CD6 and Linker of Activated T Cells are Potential Interaction Partners for T Cell‐Specific Adaptor Protein

The T cell‐specific adaptor protein (TSAd) contains several protein interaction domains, and is merging as a modulator of T cell activation. Several interaction partners for the TSAd proline‐rich region and phosphotyrosines have been identified, including the Src and Tec family kinases lymphocyte‐specific protein tyrosine kinase and interleukin 2‐inducible T cell kinase. Via its Src homology 2 (SH2) domain, TSAd may thus function as a link between these enzymes and other signalling molecules. However, few binding partners to the TSAd SH2 domain in T cells are hitherto known. Through the use of in silico ligand prediction, peptide spot arrays, pull‐down and immunoprecipitation experiments, we here report novel interactions between the TSAd SH2 domain and CD6 phosphotyrosine (pTyr)⁶²⁹ and linker of activated T cells (LAT) pTyr¹⁷¹, pTyr¹⁹¹ and pTyr²²⁶.     

In vitro characterization of major ligands for Src homology 2 domains derived from protein tyrosine kinases,from the adaptor protein SHC and from GTPase-activating protein in Ramos B cells

Antigen receptors of B lymphocytes transmit their activation signal to the cell interior by associating with and activation of specific non-receptor tyrosine kinases. Most of these kinases as well as other cytoplasmic effectors contain at least one Src homology 2 (SH2) domain, known to bind tyrosine-phosphorylated proteins. We examined the binding specificity of SH2 domains from different signaling molecules in B cells and found that each of the SH2 domains tested bound distinct subsets of stimulation-dependent phosphoproteins in vitro. SH2 domains from Src-like tyrosine kinases bound predominantly to the HS1 phosphoprotein. The tandem SH2 domains of the ZAP-70 tyrosine kinase bound to phosphorylated Ig-β but only weakly to Ig-α. Also the SHC-derived SH2 domain formed complexes with the tyrosine-phosphorylated Ig-α/β heterodimer, while the C- and N-terminal SH2 domains of GTPase-activating protein displayed completely different binding preferences. These results suggest that cytoplasmic effector molecules can be recruited to the activated B cell receptor in an SH2-phosphotyrosine-mediated manner. The data also provide a possible explanation for the notion that Ig-α and Ig-β might couple to different biochemical pathways.     

Mitogen activated protein (MAP) kinases: development of ATP and non-ATP dependent inhibitors

Extracellular signals regulate most of the body's physiological functions through the MAP kinase signaling pathways. These MAP kinase signaling pathways are normally under tight regulation such that activation and inactivation occurs only when needed. However, aberrant regulation observed with naturally occurring mutations in specific signaling proteins often results in constitutive activation of the MAP kinases and is involved in several pathophysiological conditions, such as cancer, neurodegeneration, and inflammation. As such, much effort has been expended to develop inhibitory molecules of the MAP kinase signaling pathways. Several compounds have been identified that inhibit MAP kinase signaling by targeting receptors or other proteins upstream of the MAP kinases. The development of specific inhibitors of the MAP kinases themselves has been less successful and only a few compounds, which interfere with ATP binding, have been identified. A common problem with kinase inhibitors that compete with ATP binding is their lack of specificity. Thus, alternative approaches to inhibit MAP kinase function are being sought. The MAP kinase proteins contain docking domains that direct the interactions with a variety of substrate proteins. Using the 3-dimensional structure of MAP kinases and computer modeling, molecules that target specific docking domains and selectively disrupt substrate interactions are being developed. This non-ATP interfering approach may allow the selective inhibition of MAP kinase substrates involved in disease processes while preserving MAP kinase functions associated with normal cells.     

Distinct binding patterns of HS1 to the Src SH2 and SH3 domains reflect possible mechanisms of recruitment and activation of downstream molecules

We previously identified a gene, LckBP1, which encodes a proteinthat binds to the Lck SH3 domain and is identical to murineHS1. Using unstimulated T lymphocytes, we further demonstratedthat Lck binds to HS1 in vivo and that HS1 is tyrosine phosphorylatedupon TCR stimulation. In the present report, we analyzed thebinding pattern of several src kinases and HS1 in greater detail.The Lck SH3 domain binds to HS1 constitutively, while the LckSH2 domain associates with HS1 only upon TCR stimulation. Asimilar binding pattern was observed with Lyn and HS1, but notwith Fyn and HS1, in which the Fyn SH3 region associates withHS1 upon TCR stimulation but the Fyn SH3 region does not associatewith HS1 regardless of TCR stimulation. Such distinct bindingpatterns of the src kinase SH2 and SH3 domains to HS1 may representa mechanism by which src family kinases select substrates andactivate particular downstream signaling pathways.     

