Tyrosine phosphorylation of 3BP2 is indispensable for the interaction with VAV3 in chicken DT40 cells

Adaptor protein c-Abl SH3 domain-binding protein-2 (3BP2) is known to play regulatory roles in immunoreceptor-mediated signal transduction. We have previously demonstrated that Tyr¹⁷⁴, Tyr¹⁸³ and Tyr⁴⁴⁶ in mouse 3BP2 are predominantly phosphorylated by Syk, and the phosphorylation of Tyr¹⁸³ and the Src homology 2 (SH2) domain of mouse 3BP2 are critical for B cell receptor (BCR)-induced activation of nuclear factor of activated T cells (NFAT) in human B cells. In this report, we have shown that Syk, but not Abl family protein-tyrosine kinases, is critical for BCR-mediated tyrosine phosphorylation of 3BP2 in chicken DT40 cells. Mutational analysis showed that Tyr¹⁷⁴, Tyr¹⁸³ and Tyr⁴²⁶ of chicken 3BP2 are the major phosphorylation sites by Syk and the SH2 domain of 3BP2 is critical for tyrosine phosphorylation. In addition, phosphorylation of Tyr⁴²⁶ is required for the inducible interaction with the SH2 domain of Vav3. Moreover, the expression of the mutant form of 3BP2 in which Tyr⁴²⁶ was substituted to Phe resulted in the reduction in BCR-mediated Rac1 activation, when compared with the case of wild-type. Altogether, these data suggest that 3BP2 is involved in the activation of Rac1 through the regulation of Vav3 by Syk-dependent phosphorylation of Tyr⁴²⁶ following BCR stimulation.     

Actin-binding protein 1 regulates B cell receptor-mediated antigen processing and presentation in response to B cell receptor activation

The BCR serves as both signal transducer and Ag transporter. Binding of Ags to the BCR induces signaling cascades and Ag processing and presentation, two essential cellular events for B cell activation. BCR-initiated signaling increases BCR-mediated Ag-processing efficiency by increasing the rate and specificity of Ag transport. Previous studies showed a critical role for the actin cytoskeleton in these two processes. In this study, we found that actin-binding protein 1 (Abp1/HIP-55/SH3P7) functioned as an actin-binding adaptor protein, coupling BCR signaling and Ag-processing pathways with the actin cytoskeleton. Gene knockout of Abp1 and overexpression of the Src homology 3 domain of Abp1 inhibited BCR-mediated Ag internalization, consequently reducing the rate of Ag transport to processing compartments and the efficiency of BCR-mediated Ag processing and presentation. BCR activation induced tyrosine phosphorylation of Abp1 and translocation of both Abp1 and dynamin 2 from the cytoplasm to plasma membrane, where they colocalized with the BCR and cortical F-actin. Mutations of the two tyrosine phosphorylation sites of Abp1 and depolymerization of the actin cytoskeleton interfered with BCR-induced Abp1 recruitment to the plasma membrane. The inhibitory effect of a dynamin proline-rich domain deletion mutant on the recruitment of Abp1 to the plasma membrane, coimmunoprecipitation of dynamin with Abp1, and coprecipitation of Abp1 with GST fusion of the dyanmin proline-rich domain demonstrate the interaction of Abp1 with dynamin 2. These results demonstrate that the BCR regulates the function of Abp1 by inducing Abp1 phosphorylation and actin cytoskeleton rearrangement, and that Abp1 facilitates BCR-mediated Ag processing by simultaneously interacting with dynamin and the actin cytoskeleton.     

Distinct Phosphotyrosine-dependent Functions of the ShcA Adaptor Protein Are Required for Transforming Growth Factor β (TGFβ)-induced Breast Cancer Cell Migration,Invasion, and Metastasis

