Adaptor protein Lnk associates with Tyr568 in c-Kit

2008 ◽  
Vol 415 (2) ◽  
pp. 241-245 ◽  
Author(s):  
Saskia Gueller ◽  
Sigal Gery ◽  
Verena Nowak ◽  
Liqin Liu ◽  
Hubert Serve ◽  
...  
Keyword(s):  
Binding Site ◽  
Glutathione Transferase ◽  
Critical Role ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Erythropoietin Receptor ◽  
Cytokine Receptors ◽  
Juxtamembrane Domain ◽  
Model Studies ◽  
Haemopoietic Cells

The adaptor protein Lnk is expressed in haemopoietic cells and plays a critical role in haemopoiesis. Animal model studies demonstrated that Lnk acts as a broad inhibitor of signalling pathways in haemopoietic lineages. Lnk belongs to a family of proteins sharing several structural motifs, including an SH2 (Src homology 2) domain which binds phosphotyrosine residues in various signal-transducing proteins. The SH2 domain is essential for Lnk-mediated negative regulation of several cytokine receptors [e.g. Mpl, EpoR (erythropoietin receptor), c-Kit]. Therefore inhibition of the binding of Lnk to cytokine receptors might lead to enhanced downstream signalling of the receptor and thereby to improved haemopoiesis in response to exposure to cytokines (e.g. erythropoietin in anaemic patients). This hypothesis led us to define the exact binding site of Lnk to the stem cell factor receptor c-Kit. Pull-down experiments using GST (glutathione transferase)-fusion proteins of the different domains of c-Kit showed that Lnk almost exclusively binds to the phosphorylated juxtamembrane domain. Binding of Lnk to the juxtamembrane domain was abolished by point mutation of Tyr568 and was competed by peptides with a phosphotyrosine residue at position 568. Co-immunoprecipitation with full-length wild-type or Y568F mutant c-Kit and Lnk confirmed these results, thus showing the importance of this phosphorylated tyrosine residue. Lnk bound directly to c-Kit without requiring other interacting partners. The identification of the binding site of Lnk to c-Kit will be useful to discover inhibitory molecules that prevent the binding of these two proteins, thus making haemopoietic cells more sensitive to growth factors.

The SH2-B Adaptor Protein Negatively Regulates EPO-Dependent Signalling Via Interaction with Erythropoietin Receptor Ptyr-343

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 892-892
Author(s):  
Mojib Javadi Javed ◽  
Edda Tschirch ◽  
Bryan K Beattie ◽  
Natalie Stickle ◽  
Kai Huang ◽  
...  
Keyword(s):  
Critical Role ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Erythropoietin Receptor ◽  
Negative Regulator ◽  
Erythroid Cell ◽  
Ph Domain ◽  
Murine Splenocytes ◽  
Hematopoietic Stem ◽  
Functional Studies

Abstract Erythropoiesis is a developmentally important process, whereby multipotent hematopoietic stem cells differentiate into mature erythrocytes. Erythropoietin (EPO) is a critical regulator in this process and mediates its signal via the erythropoietin receptor (EPO-R) and the primary associated tyrosine kinase, JAK2. EPO, EPO-R and JAK2 play a crucial role in erythropoiesis, as deficiency in any of these proteins results in an embryonic lethal anemia. Structural-functional studies and murine knock-in models have shown EPO-R pTyr-343 to play a critical role in EPO mediated signalling. STAT-5 is activated by EPO-R pTyr-343, but STAT5ΔN mice do not have any profound erythroid abnormalities. Such evidence has led our group to hypothesize that other SH2 containing effectors interact with EPO-R pTyr-343. Cloning of Ligand Target screening was utilized to demonstrate that EPO-R pTyr-343 binds to adaptor protein SH2-Bβ. SH2-B contains multiple protein-protein interaction domains including multiple proline-rich regions, a PH domain and an SH2 domain. Although SH2-B does play a role in a number of signaling pathways, it is not required for embryonic development. Since SH2-B is a potent regulator of JAK2 in context of Growth Hormone and Leptin signaling, and it can directly interact with EPO-R pTyr-343, we hypothesize that SH2-B functions as an important adaptor protein downstream of the EPO-R.H2-B constitutively associates to the inactive EPO-R, an interaction that is independent of JAK2 binding to the EPO-R. Upon EPO stimulation, enhanced SH2-dependent binding of SH2-B to pTyr-343 and pTyr-401 of the EPO-R was confirmed utilizing a panel of EPO-R truncation mutants. The EPO mediated interaction between SH2-B and activated EPO-R is both dose and time dependent. EPO stimulation also results in SH2-B serine and threonine phosphorylation. Importantly, the interaction of SH2-B and EPO-R was observed in erythroid cell lines and primary murine splenocytes. The function of SH2-B in EPO signaling was investigated via knocking down SH2-B in Ba/F3-EPO-R cells. Knock down of SH2-B results in hypersensitive EPO-dependent phosphorylation of multiple targets including the EPO-R, JAK2, STAT5 and Erk1/2. It is evident that SH2-B is a global negative regulator of EPO-dependent signaling via its ability to affect EPO-dependent JAK2 activation.

