Scaffold Protein SLP-76 Primes PLCγ1 for Activation by ITK-Mediated Phosphorylation

Devkota, Sujan; Joseph, Rajiv; Min, Lie; Fulton, D. Bruce; Andreotti, Amy H.

doi:10.1016/j.jmb.2015.04.012

Cited by 17 publications

(17 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, we have not addressed the subsequent recruitment to SLP-76 of Nck, Vav, and Itk. In this system Nck and Vav are thought to have a scaffolding function, and Itk, a member of the Tec family of PTKs, is required to phosphorylate PLC-γ1 on tyrosine 783, an event needed for the activation of the enzyme (39,40). In addition to the requirement for these binding events for PLC-γ1 activation, it is possible that these additional molecules might affect the interaction and dynamics of the fourmolecule complex that is the subject of the current study.…”

Section: Discussionmentioning

confidence: 99%

Cooperative assembly of a four-molecule signaling complex formed upon T cell antigen receptor activation

Manna

Zhao

Wada

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The T cell antigen receptor encounters foreign antigen during the immune response. Receptor engagement leads to activation of specific protein tyrosine kinases, which then phosphorylate multiple enzymes and adapter proteins. One such enzyme, phospholipase-Cγ1, is responsible for cleavage of a plasma membrane lipid substrate, a phosphoinositide, into two second messengers, diacylglycerol, which activates several enzymes including protein kinase C, and an inositol phosphate, which induces intracellular calcium elevation. In T cells, phospholipase-Cγ1 is recruited to the plasma membrane as part of a four-protein complex containing three adapter molecules. We have used recombinant proteins and synthetic phosphopeptides to reconstitute this quaternary complex in vitro. Extending biophysical tools to study concurrent interactions of the four protein components, we demonstrated the formation and determined the composition of the quaternary complex using multisignal analytical ultracentrifugation, and we characterized the thermodynamic driving forces of assembly by isothermal calorimetry. We demonstrate that the four proteins reversibly associate in a circular arrangement of binding interfaces, each protein interacting with two others. Three interactions are of high affinity, and the fourth is of low affinity, with the assembly of the quaternary complex exhibiting significant enthalpy-entropy compensation as in an entropic switch. Formation of this protein complex enables subsequent recruitment of additional molecules needed to activate phospholipase-Cγ1. Understanding the formation of this complex is fundamental to full characterization of a central pathway in T cell activation. Such knowledge is critical to developing ways in which this pathway can be selectively inhibited.phospholipase C | protein complex | signal transduction | T cell antigen receptor | tyrosine phosphorylation T hree decades of investigation have resulted in a description of T cell antigen receptor (TCR) engagement and the biochemical events that follow. The binding of the TCR ligand, an antigenic peptide presented by a protein encoded by the major histocompatibility complex, leads to the activation of associated and recruited protein tyrosine kinases (PTKs) (1). Critical substrates of these PTKs include the adapter molecules, linker for activation of T cells (LAT), and Src homology 2 (SH2) domaincontaining leukocyte protein of 76kDa (SLP-76) (2, 3). The phosphorylation of tyrosine residues on these adapters initiates binding of many other adapters and enzymes to generate molecular complexes of great heterogeneity (4). In this manner enzymes such as phospholipase C-γ1 (PLC-γ1) and son of sevenless homolog 1 (SOS1) are recruited to the plasma membrane where they encounter their substrates. The products that are a consequence of these enzyme activations-diacylglycerol, elevated intracellular calcium, and activated Ras-have complex intracellular effects during the process of T cell activation (5, 6).Sites of protein-protein interactions between these v...

show abstract

Section: Discussionmentioning

confidence: 99%

Cooperative assembly of a four-molecule signaling complex formed upon T cell antigen receptor activation

Manna

Zhao

Wada

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…The new report from Devkota et al addresses highly related aspects of PLCγ1 regulation but in a different signalling context [5]. Significantly, these researchers used heteronuclear NMR spectroscopy to provide evidence that is consistent with the idea that the canonical cSH2 binding site is occupied in cis by the adjacent Tyr783-containing linker segment, even in the absence of phosphorylation.…”