The ErbB2 and TGFβ signaling pathways cooperate to promote the migratory, invasive, and metastatic behavior of breast cancer cells. We previously demonstrated that ShcA is necessary for these synergistic interactions. Through a structure/function approach, we now show that the phosphotyrosine-binding, but not the Src homology 2, domain of ShcA is required for TGFβ-induced migration and invasion of ErbB2-expressing breast cancer cells. We further demonstrate that the tyrosine phosphorylation sites within ShcA (Tyr²³⁹/Tyr²⁴⁰ and Tyr³¹³) transduce distinct and non-redundant signals that promote these TGFβ-mediated effects. We demonstrate that Grb2 is required specifically downstream of Tyr³¹³, whereas the Tyr²³⁹/Tyr²⁴⁰ phosphorylation sites require the Crk adaptor proteins to augment TGFβ-induced migration and invasion. Furthermore, ShcA Tyr³¹³ phosphorylation enhances tumor cell survival, and ShcA Tyr²³⁹/Tyr²⁴⁰ signaling promotes endothelial cell recruitment into ErbB2-expressing breast tumors in vivo, whereas all three ShcA tyrosine residues are required for efficient breast cancer metastasis to the lungs. Our data uncover a novel ShcA-dependent signaling axis downstream of TGFβ and ErbB2 that requires both the Grb2 and Crk adaptor proteins to increase the migratory and invasive properties of breast cancer cells. In addition, signaling downstream of specific ShcA tyrosine residues facilitates the survival, vascularization, and metastatic spread of breast tumors.     

CD72 negatively regulates signaling through the antigen receptor of B cells

The immunoreceptor tyrosine-based inhibition motif (ITIM) is found in various membrane molecules such as CD22 and the low-affinity Fc receptor for IgG in B cells and the killer cell-inhibitory receptor and Ly-49 in NK cells. Upon tyrosine phosphorylation at the ITIMs, these molecules recruit SH2 domain-containing phosphatases such as SH2-containing tyrosine phosphatase-1 and negatively regulate cell activity. The B cell surface molecule CD72 carries an ITIM and an ITIM-like sequence. We have previously shown that CD72 is phosphorylated and recruits SH2-containing tyrosine phosphatase-1 upon cross-linking of the Ag receptor of B cells (BCR). However, whether CD72 modulates BCR signaling has not yet been elucidated. In this paper we demonstrate that expression of CD72 down-modulates both extracellular signal-related kinase (ERK) activation and Ca2+ mobilization induced by BCR ligation in the mouse B lymphoma line K46micromlambda, whereas BCR-mediated ERK activation was not reduced by the ITIM-mutated form of CD72. Moreover, coligation with CD72 with BCR reduces BCR-mediated ERK activation in spleen B cells of normal mice. These results indicate that CD72 negatively regulates BCR signaling. CD72 may play a regulatory role in B cell activation, probably by setting a threshold for BCR signaling.     

HCV NS5A Protein Containing Potential Ligands for Both Src Homology 2 and 3 Domains Enhances Autophosphorylation of Src Family Kinase Fyn in B Cells

Hepatitis C virus (HCV) infects B lymphocytes and induces mixed cryoglobulinemia and B cell non-Hodgkin''s lymphoma. The molecular mechanism for the pathogenesis of HCV infection-mediated B cell disorders remains obscure. To identify the possible role for HCV nonstructural 5A (NS5A) protein in B cells, we generated the stable B cell lines expressing Myc-His tagged NS5A. Immunoprecipitation study in the presence or absence of pervanadate (PV) implied that NS5A was tyrosine phosphorylated by pervanadate (PV) treatment of the cells. Therefore we examined pull-down assay by using glutathione S-transferase (GST)-fusion proteins of various Src homology 2 (SH2) domains, which associates with phosphotyrosine within a specific amino acid sequence. The results showed that NS5A specifically bound to SH2 domain of Fyn from PV-treated B cells in addition to Src homology 3 (SH3) domain. Substitution of Arg¹⁷⁶ to Lys in the SH2 domain of Fyn abrogated this interaction. Deletion mutational analysis demonstrated that N-terminal region of NS5A was not required for the interaction with the SH2 domain of Fyn. Tyr³³⁴ was identified as a tyrosine phosphorylation site in NS5A. Far-western analysis revealed that SH2 domain of Fyn directly bound to NS5A. Fyn and NS5A were colocalized in the lipid raft. These results suggest that NS5A directly binds to the SH2 domain of Fyn in a tyrosine phosphorylation-dependent manner. Lastly, we showed that the expression of NS5A in B cells increased phosphorylation of activation loop tyrosine in the kinase domain of Fyn. NS5A containing ligand for both SH2 and SH3 domains enhances an aberrant autophosphorylation and kinase activity of Fyn in B cells.     