抑癌基因Syk(L) 在乳腺癌中具有转录抑制因子功能

目的:研究移位到细胞核内抑癌基因Syk(L) 在乳腺癌中的生物学功能，探讨Syk(L) 抑制乳腺癌发展的可能机制。方法:将Syk(L)、失去激酶活性的Syk(L)以及Syk(L)各个功能域按正确读框插入到含有Gal4基因DNA结合功能域（Gal4-DBD）的pFA载体中，并在HEK293细胞中表达；利用Gal4荧光素酶报告基因的方法检测在乳腺癌细胞中Syk(L)、失去激酶活性的Syk(L)以及Syk(L)各个功能域对基因转录的影响。结果:在乳腺癌细胞MB231和MB435中，与Gal4-DBD结合的Syk(L)可以下调启动子上含有Gal4结合位点的萤光素酶基因的表达，这种调节与Syk(L)的激酶活性无关，是通过Syk(L)的SH2功能域和KD功能域完成的。结论:在乳腺癌中，Syk(L)具有转录抑制功能，该功能与Syk(L)的激酶活性无关，是通过Syk(L)的SH2功能域和激酶功能域实现的。Syk(L)在乳腺癌中的抑癌作用可能与其转录抑制功能有关。     

Role of Src kinases in mobilization of glycosylphosphatidylinositol-anchored decay-accelerating factor by Dr fimbria-positive adhering bacteria

Afa/Dr fimbriae constitute the major virulence factor of diffusely adhering Escherichia coli (Afa/Dr DAEC). After recognizing membrane-bound signaling receptors, they trigger cell responses. One of these receptors is the human decay-accelerating factor (hDAF). It has previously been reported that the binding of Afa/Dr fimbriae to hDAF quickly induces recruitment of hDAF around adhering bacteria. The aim of our study is to analyze the role of Src kinases in the Dr fimbria-induced recruitment of hDAF. Using biochemical methods and confocal microscopy followed by 3-dimensional (3D) analysis, we have shown that the activation and cell membrane targeting of Src kinases are necessary for the recruitment and organization of hDAF around adhering bacteria. We identified c-Src to be the specific kinase involved in this process. Using a set of Src-green fluorescent protein mutants, we showed that the catalytic activity and the Src homology 2 (SH2) and SH3 domains of the Src kinases are necessary for Dr fimbria-induced hDAF mobilization to occur. In addition, using mutated Dr fimbriae and a set of mutated hDAFs in which each of the complement control protein (CCP) domains had successively been deleted, we found that the aspartic acids at position 54 in the Dr fimbriae and in CCP domain 4 of hDAF played pivotal roles in the mobilization of the Src kinases and hDAF, respectively.     

Phosphorylation events during viral infections provide potential therapeutic targets

For many medically relevant viruses, there is now considerable evidence that both viral and cellular kinases play important roles in viral infection. Ultimately, these kinases, and the cellular signaling pathways that they exploit, may serve as therapeutic targets for treating patients. Currently, small molecule inhibitors of kinases are under investigation as therapy for herpes viral infections. Additionally, a number of cellular or host-directed tyrosine kinase inhibitors that have been previously FDA approved for cancer treatment are under study in animal models and clinical trials, as they have shown promise for the treatment of various viral infections as well. This review will highlight the wide range of viral proteins phosphorylated by viral and cellular kinases, and the potential for variability of kinase recognition sites within viral substrates to impact phosphorylation and kinase prediction. Research studying kinase-targeting prophylactic and therapeutic treatments for a number of viral infections will also be discussed.     

Platelet activation mediated through membrane glycoproteins: involvement of tyrosine kinases]

Platelet membrane glycoproteins such as GPIb and GPIa/IIa play important roles in platelet functional responses. They are the receptors for specific ligands (GPIb for von Willebrand factors, and GPIa/IIa for collagen), and the ligand-receptor interaction is the first step that elicits downstream intracellular activation signals which finally culminate in platelet aggregation. Although a variety of signal transduction pathways may be involved, tyrosine kinases appear to be most closely related to platelet activation mediated by membrane glycoproteins. Since its discovery in early 1980's, protein tyrosine phosphorylation catalyzed by tyrosine kinases has been recognized to play a role in regulating the cell function of various cells. Platelets have several Src family tyrosine kinases with an SH2 domain and an SH3 domain. Syk with two SH2 domains also appears to play an important role in platelet activation, especially in its early phase. The SH2 domain binds to a phosphorylated tyrosine residue of other proteins, and the SH3 domain recognizes proline-rich domains of target proteins, thus providing the anchoring sites for protein-protein interactions. In this article, some of the recent developments in the signal transduction pathways related with tyrosine kinases are introduced. Several signaling molecules involved in GPIb- or GPIa/IIa-mediated platelet activation have been identified. Interestingly, the members participating in these processes are distinct, suggesting a diversity of signal transduction mechanisms.     

