or indicated mutants. Anti-pY783 blot indicates extent of PLCγ1 substrate phosphorylation by Itk; Coomassie stain shows substrate levels and the anti-FLAG blot shows the Itk enzyme levels. (B) Full-length Itk enzyme (158 nM) was incubated with 5 μM PLCγ1 SH2C-linker wild-type (lane 2), SH2C-linker K749A/R753A (lane 3), SH2C-linker R748A/K751A (lane 4), SH2C-linker E709A/K711A (lane 5) in an in vitro kinase assay and initial velocity is measured as described in refs. 26 and 43. Lane 1 is enzyme alone that has been subjected to the same assay conditions. (C) CD spectra of purified substrates (wild-type PLCγ1 SH2C domain and mutants shown in A).

Structural features of the SH2C docking surface. (A) Results of the mutational analyses (Fig. 1A) are mapped onto the structure of PLCγ1 SH2C domain (45) (PDB ID code 2PLD). Blue corresponds to amino acids for which mutation to alanine disrupts docking onto the Itk kinase domain and subsequent phosphorylation of Y783 and calcium mobilization in T cell signaling. Docking site residues are labeled and are located on the CD loop and the C terminus of the SH2C domain. Orange indicates residues that, when mutated, have no effect on Y783 phosphorylation and/or calcium mobilization in T cell signaling. (B) Residues peripheral to the binding site are shown in orange and labeled. Structure figures are created using PyMol (46). (C) Overlay of HSQC spectra acquired for a 50 μM sample of PLCγ1 SH2N-SH2C alone (red) and in the presence of 30 μM unlabeled Itk kinase domain (black). Extensive line broadening is evident for a subset of the PLCγ1 SH2C residues after addition of the Itk kinase domain. Three broadened resonances are shown (K751, E708, and M750) and one representative peak that does not change upon addition of kinase is also shown (R684) for comparison. (D) PLCγ1 SH2C residues for which NMR resonances broaden on addition of Itk kinase domain are shown in red and labeled on the SH2C structure. The docking site region (as determined by mutagenesis) is circled as is the phosphotyrosine binding pocket (dashed circle).

Structural comparisons of the PLCγ1 SH2C docking site. (A) Itk binding site on PLCγ1 SH2C is highlighted in red. (B) Itk binding site on Itk SH2 (Pdb: 1LUN) in cyan (38). In (A) and (B) the orientation of the PLCγ1 and Itk SH2 domains is the same permitting comparison of the respective kinase docking sites. (C) Structure of the SAP SH2/Fyn SH3/SLAM complex (PDB ID code 1M27). The site of Fyn SH3 binding is circled (red dashes) on the SAP SH2 domain (DE loop and B helix). (D) Structure of the PLCγ1 SH2N domain (blue) bound to the FGFR1 kinase domain (black) (PDB ID code 3GQI). The classical phosphotyrosine (pY) binding of SH2N to the tail of FGFR1 is shown. The secondary site of SH2N/kinase interaction is circled (red dashes) and involves the BC and DE loops. In (C) and (D) the SH2 domains are shown in the same orientation as the PLCγ1 SH2C domain in Fig. 4A to facilitate comparison.