Identification of a novel 14-3-3ζ binding site within the cytoplasmic tail of platelet glycoprotein Ibα

Blood ◽  
2004 ◽  
Vol 104 (2) ◽  
pp. 420-427 ◽  
Author(s):  
Pierre Mangin ◽  
Tovo David ◽  
Vincent Lavaud ◽  
Susan L. Cranmer ◽  
Inna Pikovski ◽  
...  
Keyword(s):  
Binding Site ◽  
Critical Role ◽  
Chinese Hamster ◽  
Adaptor Protein ◽  
Cytoplasmic Tail ◽  
Cell Spreading ◽  
Platelet Glycoprotein ◽  
Platelet Spreading ◽  
Mutant Forms ◽  
Von Willebrand

Abstract The glycoprotein Ib-V-IX (GPIb-V-IX) complex interacts with subendothelial von Willebrand factor (VWF) to ensure recruitment of platelets at sites of vascular injury, a process that culminates in integrin αIIbβ3-dependent stable adhesion and spreading. Interaction of the 14-3-3ζ adaptor protein with the C-terminal 606-610 phosphoserine motif of the GPIbα subunit has been implicated in the control of αIIbβ3 activation and cell spreading. In this study, we have examined potentially novel 14-3-3ζ binding sites by expressing mutant forms of GPIbα in Chinese-hamster-ovary (CHO) cells. Analysis of a series of neighboring 11-12 residue deletions identified a critical role for the 580-LVAGRRPSALS-590 sequence in promoting GPIbα-14-3-3ζ interaction. Development of a phosphospecific antibody demonstrated high levels of phosphorylation of the Ser587 and Ser590 residues in resting platelets (which became dephosphorylated during platelet spreading on VWF), and peptides containing these phosphorylated residues effectively displaced 14-3-3ζ from GPIbα. Analysis of single and double alanine substitutions of Ser587 and Ser590 demonstrated a major role for these residues in promoting GPIbα-14-3-3ζ binding. Moreover, these cell lines exhibited a defect in cell spreading on immobilized VWF. These studies demonstrate the existence of a second major 14-3-3ζ binding site within the cytoplasmic tail of GPIbα that has an important functional role in regulating integrin-dependent cell spreading. (Blood. 2004;104:420-427)

Lyn Is an Important Component of the Signal Transduction Pathway Specific to FLT3/ITD and Can Be a Therapeutic Target in the Treatment of AML with FLT3/ITD.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1908-1908
Author(s):  
Fumihiko Hayakawa ◽  
Mitsunori Okamoto ◽  
Akihiro Abe ◽  
Hitoshi Kiyoi ◽  
Tomoki Naoe
Keyword(s):  
Signal Transduction ◽  
Cell Growth ◽  
Therapeutic Target ◽  
Kinase Inhibitor ◽  
Critical Role ◽  
Signal Transduction Pathway ◽  
Sh2 Domain ◽  
Transduction Pathway ◽  
Mice Model ◽  
Juxtamembrane Domain