Phenotypic profiling of the human genome reveals gene products involved in plasma membrane targeting of SRC kinases

SRC proteins are non-receptor tyrosine kinases that play key roles in regulating signal transduction by a diverse set of cell surface receptors. They contain N-terminal SH4 domains that are modified by fatty acylation and are functioning as membrane anchors. Acylated SH4 domains are both necessary and sufficient to mediate specific targeting of SRC kinases to the inner leaflet of plasma membranes. Intracellular transport of SRC kinases to the plasma membrane depends on microdomains into which SRC kinases partition upon palmitoylation. In the present study, we established a live-cell imaging screening system to identify gene products involved in plasma membrane targeting of SRC kinases. Based on siRNA arrays and a human model cell line expressing two kinds of SH4 reporter molecules, we conducted a genome-wide analysis of SH4-dependent protein targeting using an automated microscopy platform. We identified and validated 54 gene products whose down-regulation causes intracellular retention of SH4 reporter molecules. To detect and quantify this phenotype, we developed a software-based image analysis tool. Among the identified gene products, we found factors involved in lipid metabolism, intracellular transport, and cellular signaling processes. Furthermore, we identified proteins that are either associated with SRC kinases or are related to various known functions of SRC kinases such as other kinases and phosphatases potentially involved in SRC-mediated signal transduction. Finally, we identified gene products whose function is less defined or entirely unknown. Our findings provide a major resource for future studies unraveling the molecular mechanisms that underlie proper targeting of SRC kinases to the inner leaflet of plasma membranes.     

SH2 and SH3-containing adaptor proteins: redundant or independent mediators of intracellular signal transduction

Molecules which contain Src Homology 2 (SH2) and SH3 domains provide one of the principal ways by which signals are transduced in cells using protein–protein interactions between proline-rich motifs and SH3 domains and induced interactions between phosphotyrosine residues and SH2 domains. The simplest of SH2/SH3-containing proteins are the Crk, Grb2 and Nck adaptor proteins which contain SH2 and SH3 domains but no intrinsic catalytic activity. Whereas Grb2 connects activated receptor tyrosine kinases with Sos and activates p21^ras, recent evidence suggests that this may not be the major mechanism by which Crk and Nck signal to downstream effectors. Identification of novel binding partners for Crk, Grb2 and Nck indicate that these adaptor proteins control distinct aspects of tyrosine kinase signalling.     

Ternary complex formation of EphA4, FGFR and FRS2α plays an important role in the proliferation of embryonic neural stem/progenitor cells

EphA4 belongs to a superfamily of receptor tyrosine kinases and interacts with several molecules including fibroblast growth factor receptors (FGFRs) as we reported earlier. Several receptor tyrosine kinases, FGFRs, Trks, Alk and Ret, are currently known to transduce a signal through a docking protein, fibroblast growth factor receptor substrate 2α (FRS2α). However, nothing has been reported about the interaction of FRS2α with EphA4. Using the yeast two‐hybrid system and the in vitro binding and kinase assays, we found that the mid‐kinase region of EphA4 directly interacts with the FRS2α PTB domain upon tyrosine phosphorylation of the EphA4 juxtamembrane (JM) domain and EphA4 directly phosphorylates FRS2α. We also found that the FRS2α PTB domain and the amino‐terminal region of EphA4 bind to the amino‐ and carboxy‐terminal regions of the FGFR JM domain, respectively, suggesting that FRS2α and EphA4 interact with FGFR simultaneously. Furthermore, a kinase‐dead EphA4 mutant that constitutively binds to FGFR functions as a dominant‐negative molecule for signaling through both EphA4 and FGFR, and so does the truncated FRS2α lacking multiple tyrosine phosphorylation sites. These dominant‐negative mutants similarly inhibit the ligand‐dependent proliferation of the mouse embryonic neural stem/progenitor cells. These results suggest the formation of a ternary complex comprising EphA4, FGFR and FRS2α. The signaling complex appears to integrate the input from FGFR and EphA4, and release the output signal through FRS2α.     

Two-step TCRζ/CD3-CD4 and CD28 signaling in T cells: SH2/SH3 domains,protein-tyrosine and lipid kinases

A central question in T-cell immunity concerns the nature of intracellular signaling from the antigen receptor, the CD4/CD8 co-receptors and the CD28 antigen. Since the original discovery that T-cell receptors such as CD4 can interact with intracellular protein-tyrosine kinases such as p56^1ck, remarkable progress has been made in deciribing the signaling pathways that control T-cell growth and immune function. Here, Christopher Rudd and colleagues examine the role of protein-tyrosine kinases, SH2/SH3 domains and lipid kinases in the generation of signals from the TCRζ/CD3 complex and the CD22 antigen.     