mentioning

confidence: 92%

“…As that work has progressed and inexorably expanded away from modular domains towards multiprotein signalling assemblies, it has become clear that the simple plug-in-socket picture fails to completely capture a much more nuanced picture for the regulatory role that SH2 domains play, both in three dimensions (structure) and the fourth, time dimension (function) [4]. It is in that vein that the work of Devkota et al, reported in this issue [5], describes aspects of the interaction between the C-terminal SH2 domain of phosphatidylinositol PLCγ1 (phospholipase C γ 1) and the scaffolding protein SLP-76 (SH2 domaincontaining leukocyte protein, 76 kDa).Phospholipases C (PLCs) act to convert membranebound phosphatidylinositol-4,5-bisphosphate PIP 2 phospholipids into the second messengers diacylglycerol and inositol-1,4,5-trisphosphate IP 3 that effect activation of protein kinases C and calcium signalling, respectively [6][7][8]. In mammals, there are six families of PLCs (β, γ, δ, ε, η and ζ).…”

mentioning

confidence: 98%

mentioning

confidence: 98%

See 1 more Smart Citation

Exposed: The Many and Varied Roles of Phospholipase C γ SH2 Domains

Driscoll

2015

Journal of Molecular Biology

View full text Add to dashboard Cite

“…In the PLC γ resting state, the extent to which the cSH2 domain is “available” for contact with the enzyme core and upstream agonists is unclear. In this context, the binding status of the cSH2 C-terminus, which is equivalent to the cSH2-SH3 linker and harbors the Tyr771, Tyr775, and Tyr783 phosphorylation sites, plays a particular role; recent studies proposed that Tyr783 phosphorylation by ITK kinase and Tyr771 phosphorylation by FGFR2 kinase would occur because of an enhancement of C-terminus availability ( 12 , 16 ). Although tandem SH2 crystal structures suggest that the cSH2 C-terminus might indeed be unbound in the nonphosphorylated state ( 10 ), a recent NMR study comparing constructs with and without an unmodified C-terminal extension suggested that the C-terminus is bound even in the nonphosphorylated state ( 16 ).…”

Section: Introductionmentioning

confidence: 99%

Dynamic Allostery in PLCγ1 and Its Modulation by a Cancer Mutation Revealed by MD Simulation and NMR

Koss

Bunney

Esposito

et al. 2018

Biophysical Journal

View full text Add to dashboard Cite

Phosphatidylinositol phospholipase Cγ (PLCγ) is an intracellular membrane-associated second-messenger signaling protein activated by tyrosine kinases such as fibroblast growth factor receptor 1. PLCγ contains the regulatory γ-specific array (γSA) comprising a tandem Src homology 2 (SH2) pair, an SH3 domain, and a split pleckstrin homology domain. Binding of an activated growth factor receptor to γSA leads to Tyr783 phosphorylation and consequent PLCγ activation. Several disease-relevant mutations in γSA have been identified; all lead to elevated phospholipase activity. In this work, we describe an allosteric mechanism that connects the Tyr783 phosphorylation site to the nSH2-cSH2 junction and involves dynamic interactions between the cSH2-SH3 linker and cSH2. Molecular dynamics simulations of the tandem SH2 protein suggest that Tyr783 phosphorylation is communicated to the nSH2-cSH2 junction by modulating cSH2 binding to sections of the cSH2-SH3 linker. NMR chemical shift perturbation analyses for designed tandem SH2 constructs reveal combined fast and slow dynamic processes that can be attributed to allosteric communication involving these regions of the protein, establishing an example in which complex N-site exchange can be directly inferred from 1H,15N-HSQC spectra. Furthermore, in tandem SH2 and γSA constructs, molecular dynamics and NMR results show that the Arg687Trp mutant in PLCγ1 (equivalent to the cancer mutation Arg665Trp in PLCγ2) perturbs the dynamic allosteric pathway. This combined experimental and computational study reveals a rare example of multistate kinetics involved in a dynamic allosteric process that is modulated in the context of a disease-relevant mutation. The allosteric influences and the weakened binding of the cSH2-SH3 linker to cSH2 should be taken into account in any more holistic investigation of PLCγ regulation.

show abstract

Scaffold Protein SLP-76 Primes PLCγ1 for Activation by ITK-Mediated Phosphorylation

Cited by 17 publications

References 47 publications

Cooperative assembly of a four-molecule signaling complex formed upon T cell antigen receptor activation

Cooperative assembly of a four-molecule signaling complex formed upon T cell antigen receptor activation

Exposed: The Many and Varied Roles of Phospholipase C γ SH2 Domains

Dynamic Allostery in PLCγ1 and Its Modulation by a Cancer Mutation Revealed by MD Simulation and NMR

Contact Info

Product

Resources

About