Abstract Fms-like tyrosine kinase 3 (FLT3) is expressed in hematopoietic progenitor cells. An internal tandem duplication (ITD) of the FLT3 juxtamembrane domain (FLT3/ITD) is the most frequent mutation in human adult acute myeloid leukemia (AML). Being transfected into IL-3 dependent cell line, 32D, FLT3/ITD causes constitutive activation of FLT3 itself and its downstream signal components, which leads to IL-3 independent cell growth. In particular, STAT5 activation is thought to be a key signaling event since it is induced only by FLT3/ITD not by wild type FLT3. However the signal component which mediates FLT3/ITD to STAT5 is yet to be clarified. In the present study, we showed the mutant- specific association of FLT3/ITD with Lyn, which led to the phosphorylation of Lyn in vivo. We also demonstrated that Lyn directly bound to FLT3 through its SH2 domain in vitro and that the association depended on tyrosyl-phosphorylation at juxtamembrane domain of FLT3. We revealed the critical role of Lyn for STAT5 activation and the autonomous cell growth in FLT3/ITD transfectant of 32D by using anti-Lyn siRNA and the Src family kinase inhibitor PP2. More importantly, we demonstrated successful treatment of FLT3/ITD induced tumors with PP2 in the mice model. These results demonstrate that Lyn is a critical component of the signal transduction pathway specific to FLT3/ITD and can be a therapeutic target in the treatment of AML with FLT3/ITD.

scholarly journals The Vav Binding Site (Y315) in ZAP-70 Is Critical for Antigen Receptor–mediated Signal Transduction

1997 ◽  
Vol 185 (10) ◽  
pp. 1877-1882 ◽  
Author(s):  
Jun Wu ◽  
Qihong Zhao ◽  
Tomohiro Kurosaki ◽  
Arthur Weiss
Keyword(s):  
Signal Transduction ◽  
B Cell ◽  
Binding Site ◽  
Tyrosine Kinases ◽  
Critical Role ◽  
Antigen Receptor ◽  
Sh2 Domain ◽  
Antigen Receptors ◽  
Antigen Receptor Signaling ◽  
Receptor Ligation

Stimulation of antigen receptors in T and B cells leads to the activation of the Src and Syk families of protein tyrosine kinases (PTK). These PTKs subsequently phosphorylate numerous intracellular substrates, including the 95-kD protooncogene product Vav. Vav is essential for both T and B cell development and T and B cell antigen receptor–mediated signal transduction. After receptor ligation, Vav associates with phosphorylated Syk and ZAP-70 PTKs, an interaction that depends upon its SH2 domain. Here we demonstrate that a point mutation of tyrosine 315 (Y315F) in ZAP-70, a putative Vav SH2 domain binding site, eliminated the Vav– ZAP-70 interaction. Moreover, the Y315 mutation impaired the function of ZAP-70 in antigen receptor signaling. Surprisingly, this mutation also resulted in marked reduction in the tyrosine phosphorylation of ZAP-70, Vav, SLP-76, and Shc. These data demonstrate that the Vav binding site in ZAP-70 plays a critical role in antigen receptor–mediated signal transduction.

scholarly journals Phosphorylation of Tyrosine Residues 31 and 118 on Paxillin Regulates Cell Migration through an Association with Crk in Nbt-II Cells

2000 ◽  
Vol 148 (5) ◽  
pp. 957-970 ◽  
Author(s):  
Valérie Petit ◽  
Brigitte Boyer ◽  
Delphine Lentz ◽  
Christopher E. Turner ◽  
Jean Paul Thiery ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cell Motility ◽  
Critical Role ◽  
Adaptor Protein ◽  
Signaling Molecules ◽  
Sh2 Domain ◽  
Wild Type ◽  
Tyrosine Residues ◽  
Pathological Conditions