SH2 docking in full length PLCγ1 (A) Full-length PLCγ1 was subjected to an in vitro kinase assay using Itk full-length enzyme alone (lane 1), or with PLCγ1 SH2C domain (10 and 20 μM; lanes 2 and 3, respectively), or the Grb2 SH2 domain (10 and 20 μM; lanes 4 and 5). Anti-pY783 antibody is used to detect levels of Itk induced phosphorylation of Y783, anti-strep tag antibody indicates the level of the full-length PLCγ1 substrate and anti-Itk antibody is used to confirm Itk enzyme levels. (B) In lane 1: Strep-tagged, wild-type full-length PLCγ1 is incubated with Itk enzyme. Lanes 2, 4, and 6 are no Itk enzyme controls. Lanes 3 and 5 are the same experiment as lane 1 using either the K749A/R753A mutant of full-length PLCγ1 (lane 3) or the R748A/K751A mutant of full-length PLCγ1 (lane 5) as substrate. Blots are probed as described in A. (C, D, F, and G) Jurkat Jγ1 cells were transfected with full-length PLCγ1 wild-type or different mutants; histograms of GFP fluorescence after sorting for all cell lines are provided as (Fig. S2). Jurkat Jγ1 cells transfected with the indicated PLCγ1 constructs [wild-type PLCγ1, PLCγ1 mutants (C: R748A/K749A/K751A/R753A; D: E709A/K711A; F: P686A/R687A/D688A; G: K713A/Y747A and Y783F control)]. The two variants that target the basic C-terminal region of SH2C (see data in B) were combined in subsequent experiments (see C). After stimulation [Ca²⁺]_i change is measured as fluorescence emission ratio (FER) at 398 nm (Ca²⁺ bound Indo-1) and 490 nm (Ca²⁺ unbound Indo-1) over a 200-s period. In C, D, F, and G [Ca²⁺]_i change for Jurkat Jγ1 cells transfected with PLCγ1 wild-type after TCR stimulation is shown in black, [Ca²⁺]_i change for Jurkat Jγ1 cells transfected with PLCγ1 Y783F after TCR stimulation is shown in light gray, and [Ca²⁺]_i change for Jurkat Jγ1 cells transfected with the indicated mutation is shown in medium gray. The [Ca²⁺]_i change for Jurkat Jγ1 cells transfected with PLCγ1 wild-type after mouse IgG isotype stimulation is provided as (Fig. S2). (E) Superposition of NMR data for wild-type or specified mutant PLCγ1 SH2C domain before (spectra of free SH2C shown in black in A–H) and after addition of phospholigand (red in each). The ¹⁵N-labeled wild-type SH2C, R784A/K749A/K751A/R753A SH2C mutant and E709A/K711A SH2C mutant were all concentrated to 0.3 mM (black spectrum) and an equimolar amount of phosphopeptide (PGF(pY)VEAN) was added before acquisition of the second spectrum in each case (red). Arrow indicates chemical shift change induced by phospholigand binding to each SH2C domain. NMR data here and in Fig. 3 were acquired on a Bruker AV700 spectrometer. (H) Histogram summarizing the data in C, D, F. and G, which are representative of at least three independent experiments. Data are normalized as described in SI Text. The Y783F data represents the extent of calcium flux with no phosphorylation at position 783 in PLCγ1.

Doubly phosphorylated peptide alters docking surface. (A) Structure of the PLCγ1 SH2C domain bound to a classical phosphotyrosine containing ligand. Ligand is depicted in black and the docking site residues that bind the Itk kinase domain are red and circled. (B) Binding assay showing the interaction between purified Itk kinase domain (0.22 μM) and the GST-PLCγ1 SH2C fusion protein (3.8 μM added to beads). Anti-flag is used to detect Itk kinase domain that binds to immobilized SH2C (Top). Lane 1 is purified Itk kinase domain, lane 2 is the GST control, lane 3 shows the SH2C/kinase interaction with no added peptide, lanes 4–7 are the same experiment with increasing concentration of phosphopeptide, PGF(pY)VEAN (peptide concentrations are: 0.19, 0.38, 3.84, and 19.22 μM in lanes 4–7, respectively). The Bottom shows GST and GST-SH2C levels. (C) Structure of the PLCγ1 SH2C domain bound to a doubly phosphorylated peptide derived from Syk showing the structural rearrangement within SH2C (31). Peptide ligand is shown in black and the SH2C residues required for binding to Itk kinase domain are in red and circled. (D) Same binding experiment as in (B). Increasing concentration of the doubly phosphorylated peptide ligand, DTEV(pY)ESP(pY)ADPE (peptide concentrations are: 0.38, 0.96, 1.92, 3.84, and 19.22 μM in lanes 11–15, respectively).