An Autoinhibitory Role for the Pleckstrin Homology Domain of Interleukin-2-Inducible Tyrosine Kinase and Its Interplay with Canonical Phospholipid Recognition

Abstract

Pleckstrin homology (PH) domains are well known as phospholipid binding modules yet evidence is mounting that PH domain function extends beyond lipid recognition. In the current work, we characterize a protein binding function for the PH domain of Interleukin-2-inducible tyrosine kinase (ITK), an immune cell specific signaling protein that belongs to the TEC family of non-receptor tyrosine kinases. Its N-terminal Pleckstrin homology (PH) domain is a well characterized lipid-binding module that localizes ITK to the membrane via phosphatidylinositol (3,4,5)-trisphosphate (PIP3) binding. Using a combination of NMR spectroscopy and mutagenesis, we have mapped an autoregulatory protein interaction site on the ITK PH domain that makes direct contact with the catalytic kinase domain of ITK inhibiting the phospho-transfer reaction. Moreover, we have elucidated an important interplay between lipid binding by the ITK PH domain and the stability of the autoinhibitory complex formed by full length ITK. The ITK activation loop in the kinase domain becomes accessible to phosphorylation to the exogenous kinase LCK upon binding of the ITK PH domain to PIP3. By clarifying the allosteric role of the ITK PH domain in controlling ITK function we have expanded the functional repertoire of the PH domain generally and opened the door to alternative strategies to target this specific kinase in the context of immune cell signaling.

The Src module: an ancient scaffold in the evolution of cytoplasmic tyrosine kinases

“…The NMR analysis indicates that, in solution, the PHTH module can dock in an alternative conformation that directly blocks the kinase active site (Figure 3(C)) (Joseph et al 2017). This configuration was first observed by NMR for Itk (Devkota et al 2017), and it has a general resemblance to the auto-inhibited structure of the Ser/Thr kinase Akt, in which the PH domain of Akt is docked onto the catalytic face of the kinase domain (PDB code 3O96) (Wu et al 2010). It is possible that there are two states of auto-inhibited Btk.…”

“…All five domains of the Tec-family kinases (PH-TH-SH3-SH2-kinase) participate in auto-inhibitory interactions to reduce kinase activity in the cytosol, as described above (Figure 3(C)) (Wang et al 2015; Devkota et al 2017; Joseph et al 2017; Andreotti et al 2018). Unlike Src-family kinases, however, the kinase domains of Btk and Itk alone have very little intrinsic activity.…”

HIV-1 Nef dimers short-circuit immune receptor signaling by activating Tec-family kinases at the host cell membrane

“…In this way, Nef-mediated PI3K activation may also contribute to the relocalization of Tec family kinases from the cytoplasm to the membrane through their PH domains. Structural work has established that the PH domain of Btk interacts with the kinase domain in an autoinhibitory fashion that is relieved by phosphoinositide binding (39). Interaction with Nef may relieve this negative regulatory influence by displacing the PH domain, in a manner analogous to regulatory SH3 domain displacement associated with Nef-dependent activation of the Src-family kinase, Hck (16,40).…”

Itk promotes the integration of TCR and CD28 costimulation, through its direct substrates, SLP-76 and Gads

“…Structural studies suggest that an inactive conformation of Itk is stabilized by intramolecular interactions of its SH2 and SH3 domains, as well as by an inhibitory interaction between the N-terminal PH domain and the kinase domain [24][25][26]. Upon TCR or CD28 stimulation, PIP3 in the plasma membrane is increased.…”

“…Upon TCR or CD28 stimulation, PIP3 in the plasma membrane is increased. Elevated PIP3 binds to the Itk PH domain, relieving the inhibitory influence of the PH domain on the kinase domain [26]; moreover, bivalent binding of the SH2 and The SH2 domain of Itk is thought to bind to SLP-76 p-Y145 [25]; however, competitive binding studies suggested that it may also bind to SLP-76 p-Y113 [28], which is equivalent to murine p-Y112.…”