Binding and Function of Phosphotyrosines of the Ephrin A2 (EphA2) Receptor Using Synthetic Sterile α Motif (SAM) Domains

The sterile α motif (SAM) domain of the ephrin receptor tyrosine kinase, EphA2, undergoes tyrosine phosphorylation, but the effect of phosphorylation on the structure and interactions of the receptor is unknown. Studies to address these questions have been hindered by the difficulty of obtaining site-specifically phosphorylated proteins in adequate amounts. Here, we describe the use of chemically synthesized and specifically modified domain-length peptides to study the behavior of phosphorylated EphA2 SAM domains. We show that tyrosine phosphorylation of any of the three tyrosines, Tyr⁹²¹, Tyr⁹³⁰, and Tyr⁹⁶⁰, has a surprisingly small effect on the EphA2 SAM structure and stability. However, phosphorylation at Tyr⁹²¹ and Tyr⁹³⁰ enables differential binding to the Src homology 2 domain of the adaptor protein Grb7, which we propose will lead to distinct functional outcomes. Setting up different signaling platforms defined by selective interactions with adaptor proteins thus adds another level of regulation to EphA2 signaling.     

Tyrosine phosphorylation of BCR by FPS/FES protein-tyrosine kinases induces association of BCR with GRB-2/SOS.

The human bcr gene encodes a protein with serine/threonine kinase activity, CDC24/dbl homology, a GAP domain, and an SH2-binding region. However, the precise physiological functions of BCR are unknown. Coexpression of BCR with the cytoplasmic protein-tyrosine kinase encoded by the c-fes proto-oncogene in Sf-9 cells resulted in stable BCR-FES protein complex formation and tyrosine phosphorylation of BCR. Association involves the SH2 domain of FES and a novel binding domain localized to the first 347 amino acids of the FES N-terminal region. Deletion of the homologous N-terminal BCR-binding domain from v-fps, a fes-related transforming oncogene, abolished transforming activity and tyrosine phosphorylation of BCR in vivo. Tyrosine phosphorylation of BCR in v-fps-transformed cells induced its association with GRB-2/SOS, the RAS guanine nucleotide exchange factor complex. These data provide evidence that BCR couples the cytoplasmic protein-tyrosine kinase and RAS signaling pathways.     

Involvement of Grb2 Adaptor Protein in Nucleophosmin-Anaplastic Lymphoma Kinase (NPM-ALK)-mediated Signaling and Anaplastic Large Cell Lymphoma Growth

Most anaplastic large cell lymphomas (ALCL) express oncogenic fusion proteins derived from chromosomal translocations or inversions of the anaplastic lymphoma kinase (ALK) gene. Frequently ALCL carry the t(2;5) translocation, which fuses the ALK gene to the nucleophosmin (NPM1) gene. The transforming activity mediated by NPM-ALK fusion induces different pathways that control proliferation and survival of lymphoma cells. Grb2 is an adaptor protein thought to play an important role in ALK-mediated transformation, but its interaction with NPM-ALK, as well as its function in regulating ALCL signaling pathways and cell growth, has never been elucidated. Here we show that active NPM-ALK, but not a kinase-dead mutant, bound and induced Grb2 phosphorylation in tyrosine 160. An intact SH3 domain at the C terminus of Grb2 was required for Tyr¹⁶⁰ phosphorylation. Furthermore, Grb2 did not bind to a single region but rather to different regions of NPM-ALK, mainly Tyr^152–156, Tyr⁵⁶⁷, and a proline-rich region, Pro^415–417. Finally, shRNA knockdown experiments showed that Grb2 regulates primarily the NPM-ALK-mediated phosphorylation of SHP2 and plays a key role in ALCL cell growth.     