Identification of signaling molecules that regulate cell migration is important for understanding fundamental processes in development and the origin of various pathological conditions. The migration of Nara Bladder Tumor II (NBT-II) cells was used to determine which signaling molecules are specifically involved in the collagen-mediated locomotion. We show here that paxillin is tyrosine phosphorylated after induction of motility on collagen. Overexpression of paxillin mutants in which tyrosine 31 and/or tyrosine 118 were replaced by phenylalanine effectively impaired cell motility. Moreover, stimulation of motility by collagen preferentially enhanced the association of paxillin with the SH2 domain of the adaptor protein CrkII. Mutations in both tyrosine 31 and 118 diminished the phosphotyrosine content of paxillin and prevented the formation of the paxillin–Crk complex, suggesting that this association is necessary for collagen-mediated NBT-II cell migration. Other responses to collagen, such as cell adhesion and spreading, were not affected by these mutations. Overexpression of wild-type paxillin or Crk could bypass the migration-deficient phenotype. Both the SH2 and the SH3 domains of CrkII are shown to play a critical role in this collagen-mediated migration. These results demonstrate the important role of the paxillin–Crk complex in the collagen-induced cell motility.

scholarly journals A Drosophila shc gene product is implicated in signaling by the DER receptor tyrosine kinase.

1995 ◽  
Vol 15 (9) ◽  
pp. 4810-4818 ◽  
Author(s):  
K M Lai ◽  
J P Olivier ◽  
G D Gish ◽  
M Henkemeyer ◽  
J McGlade ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Binding Site ◽  
Receptor Tyrosine Kinase ◽  
Growth Factor Receptor ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Expression Library ◽  
Close Relationship ◽  
Ptb Domain

Antibodies to the human Shc adaptor protein were used to isolate a cDNA encoding a Drosophila Shc protein (dShc) by screening an expression library. The dshc gene, which maps to position 67B-C on the third chromosome, encodes a 45-kDa protein that is widely expressed throughout the Drosophila life cycle. In flies, the dShc protein physically associates with the activated Drosophila epidermal growth factor receptor homolog (DER) and is inducibly phosphorylated on tyrosine by DER. The 45-kDa dShc protein is closely related both in overall organization and in amino acid sequence (46% identity) to the 52-kDa mammalian Shc isoform. In addition to a C-terminal Src homology 2 (SH2) domain, dShc contains an N-terminal phosphotyrosine-binding (PTB) domain, which associates in vitro with the autophosphorylated DER receptor tyrosine kinase and with phosphopeptides containing an Asn-Pro-X-pTyr motif, where pTyr stands for phosphotyrosine. A potential binding site for the dShc PTB domain is located at Tyr-1228 of DER. These results indicate that the shc gene has been conserved in evolution, as have the binding properties of the Shc PTB and SH2 domains. Despite the close relationship between the Drosophila and mammalian Shc proteins, dShc lacks the high-affinity Grb2-binding site found in mammalian Shc, suggesting that Shc proteins may have functions in addition to regulation of the Ras pathway.

scholarly journals Mutational Analysis of the Binding of Alternative Substrates and Inhibitors to the Active Site of Human Glutathione Transferase P1–1

Processes ◽  
2020 ◽  
Vol 8 (10) ◽  
pp. 1232
Author(s):  
Abeer Shokeer ◽  
Aram Ismail ◽  
Usama M. Hegazy ◽  
Rüdiger H. Kolm ◽  
Bengt Mannervik
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Glutathione Transferase ◽  
Mutational Analysis ◽  
Critical Role ◽  
Catalytic Efficiency ◽  
Binding Energies ◽  
Nucleophilic Attack ◽  
Therapeutic Drugs ◽  
Glutathione Binding

Glutathione transferases (GSTs) are enzymes that play a critical role in cellular detoxication by catalyzing the nucleophilic attack of glutathione on the electrophilic center of a number of xenobiotic compounds, including many therapeutic drugs. Mutations of amino acid residues in the glutathione-binding site of human glutathione transferase P1–1, namely W39C, K45A, Q52A, Q52K, and Q52E, have been engineered. The recombinant mutant proteins were expressed in Escherichia coli, but only mutants K45A, Q52A, and Q52K showed measurable activity. Steady-state kinetics comparing glutathione with the alternative thiol substrate γ-glutamylcysteine demonstrated the importance of the glycine residue in glutathione for high catalytic efficiency. Inhibition experiments with a set of glutathione analogs structurally related to the therapeutic drugs Telintra and Telcyta enabled determination of binding energies that were contributed by different substituents. The effects of substituting amino acid side chains in the glutathione-binding site of the enzyme on binding the glutathione derivatives and catalysis were evaluated.

