Itk tyrosine kinase substrate docking is mediated by a nonclassical SH2 domain surface of PLC␥1 Lie Min, Raji E. Joseph, D. Bruce Fulton, and Amy H. Andreotti1 Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011 Edited by Susan S. Taylor, University of California at San Diego, La Jolla, CA, and approved October 20, 2009 (received for review October 1, 2009) Interleukin-2 tyrosine kinase (Itk) is a Tec family tyrosine kinase that We have previously shown that the PLC␥1 SH2C domain mediates signaling processes after T cell receptor engagement. Acti- (spanning residues 659–756 within full-length PLC␥1) binds di- vation of Itk requires recruitment to the membrane via its pleckstrin rectly to the Itk kinase domain and is required for efficient homology domain, phosphorylation of Itk by the Src kinase, Lck, and phosphorylation of Y783 by Itk (26). Fragments of PLC␥1 that binding of Itk to the SLP-76/LAT adapter complex. After activation, Itk contain Y783 but not the SH2C domain are not efficiently phos- phosphorylates and activates phospholipase C-␥1 (PLC-␥1), leading to phorylated by Itk. Moreover, phosphorylation of PLC␥1 substrate production of two second messengers, DAG and IP3. We have previ- fragments that contain both SH2C and Y783 (spanning 659–789, ously shown that phosphorylation of PLC-␥1 by Itk requires a direct, hereafter referred to as PLC␥1 SH2C-linker) can be inhibited by phosphotyrosine-independent interaction between the Src homol- titration with isolated PLC␥1 SH2C domain (26). The excess, free ogy 2 (SH2) domain of PLC-␥1 and the kinase domain of Itk. We now SH2C domain binds to the Itk kinase domain preventing association define this docking interface using a combination of mutagenesis and between Itk kinase domain and the PLC␥1 SH2C-linker substrate. NMR spectroscopy and show that disruption of the Itk/PLC␥1 docking Competition by free SH2C domain is specific since the SH2 interaction attenuates T cell signaling. The binding surface on PLC␥1 domains from Grb2 and PI3K have no effect on Itk mediated that mediates recognition by Itk highlights a nonclassical binding phosphorylation of PLC␥1 (26). Thus, the PLC␥1 SH2C domain activity of the well-studied SH2 domain providing further evidence contains a recognition motif or docking site for Itk that mediates that SH2 domains participate in important signaling interactions specific phosphorylation of the target tyrosine that is located beyond recognition of phosphotyrosine. outside of the SH2C domain at position 783. Despite their fame as phosphotyrosine recognition modules, we substrate recognition ͉ T cell signaling ͉ Tec kinases find that mutation of the conserved phosphotyrosine binding pocket in PLC␥1 SH2C (R694A/R696A) has no effect on the nterleukin-2 tyrosine kinase (Itk) is a nonreceptor protein tyrosine PLC␥1/Itk docking interaction. Equal levels of Y783 phosphory- Ikinase that is expressed in T cells, mast cells and NK cells (1–5). lation by Itk are measured for both wildtype and the R694A/R696A Previous studies have shown that activation of Itk after T cell receptor mutant PLC␥1 SH2C-linker substrates suggesting that the specific engagement requires Itk recruitment to PIP3 in the membrane via its SH2C residues involved in docking onto the Itk kinase are located PH domain, binding of Itk to the SLP-76/LAT adapter complex, and outside of the classical SH2 ligand-binding surface (26). phosphorylation of Itk by Lck at the activation loop tyrosine in its kinase We now define the Itk-docking element within the PLC␥1 SH2C domain (6). Activated Itk then phosphorylates its substrate, phospho- domain and confirm that the site of interaction is distinct from the lipase C-␥1 (PLC-␥1), resulting in activation of PLC␥1 lipase activity classical phosphotyrosine ligand binding surface common to all and subsequent hydrolysis of PIP2 to IP3 and DAG. IP3 and DAG SH2 domains. Mutation of this nonclassical binding surface in the stimulate the release of calcium ions from the endoplasmic reticulum SH2C domain within full-length PLC␥1 leads to loss of Y783 and activate Protein Kinase C, respectively (7–11). The overall signaling phosphorylation by Itk and attenuation of calcium flux upon TCR pathway has been clearly delineated but many of the precise molecular stimulation in T cells. A comparison of these data to other SH2 details of the protein–protein interactions and enzyme/substrate inter- mediated protein kinase docking interactions characterized to date BIOCHEMISTRY actions that control signal transduction after TCR engagement remain suggests a wide versatility among SH2 binding interactions. to be determined. Itk belongs to the Tec family of nonreceptor tyrosine kinases that Results also