Tyrosine phosphorylation of shc in response to B cell antigen receptor engagement depends on the SHIP inositol phosphatase

Tyrosine phosphorylation of Shc in response to B cell Ag receptor (BCR) engagement creates binding sites for the Src homology 2 (SH2) domain of Grb2. This facilitates the recruitment of both Grb2. Sos complexes and Grb2. SHIP complexes to the plasma membrane where Sos can activate Ras and SH2 domain-containing inositol phosphatase (SHIP) can dephosphorylate phosphatidylinositol 3,4,5-trisphosphate. Given the importance of Shc phosphorylation, we investigated the mechanism by which the BCR stimulates this response. We found that both the SH2 domain and phosphotyrosine-binding (PTB) domain of Shc are important for BCR-induced tyrosine phosphorylation of Shc and the subsequent binding of Grb2 to Shc. The unexpected finding that the PTB domain of Shc is required for Shc phosphorylation was investigated further. Because the major ligand for the Shc PTB domain is SHIP, we asked whether the interaction of Shc with SHIP was required for BCR-induced tyrosine phosphorylation of Shc. Using SHIP-deficient DT40 cells, we show that SHIP is necessary for the BCR to induce significant levels of Shc tyrosine phosphorylation. BCR-induced tyrosine phosphorylation of Shc could be restored in the these cells by expressing wild-type SHIP but not by expressing a mutant form of SHIP that cannot bind to Shc. This suggests that BCR-induced tyrosine phosphorylation of Shc may depend on the binding of SHIP to the Shc PTB domain. Thus, we have described a novel role for SHIP in BCR signaling, promoting the tyrosine phosphorylation of Shc.     

14-3-3:Shc Scaffolds Integrate Phosphoserine and Phosphotyrosine Signaling to Regulate Phosphatidylinositol 3-Kinase Activation and Cell Survival

Integrated cascades of protein tyrosine and serine/threonine phosphorylation play essential roles in transducing signals in response to growth factors and cytokines. How adaptor or scaffold proteins assemble signaling complexes through both phosphotyrosine and phosphoserine/threonine residues to regulate specific signaling pathways and biological responses is unclear. We show in multiple cell types that endogenous 14-3-3ζ is phosphorylated on Tyr¹⁷⁹ in response to granulocyte macrophage colony-stimulating factor. Importantly, 14-3-3ζ can function as an intermolecular bridge that couples to phosphoserine residues and also directly binds the SH2 domain of Shc via Tyr¹⁷⁹. The assembly of these 14-3-3:Shc scaffolds is specifically required for the recruitment of a phosphatidylinositol 3-kinase signaling complex and the regulation of CTL-EN cell survival in response to cytokine. The biological significance of these findings was further demonstrated using primary bone marrow-derived mast cells from 14-3-3ζ^-/- mice. We show that cytokine was able to promote Akt phosphorylation and viability of primary mast cells derived from 14-3-3ζ^-/- mice when reconstituted with wild type 14-3-3ζ, but the Akt phosphorylation and survival response was reduced in cells reconstituted with the Y179F mutant. Together, these results show that 14-3-3:Shc scaffolds can act as multivalent signaling nodes for the integration of both phosphoserine/threonine and phosphotyrosine pathways to regulate specific cellular responses.The ability of a cell to respond to extrinsic stimuli critically hinges on its ability to regulate specific intracellular protein-protein interactions in a reversible manner. Such signals are relayed within the cell through the assembly of signaling complexes that are built using protein scaffolds. One important mechanism by which this occurs is via the binding of Src homology 2 (SH2)⁵ or phosphotyrosine-binding (PTB) domains to phosphotyrosine residues (1, 2). Importantly, the ability of individual SH2 or PTB domains to recognize specific phosphotyrosine motifs in different proteins enables the assembly of purpose-built signaling complexes that promote signaling via specific pathways (3). In some cases, signaling proteins not only contain more than one SH2 and/or PTB domain but are also themselves tyrosine-phosphorylated, leading to a network of phosphotyrosine-mediated protein-protein interactions.Although less well studied, phosphoserine/threonine-binding proteins are also important for the assembly of signaling complexes. For example, the 14-3-3 family of proteins is able to bind phosphoserine/threonine residues in a sequence-specific context (RSX(S/T)XP and RXXX(S/T)XP, where (S/T) is phosphoserine/threonine) (4, 5). The 14-3-3 proteins have been proposed to function as “modifiers” or “sequestrators”; however, because of their dimeric structure, they have also been proposed to function as “adaptor” or “scaffold” proteins through their ability to bring together two serine/threonine phosphorylated proteins (4–7). Additionally, a number of phosphoserine/threonine-binding modules such as tryptophan-tryptophan (WW), Forkhead-associated (FHA), Polo box (PBD), and BRCA1 C-terminal (BRCT) domains have been shown to interact with phosphoserine/threonine residues within a sequence-specific context and have also been proposed to be important for the assembly of multi-protein signaling complexes (8).The genes/cassettes encoding each phosphotyrosine- and phosphoserine/threonine-binding protein/module arose as a separate evolutionary event, and the DNA encoding these modules has been subject to frequent duplication and shuffling. For example, the 14-3-3 family of proteins is ubiquitously expressed in mammalian tissues and is composed of seven different isoforms, each encoded by a separate gene (6). In addition, duplication and shuffling of SH2, PTB, WW, FHA, PBD, and BCRT cassettes has led to their wide distribution among signaling proteins. Yet, despite the frequent duplication and shuffling of the DNA encoding these domains throughout evolution, proteins that contain both a phosphotyrosine-binding cassette (e.g. SH2 or PTB) and a phosphoserine/threonine-binding cassette (e.g. 14-3-3, WW, FHA, PBD, and BCRT) have not been identified. This is perhaps surprising given the highly integrated nature of phosphotyrosine and phosphoserine/threonine signaling and would suggest that alternative strategies to regulate integration are at play.We show here that 14-3-3ζ is tyrosine-phosphorylated, enabling it to interact with Shc and provide a scaffold for the assembly of signaling complexes via both phosphoserine/threonine and phosphotyrosine residues. Our results show that Tyr¹⁷⁹ of 14-3-3ζ directly binds to the SH2 domain of Shc and that this interaction is critical for the assembly of a phosphatidylinositol (PI) 3-kinase signaling complex in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation. Moreover, we show that Tyr¹⁷⁹ of 14-3-3ζ is necessary and sufficient for the ability of GM-CSF to regulate PI 3-kinase and cell survival in the CTL-EN line. Furthermore, reconstitution of primary mast cells derived from 14-3-3ζ^-/- mice with wild type (wt) or mutant 14-3-3ζ demonstrated an important role for Tyr¹⁷⁹ in cytokine-mediated Akt phosphorylation and cell survival. These multivalent 14-3-3:Shc scaffolds provide a novel mechanism by which phosphoserine/threonine and phosphotyrosine pathways can be integrated for the regulation of specific cellular responses.     

