SH2-Dependent Autophosphorylation within the Tec Family Kinase Itk

Joseph, Rajiv; Severin, Andrew J.; Min, Lie; Fulton, D. Bruce; Andreotti, Amy H.

doi:10.1016/j.jmb.2009.06.023

Cited by 17 publications

(18 citation statements)

References 50 publications

(101 reference statements)

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“…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.…”

Section: Discussionmentioning

confidence: 78%

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

Chen

Martin

Gorenstein

et al. 2011

Molecular and Cellular Biology

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Syk is a cytoplasmic protein tyrosine kinase that regulates cellular responses mediated by a variety of membrane receptors with intracellular signaling modules known as immunoreceptor tyrosine-based activation motifs (ITAMs) (23, 52). Examples of ITAM/Syk receptors include the B and T cell receptors for antigen, the immunoglobulin receptors FcεRI, Fc␥RI, and Fc␥RIIa, the activating NK cell receptors, and integrins. When these receptors are engaged, a pair of tyrosines within the ITAM become phosphorylated to create a docking site for Syk's N-terminal pair of SH2 domains. Syk binding to the ITAM leads to Syk activation and phosphorylation on several tyrosines to generate sites that participate in protein-protein interactions. Thus, activated Syk at the site of the clustered receptor participates in the formation of a signaling complex. This "signalosome" contains multiple effector proteins that include members of the Vav family of guanine nucleotide exchange factors (GEFs) (13,14).Among the three members of the Vav family, Vav1 is restricted largely to hematopoietic cells while Vav2 and Vav3 are more widely distributed (8,55,67,77). Vav proteins have both unique and redundant functions in the immune system, as a loss of Vav1 produces severe defects in T cell development and signaling, while the loss of more than one Vav family member is needed to produce similar defects in B cells (12,17,21,75,76). Vav proteins enhance many signaling pathways mediated by Syk-associated receptors, including the activation of phospholipase C-␥ (PLC-␥) and the mobilization of intracellular calcium, the activation of phosphoinositide 3-kinase (PI3K) and Akt, the activation of the Ras/Erk pathway, the promotion of cytoskeletal rearrangements, and the inside-out activation of integrins. While Vav proteins serve as GEFs for Rac/Rho family GTPases, they also act as scaffolds, a function that promotes the assembly of signaling complexes but does not require GEF activity (66).Syk associates with Vav family proteins and phosphorylates them on tyrosines that regulate their activity (13,43,50,53,59,69,74,75). This physical association occurs between the Vav SH2 domain and the Syk linker B region, which separates the tandem SH2 domains from the catalytic domain and contains phosphotyrosines 342 and 346 (based on the numbering system for murine Syk). Syk linker B interacts with multiple binding partners depending on which tyrosines in linker B are phosphorylated and the stoichiometry of this phosphorylation (23). As such, Syk linker B harbors the unusual site of two closely space tyrosines, which are recognized by a single SH2 domain of particular Syk binding partners. These interactions influence the ability of the kinase to couple ITAM-bearing receptors to the many different intracellular signaling pathways that are regulated following receptor engagement (23,52).Vav1 SH2 adopts the typical fold of an SH2 domain, including a central ␤-sheet flanked by two ␣-helices (Protein Data Bank [PDB] designation: 2CRH). The nonaromatic hydrophobic Ile r...

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Section: Discussionmentioning

confidence: 78%

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

Chen

Martin

Gorenstein

et al. 2011

Molecular and Cellular Biology

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“…The samples were boiled, separated by SDS-PAGE and western blotted with the Anti Btk phosphoY551 antibody (BD Biosciences), anti-FLAG (Sigma) antibody or anti-His (Upstate) antibody as described previously (23). The Anti Btk phosphoY551 antibody is also used for detection of phosphorylation on Itk Y511 .…”

Section: Methodsmentioning

confidence: 99%

Controlling the Activity of the Tec Kinase Itk by Mutation of the Phenylalanine Gatekeeper Residue

Joseph

Andreotti

2010

Biochemistry

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The regulatory spine is a set of conserved residues that are assembled and disassembled upon activation and inactivation of kinases. We recently identified the regulatory spine within the immunologically important Tec family kinases and have shown that in addition to the core spine residues within the kinase domain itself, contributions from the SH2-kinase linker region result in an extended spine structure for this kinase family. Disruption of the regulatory spine, either by mutation or removal of the amino-terminal SH2-kinase linker region or mutation of core spine residues, leads to inactivation of the Tec kinases. With a focus on the Tec family members, Itk and Btk, we now show that the gatekeeper residue is also critical for the assembly of the regulatory spine. Mutation of the bulky Itk F434 gatekeeper residue to alanine or glycine inactivates Itk. The activity of the Itk F434A mutant can be recovered by a secondary-site mutation within the N-terminal lobe, specifically L432I. The Itk L432I mutation likely rescues the activity of the gatekeeper F434A mutation by promoting the assembly of the regulatory spine. We also show that mutation of the Itk and Btk gatekeeper residues to methionine is sufficient to activate the isolated kinase domains of Tec kinases in the absence of the amino-terminal SH2-kinase linker. Thus, shifting the conformational equilibrium between the assembled and disassembled states of the regulatory spine by changing the nature of the gatekeeper residue is key to regulating the activity of Tec kinases.

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“…Circular dichroism (CD) measurements of the PLC␥1 SH2C-linker variants were carried out as described in ref. 38 and NMR data acquisition followed procedures described in ref. 44.…”

Section: Methodsmentioning

confidence: 99%

“…Orange indicates residues that, when mutated, have no effect on Y783 phosphorylation and/or calcium mobilization in T cell signaling. rylation of the neighboring Y180 (38). The chemical similarities between the docking sites on the PLC␥1 and Itk SH2 domains, and the fact that both domains are the target of the same kinase, suggest the possibility that both substrates interact with the same or closely related surfaces on the Itk kinase domain.…”

Section: Mutation Of the Sh2c Domain In Full-length Plc␥1 Attenuates mentioning

confidence: 99%

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

Min

Joseph

Fulton

et al. 2009

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

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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...

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SH2-Dependent Autophosphorylation within the Tec Family Kinase Itk

Cited by 17 publications

References 50 publications

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

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

Controlling the Activity of the Tec Kinase Itk by Mutation of the Phenylalanine Gatekeeper Residue

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

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