Receptor-interacting protein (RIP) kinase family

Receptor-interacting protein (RIP) kinases are a group of threonine/serine protein kinases with a relatively conserved kinase domain but distinct non-kinase regions. A number of different domain structures, such as death and caspase activation and recruitment domain (CARD) domains, were found in different RIP family members, and these domains should be keys in determining the specific function of each RIP kinase. It is known that RIP kinases participate in different biological processes, including those in innate immunity, but their downstream substrates are largely unknown. This review will give an overview of the structures and functions of RIP family members, and an update of recent progress in RIP kinase research.     

Regulation of the interaction of Disabled-1 with CIN85 by phosphorylation with Cyclin-dependent kinase 5

Disabled-1 (Dab1) is an adaptor protein mediating Reelin signaling in neuronal migration during brain development. Cyclin-dependent kinase 5 (Cdk5)-p35 is a proline-directed Ser/Thr kinase also involved in neuronal migration. The interaction between Dab1 and Cdk5 is in need of investigation. Dab1 was phosphorylated at Ser400 and Ser491 by Cdk5 in vivo. We search for proteins that interact with Dab1 in a phosphorylation-dependent manner at these sites, and identified CIN85, an SH3-containing adaptor protein involved in endocytosis, and CPalpha/CPbeta, which are subunits of barbed end F-actin-capping proteins (CP), as proteins bound to unphosphorylated Dab1 by mass spectrometric analysis. It was shown that the PTPAPR sequence of Dab1, conforming to the PxxxPR atypical SH3-binding motif, was the binding site for SH3 domains of CIN85. The results that phosphorylation at Ser491 close to the PTPAPR sequence inhibited association with CIN85 may provide a mechanism regulating the interaction between the PxxxPR motif proteins and SH3 domains of CIN85 family proteins. Together with previous results that CIN85 regulates actin assembly, present results raise the possibility that Cdk5 modulates actin dynamics through regulation of CIN85-Dab1 interaction by the Dab1 phosphorylation.     

酪氨酸激酶Btk家族的结构和功能

Btk(Bruton'styrosinekinase,Bruton酪氨酸蛋白激酶)家族是新近发现的一类非受体酪氨酸蛋白激酶,包括Btk等5个成员。该家族成员同源程度高,包括PH、TH、SH2、SH3B及Src1等5个结构功能域,与PI3(磷脂酰肌醇-3-激酶K)、G-蛋白双受体等结合后发生激活,通过PLCg2(磷脂酶C-g2)、PKCbI(丝氨酸-苏氨酸激酶bI)等下游信号分子,参与对血管生成、细胞增殖和凋亡以及细胞运动的调节。     

SDF1α‐induced interaction of the adapter proteins Nck and HS1 facilitates actin polymerization and migration in T cells

Noncatalytic region of tyrosine kinase (Nck) is an adapter protein that comprises one SH2 (Src homology) domain and three SH3 domains. Nck links receptors and receptor‐associated tyrosine kinases or adapter proteins to proteins that regulate the actin cytoskeleton. Whereas the SH2 domain binds to phosphorylated receptors or associated phosphoproteins, individual interactions of the SH3 domains with proline‐based recognition motifs result in the formation of larger protein complexes. In T cells, changes in cell polarity and morphology during T‐cell activation and effector function require the T‐cell receptor‐mediated recruitment and activation of actin‐regulatory proteins to initiate cytoskeletal reorganization at the immunological synapse. We previously identified the adapter protein HS1 as a putative Nck‐interacting protein. We now demonstrate that the SH2 domain of Nck specifically interacts with HS1 upon phosphorylation of its tyrosine residue 378. We report that in human T cells, ligation of the chemokine receptor CXCR4 by stromal cell‐derived factor 1α (SDF1α) induces a rapid and transient phosphorylation of tyrosine 378 of HS1 resulting in an increased association with Nck. Consequently, siRNA‐mediated downregulation of HS1 and/or Nck impairs SDF1α‐induced actin polymerization and T‐cell migration.     

Genetic evidence for a role for Src family kinases in TNF family receptor signaling and cell survival

Mutant src(-/-) mice have osteopetrosis resulting from defective osteoclasts, the cells that resorb bone. However, signaling pathways involving Src family members in osteoclasts remain unclear. We demonstrate that expression of a truncated Src molecule, Src251, lacking the kinase domain, induces osteopetrosis in wild-type and src(+/-) mice and worsens osteopetrosis in src(-/-) mice by a novel mechanism, increased osteoclast apoptosis. Induction of apoptosis by Src251 requires a functional SH2, but not an SH3, domain and is associated with reduced AKT kinase activity. Expression of Src251 dramatically reduces osteoclast survival in response to RANKL/TRANCE/OPGL, providing evidence that Src family kinases are required in vivo for survival signaling pathways downstream from TNF family receptors.