MIST functions through distinct domains in immunoreceptor signaling in the presence and absence of LAT.

MIST (also termed Clnk) is an adaptor protein structurally related to SLP-76 and BLNK/BASH/SLP-65 hematopoietic cell-specific adaptor proteins. By using the BLNK-deficient DT40 chicken B cell system, we demonstrated MIST functions through distinct intramolecular domains in immunoreceptor signaling depending on the availability of linker for activation of T cells (LAT). MIST can partially restore the B cell antigen receptor (BCR) signaling in the BLNK-deficient cells, which requires phosphorylation of the two N-terminal tyrosine residues. Co-expression of LAT with MIST fully restored the BCR signaling and dispenses with the requirement of the two tyrosines in MIST for BCR signaling. However, some other tyrosine(s), as well as the Src homology (SH) 2 domain and the two proline-rich regions in MIST, is still required for full reconstitution of the BCR signaling, in cooperation with LAT. The C-terminal proline-rich region of MIST is dispensable for the LAT-aided full restoration of MAP kinase activation, although it is responsible for the interaction with LAT and for the localization in glycolipid-enriched microdomains. On the other hand, the N-terminal proline-rich region, which is a binding site of the SH3 domain of phospholipase Cgamma, is essential for BCR signaling. These results revealed a marked plasticity of MIST function as an adaptor in the cell contexts with or without LAT.     

PTK6 Inhibits Down-regulation of EGF Receptor through Phosphorylation of ARAP1

PTK6 (also known as Brk) is a non-receptor-tyrosine kinase containing SH3, SH2, and catalytic domains, that is expressed in more than 60% of breast carcinomas but not in normal mammary tissues. To analyze PTK6-interacting proteins, we have expressed Flag-tagged PTK6 in HEK293 cells and performed co-immunoprecipitation assays with Flag antibody-conjugated agarose. A 164-kDa protein in the precipitated fraction was identified as ARAP1 (also known as centaurin δ-2) by MALDI-TOF mass analysis. ARAP1 associated with PTK6 in an EGF/EGF receptor (EGFR)-dependent manner. In addition, the SH2 domain of PTK6, particularly the Arg¹⁰⁵ residue that contacts the phosphate group of the tyrosine residue, was essential for the association. Moreover, PTK6 phosphorylated residue Tyr²³¹ in the N-terminal domain of ARAP1. Expression of ARAP1, but not of the Y231F mutant, inhibited the down-regulation of EGFR in HEK293 cells expressing PTK6. Silencing of endogenous PTK6 expression in breast carcinoma cells decreased EGFR levels. These results demonstrate that PTK6 enhances EGFR signaling by inhibition of EGFR down-regulation through phosphorylation of ARAP1 in breast cancer cells.     