scholarly journals The SH3 domain of Src tyrosyl protein kinase interacts with the N-terminal splice region of the PDE4A cAMP-specific phosphodiesterase RPDE-6 (RNPDE4A5)

1996 ◽  
Vol 318 (1) ◽  
pp. 255-261 ◽  
Author(s):  
Jonathan C. O'CONNELL ◽  
J. Fraser McCALLUM ◽  
Ian McPHEE ◽  
Jill WAKEFIELD ◽  
Emma S HOUSLAY ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Binding Site ◽  
Splice Variant ◽  
Src Kinase ◽  
Adaptor Protein ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Glutathione S Transferase ◽  
Sh3 Domains ◽  
Type Iv

The PDE4A (type IV) cAMP-specific, rolipram-inhibited phosphodiesterase RPDE-6 (RNPDE4A5), when transiently expressed in COS7 cells, could be complexed with the v-Src-SH3 domain expressed as a glutathione S-transferase (GST) fusion protein. RPDE-6 did not interact with GST itself. This complex was not disrupted by treatment with high NaCl concentration together with Triton X-100. Interaction was apparently determined by the N-terminal splice region of RPDE-6, as the PDE4A splice variant RPDE-39, which differs from RPDE-6 at the extreme N-terminus, failed to associate with v-Src-SH3; met26RD1 (where RD1 is rat ‘dunc-like’ PDE), which has the N-terminal splice region deleted, failed to associate with v-Src-SH3, and the association of RPDE-6 and v-Src-SH3 was blocked by a fusion protein formed from the N-terminal splice region. RDPE-6 showed binding to GST fusion proteins of both the intact Src kinase and an SH2–SH3 construct but did not bind to the Src-SH2 domain or to the adaptor protein Grb-2. RPDE-6 could be co-immunoprecipitated from cytosol extracts of transfected cells by using anti-Src antiserum. RPDE-6 exhibited selectivity in binding to the SH3 domains of c-Abl, Crk, Csk, Lck, Lyn, Fyn and v-Src, with binding to the SH3 regions of the Src-related tyrosyl kinases Lyn and Fyn being the most effective. The binding of RPDE-6 to the SH3 domains of Crk, Csk and Lck led to a marked reduction in PDE activity, but no change was apparent in complexes with other species. Endogenous RPDE-6 from brain, but not endogenous RPDE-39 from testis, bound to the Src-SH3 domain. We suggest that the PDE4A splice variant RPDE-6 has a propensity for interaction with selective SH3 domains, in particular those from Src and the Src-related tyrosyl kinases Lyn and Fyn. This interaction seems to be governed by alternative splicing of the PDE4A gene, because RPDE-39, a splice variant that lacks the proline-rich N-terminal splice region of RPDE-6, does not interact with these SH3 domains. It is proposed that the binding site on RPDE-6 for SH3 domains lies within the unique first 102 residues of its N-terminal splice domain, where two motifs representing Class I SH3 binding sites with selectivity for Src kinase SH3 domains can be identified and one motif for a putative Class II SH3 binding site.

scholarly journals Signaling for NKT cell development

2005 ◽  
Vol 201 (6) ◽  
pp. 833-836 ◽  
Author(s):  
Christine Borowski ◽  
Albert Bendelac
Keyword(s):  
T Cell ◽  
Critical Role ◽  
Hematopoietic Cells ◽  
Adaptor Protein ◽  
Cell Development ◽  
Thymic Selection ◽  
Nkt Cell ◽  
Cell Lineages ◽  
Receptor Interactions ◽  
Slam Family

New studies demonstrate a critical role for the adaptor protein SAP (SLAM-associated protein) during NKT cell development. By connecting homotypic SLAM family receptor interactions with the FynT Src kinase, SAP may integrate a set of long-standing yet seemingly disparate observations characterizing NKT cell development. In fact, SAP-dependent signaling may underlie the development of multiple unconventional T cell lineages whose thymic selection relies on homotypic interactions between hematopoietic cells.

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