includes Btk, Tec, Rlk, and Bmx (12). The Tec kinases, like the Mapping the Docking Site on PLC␥1. To identify the PLC␥1 SH2C larger protein kinase superfamily, control numerous cellular sig- residues involved in substrate recognition by the Itk kinase domain, naling networks by phosphorylating target amino acid side chains we compared the primary sequences of the PLC␥1 SH2C domain in a stringently specific manner. Based on results of combinatorial derived from several species with the primary sequences of two SH2 peptide library screens and structures of kinase/peptide substrate domains that do not compete with Itk mediated substrate phos- complexes, the view has emerged that the active sites of most phorylation (PI3K and Grb2). Surface exposed residues that are kinases tolerate different sequences and are therefore not neces- conserved across the PLC␥1 SH2 domains from different species sarily stringently specific for short peptide sequences (13, 14). but differ in either PI3K or Grb2 (or both) were targeted for Stringent specificity is, however, a strict requirement of cellular mutation. A total of 23 PLC␥1 SH2C candidate residues (see Fig. signaling cascades and so these enzymes must have mechanisms to S1) were identified as potential substrate docking site residues. We control substrate fidelity (14–22). expect that mutation of the docking motif that mediates the direct The physiological substrate of Itk, PLC␥1, is a phospholipase that contains an amino-terminal PH domain, EF hand motif, a split catalytic domain, a split PH domain, two tandem Src homology 2 Author contributions: L.M. and A.H.A. designed research; L.M., R.E.J., and D.B.F. performed (SH2) domains (SH2N for amino-terminal and SH2C for carboxy- research; L.M., R.E.J., and A.H.A. analyzed data; and L.M. and A.H.A. wrote the paper. terminal, respectively), a 33-amino acid linker, an SH3 domain, and The authors declare no conflict of interest. a C2 domain. The tyrosine at position 783, located within the This article is a PNAS Direct Submission. 33-amino acid linker region between the SH2C and SH3 domains, Freely available online through the PNAS open access option. is the site of PLC␥1 phosphorylation by Itk (23–25). How does Itk 1To whom correspondence should be addressed. E-mail: [email protected]. achieve selectivity for this particular tyrosine when multiple poten- This article contains supporting information online at www.pnas.org/cgi/content/full/ tial sites of phosphohrylation are present in the substrate molecule? 0911309106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0911309106 PNAS ͉ December 15, 2009 ͉ vol. 106 ͉ no. 50 ͉ 21143–21148 Downloaded by guest on September 28, 2021 Fig. 1. PLC␥1 SH2C domain mediates phosphorylation of K695A A B remote tyrosine. (A) One and five micromolar PLC␥1 SH2C- N728A R696A S739A K743A K749A ) -1 linker substrate (wild-type: lanes 1–3) or indicated mutants WT E730A S701A D732A E742A H744A R753A 0.16 (lanes 4–39) were subjected to in vitro phosphorylation by 0.12 anti-pY783 WT 475 nM full-length FLAG-tagged Itk enzyme. Lane 3, 6, 9, 12, 0.08 15, 18, 21, 24, 27, 30, 33, 36, and 39 are no Itk enzyme controls Coomassie 0.04 for the wild-type substrate or indicated mutants. Anti-pY783 0 anti-FLAG 1 2 3 4 5 blot indicates extent of PLC␥1 substrate phosphorylation by 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Itk; Coomassie stain shows substrate levels and the anti-FLAG Initial velocity (min blot shows the Itk enzyme levels. (B) Full-length Itk enzyme N697A C715A 12000 R748A E709A M681A C ␮ ␥ ) (158 nM) was incubated with 5 M PLC 1 SH2C-linker wild- K751A K711A T674A M683A E698A R716A -1 7000 type (lane 2), SH2C-linker K749A/R753A (lane 3), SH2C-linker anti-pY783 2000 dmol R748A/K751A (lane 4), SH2C-linker E709A/K711A (lane 5) in 2 -3000 an in vitro kinase assay and initial velocity is measured as Coomassie -8000 described in refs. 26 and 43. Lane 1 is enzyme alone that has anti-FLAG (deg cm been subjected to the same assay conditions. (C) CD spectra of -13000 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 200 220 240 260 purified substrates (wild-type PLC␥1 SH2C domain and mu- Molar residue ellipticity wavelength (nm) tants shown in A). Itk/PLC␥1 substrate interaction will result in loss or reduction of full-length PLC␥1 as substrate. First, we find that full-length Itk substrate phosphorylation by Itk. enzyme readily phosphorylates Y783 in full-length PLC␥1 (Fig. 2A; In the context of the PLC␥1 SH2C-linker substrate, the 23 lane 1). Consistent with the earlier experiments using the PLC␥1 conserved SH2C residues were mutated to alanine either separately substrate fragment (26), we find that addition of isolated PLC␥1 or in groups.