Recruitment of phospholipase Cγ1 to the non-structural membrane protein pK15 of Kaposi Sarcoma-associated herpesvirus promotes its Src-dependent phosphorylation

Kaposi Sarcoma-associated herpesvirus (KSHV) causes three human malignancies, Kaposi Sarcoma (KS), Primary Effusion Lymphoma (PEL) and the plasma cell variant of multicentric Castleman’s Disease (MCD), as well as an inflammatory cytokine syndrome (KICS). Its non-structural membrane protein, pK15, is among a limited set of viral proteins expressed in KSHV-infected KS tumor cells. Following its phosphorylation by Src family tyrosine kinases, pK15 recruits phospholipase C gamma 1 (PLCγ1) to activate downstream signaling cascades such as the MEK/ERK, NFkB and PI3K pathway, and thereby contributes to the increased proliferation and migration as well as the spindle cell morphology of KSHV-infected endothelial cells. Here, we show that a phosphorylated Y⁴⁸¹EEVL motif in pK15 preferentially binds into the PLCγ1 C-terminal SH2 domain (cSH2), which is involved in conformational changes occurring during the activation of PLCγ1 by receptor tyrosine kinases. We determined the crystal structure of a pK15 12mer peptide containing the phosphorylated pK15 Y⁴⁸¹EEVL motif in complex with a shortened PLCγ1 tandem SH2 (tSH2) domain. This structure demonstrates that the pK15 peptide binds to the PLCγ1 cSH2 domain in a position that is normally occupied by the linker region connecting the PLCγ1 cSH2 and SH3 domains. We also show that longer pK15 peptides containing the phosphorylated pK15 Y⁴⁸¹EEVL motif can increase the Src-mediated phosphorylation of the PLCγ1 tSH2 region in vitro. This pK15-induced increase in Src-mediated phosphorylation of PLCγ1 can be inhibited with the small pK15-derived peptide which occupies the PLCγ1 cSH2 domain. Our findings thus suggest that pK15 may act as a scaffold protein to promote PLCγ1 activation in a manner similar to the cellular scaffold protein SLP-76, which has been shown to promote PLCγ1 activation in the context of T-cell receptor signaling. Reminiscent of its positional homologue in Epstein-Barr Virus, LMP2A, pK15 may therefore mimic aspects of antigen-receptor signaling. Our findings also suggest that it may be possible to inhibit the recruitment and activation of PLCγ1 pharmacologically.     

Tyrosine Phosphorylation of the Lyn Src Homology 2 (SH2) Domain Modulates Its Binding Affinity and Specificity

Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y¹⁹⁴ impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y¹⁹⁴ on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases.Src homology 2 (SH2) domains are modular protein structures that are important for signal transduction due to their ability to bind phosphotyrosine (pY)-containing polypeptides within defined amino acid sequence motifs (1). SH2 domains are found in various signaling enzymes and adaptor proteins. Given the reversibility of protein tyrosine phosphorylation and the affinity of SH2-pY binding, the interactions of SH2 domains are inherently dynamic and diverse. Indeed, selective, transient binding to pY motifs is a key mechanism through which intracellular signaling networks are dynamically assembled, localized, and regulated. In addition to mediating protein interactions in trans, SH2 domains bind intramolecularly (2). For example, in Src family kinases (SFKs), the SH2 domain binds in cis to the phosphorylated C-terminal tail as a mechanism to constrain and thereby auto-inhibit the intervening tyrosine kinase domain (3, 4). As well, SH2 domains of cytoplasmic tyrosine kinases have been shown to affect the kinase activity of adjacent kinase domains through allosteric interactions (5). The SFKs are therefore highly regulated as a function of their SH2 domains, which exist in dynamic equilibrium between intra- and intermolecular interactions (6). Hence, as discussed by Pawson (7), the transient and diverse interactions of an SH2 domain can regulate signaling enzymes and constitutes a major mechanism of signal transduction in response to extracellular signals.The structure of the SH2 domain has been extensively characterized. At its core is a conserved antiparallel β-sheet sandwiched between two α-helices (8). SH2 domains bind phosphotyrosine-containing peptides in an extended conformation across the central β-sheet, with the pY residue inserted in a deep recognition pocket formed by conserved residues from strands βB, βC, and βD, helix αA, and the phosphate binding loop. Peptide binding specificity is determined by more variable binding surfaces on the SH2 domain, which recognize residues C-terminal to the pY residue. For the SFK SH2 domains, the three residues C-terminal to the pY residue (pY+1,+2,+3) are dominant determinants of specificity (9, 10), with the domain binding most tightly to sequences containing the motif pYEEI (11, 12). The hydrophobic pY+3 residue inserts in a deep hydrophobic specificity pocket defined by residues of the EF and BG loops (8, 13, 14). Indeed, structural analysis of the SH2 domain revealed that the configuration of the EF and BG loops is critical in dictating SH2 domain specificity by shaping the ligand-binding surface and controlling accessibility of the pY+3 binding pocket (15). Mutation of a single residue of the EF loop can drastically impact peptide binding specificity by altering the pY+3 pocket (15–17), indicating the importance of the pY+3 pocket in substrate selectivity for the SFK SH2 domains.In addition to binding pY-containing polypeptides, SH2 domains themselves may be modulated by phosphorylation. For example, phosphorylation of the Src SH2 domain at conserved Y²¹³ resulted in activation of the cognate kinase domain, possibly by impairing SH2 binding to the phosphorylated C-terminal tail (18). Similarly, phosphorylation of Lck at the equivalent SH2 residue (Y¹⁹²) generally reduced binding to pY-peptides and proteins (19). Phosphorylation at S⁶⁹⁰ in the SH2 domain of the p85α subunit of PI 3-kinase decreased its affinity for pY-containing proteins and promoted feedback inhibition of PI 3-kinase and Akt in response to cellular starvation (20). Conversely, tyrosine phosphorylation of the tensin-3 SH2 domain stimulated substrate binding and biological activity (21). Therefore, phosphorylation of SH2 domains appears to be a general mechanism for modulating their binding properties.Here, we report that Y¹⁹⁴ in the SH2 domain of the SFK Lyn, a residue conserved in SFK SH2 domains, is frequently phosphorylated in hematopoietic and other cancers. In vitro protein and peptide interactions with the Lyn SH2 domain were affected by this phosphorylation. Our results suggest that tyrosine phosphorylation of the SFK SH2 domain modulates both its binding affinity and specificity and may constitute another layer of regulation in signaling networks.     

Two closely spaced tyrosines regulate NFAT signaling in B cells via Syk association with Vav

Activated Syk, an essential tyrosine kinase in B cell signaling, interacts with Vav guanine nucleotide exchange factors and regulates Vav activity through tyrosine phosphorylation. The Vav SH2 domain binds Syk linker B by an unusual recognition of two closely spaced Syk tyrosines: Y342 and Y346. The binding affinity is highest when both Y342 and Y346 are phosphorylated. An investigation in B cells of the dependence of Vav phosphorylation and NFAT activation on phosphorylation of Y342 and Y346 finds that cellular response levels match the relative binding affinities of the Vav1 SH2 domain for singly and doubly phosphorylated linker B peptides. This key result suggests that the uncommon recognition determinant of these two closely spaced tyrosines is a limiting factor in signaling. Interestingly, differences in affinities for binding singly and doubly phosphorylated peptides are reflected in the on rate, not the off rate. Such a control mechanism would be highly effective for regulating binding among competing Syk binding partners. The nuclear magnetic resonance (NMR) structure of Vav1 SH2 in complex with a doubly phosphorylated linker B peptide reveals diverse conformations associated with the unusual SH2 recognition of two phosphotyrosines. NMR relaxation indicates compensatory changes in loop fluctuations upon binding, with implications for nonphosphotyrosine interactions of Vav1 SH2.