Itk tyrosine kinase substrate docking is mediated by a nonclassical SH2 domain surface of PLCγ1

Abstract

Interleukin-2 tyrosine kinase (Itk) is a Tec family tyrosine kinase that mediates signaling processes after T cell receptor engagement. Activation of Itk requires recruitment to the membrane via its pleckstrin homology domain, phosphorylation of Itk by the Src kinase, Lck, and binding of Itk to the SLP-76/LAT adapter complex. After activation, Itk phosphorylates and activates phospholipase C-␥1 (PLC-␥1), leading to production of two second messengers, DAG and IP3. We have previously shown that phosphorylation of PLC-␥1 by Itk requires a direct, phosphotyrosine-independent interaction between the Src homology 2 (SH2) domain of PLC-␥1 and the kinase domain of Itk. We now define this docking interface using a combination of mutagenesis and NMR spectroscopy and show that disruption of the Itk/PLC␥1 docking interaction attenuates T cell signaling. The binding surface on PLC␥1 that mediates recognition by Itk highlights a nonclassical binding activity of the well-studied SH2 domain providing further evidence that SH2 domains participate in important signaling interactions beyond recognition of phosphotyrosine.substrate recognition ͉ T cell signaling ͉ Tec kinases I nterleukin-2 tyrosine kinase (Itk) is a nonreceptor protein tyrosine kinase that is expressed in T cells, mast cells and NK cells (1)(2)(3)(4)(5). Previous studies have shown that activation of Itk after T cell receptor engagement requires Itk recruitment to PIP3 in the membrane via its PH domain, binding of Itk to the SLP-76/LAT adapter complex, and phosphorylation of Itk by Lck at the activation loop tyrosine in its kinase domain (6). Activated Itk then phosphorylates its substrate, phospholipase C-␥1 (PLC-␥1), resulting in activation of PLC␥1 lipase activity and subsequent hydrolysis of PIP 2 to IP 3 and DAG. IP 3 and DAG stimulate the release of calcium ions from the endoplasmic reticulum and activate Protein Kinase C, respectively (7-11). The overall signaling pathway has been clearly delineated but many of the precise molecular details of the protein-protein interactions and enzyme/substrate interactions that control signal transduction after TCR engagement remain to be determined.Itk belongs to the Tec family of nonreceptor tyrosine kinases that also includes Btk, Tec, Rlk, and Bmx (12). The Tec kinases, like the larger protein kinase superfamily, control numerous cellular signaling networks by phosphorylating target amino acid side chains in a stringently specific manner. Based on results of combinatorial peptide library screens and structures of kinase/peptide substrate complexes, the view has emerged that the active sites of most kinases tolerate different sequences and are therefore not necessarily stringently specific for short peptide sequences (13,14). Stringent specificity is, however, a strict requirement of cellular signaling cascades and so these enzymes must have mechanisms to control substrate fidelity (14-22).The physiological substrate of Itk, PLC␥1, is a phospholipase that contains an amino-terminal PH domain, EF hand motif, a split c...

Two Closely Spaced Tyrosines Regulate NFAT Signaling in B Cells via Syk Association with Vav

“…This region has been reported to be an alternative site for phosphotyrosine-independent binding in other SH2 domains (16,29). Growing evidence supports the notion that nonclassical binding outside the pTyr and specificity pockets of as SH2 domain can be significant for inter-or intramolecular interactions (1,9,34,49,70). Future mutagenesis study might provide insight into the possibility of multiple domain interactions between Vav1 and Syk that affect downstream signaling.…”

DEF6, a Novel Substrate for the Tec Kinase ITK, Contains a Glutamine-rich Aggregation-prone Region and Forms Cytoplasmic Granules that Co-localize with P-bodies

“…Significantly, fulllength DEF6, or truncated versions of DEF6 that contained the PH domain, were necessary for mediating the interaction with ITK, associated with both active and kinase-dead ITK. Furthermore, interaction of the PH domain of DEF6 with ITK is mediated through the kinase domain of ITK, which has also been found to be the case for the best characterized substrates of ITK (28,47). Since LCK is both responsible for ITK activation by phosphorylating it at tyrosine 511 (16) and targeting DEF6 to the cell membrane by phosphorylation of tyrosines 133 and 144 (5) this places LCK as the key upstream regulator of DEF6 phosphorylation at tyrosine residues 210 and 222 by ITK.…”

“…Phosphorylation by ITK-Current evidence suggests that substrate recognition by ITK is mediated by interactions at sites that are remote from its catalytic site (27)(28)(29). Analysis of the interaction between DEF6 and ITK was initially carried out in COS-7 cells to minimize the possibility of interfering associations with other T cell proteins.…”

SH2 Domains Recognize Contextual Peptide Sequence Information to Determine Selectivity

“…Such low affinity secondary contacts play a role in promoting functional signaling but are not sufficient for the initial complex formation (16). Although it is well established that additional specificity may be provided by longer range interactions, secondary contacts, avidity, or allosteric mechanisms (17,18), the interaction of the SH2 domain with a linear phosphotyrosinecontaining motif within the ligand is essential for complex formation and represents the primary basis for selectivity. In the context of ongoing efforts to understand the mechanisms underlying interaction selectivity, we postulate that the SH2 domain achieves selectivity for physiologically relevant peptide ligands by making use of additional information content embedded within the short phosphotyrosine peptide sequences that constitute their primary ligands.…”