The Shc adaptor protein forms interdependent phosphotyrosine-mediated protein complexes in mast cells stimulated with interleukin 3

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 132-138 ◽  
Author(s):  
Laura Velazquez ◽  
Gerald D. Gish ◽  
Peter van der Geer ◽  
Lorne Taylor ◽  
Johanna Shulman ◽  
...  
Keyword(s):  
Mast Cells ◽  
Protein Complexes ◽  
Adaptor Protein ◽  
Phospholipid Metabolism ◽  
Sh2 Domain ◽  
Interleukin 3 ◽  
Inositol Phosphatase ◽  
Ptb Domain

Abstract The Shc adaptor protein possesses 2 distinct phosphotyrosine (pTyr) recognition modules—the pTyr binding (PTB) domain and the Src homology 2 (SH2) domain—and multiple potential sites for tyrosine (Tyr) phosphorylation (Tyr residues 239, 240, and 317). On stimulation of hematopoietic cells with interleukin 3 (IL-3), Shc becomes phosphorylated and may therefore contribute to IL-3 signaling. We investigated the interactions mediated by the Shc modular domains and pTyr sites in IL-3–dependent IC2 premast cells. The Shc PTB domain, rather than the SH2 domain, associated both in vitro and in vivo with the Tyr-phosphorylated β subunit of the IL-3 receptor and with the SH2-containing 5′ inositol phosphatase (SHIP), and it recognized specific NXXpY phosphopeptides from these binding partners. In IL-3–stimulated mast cells, Shc phosphorylation occurred primarily on Tyr239 and 317 and was dependent on a functional PTB domain. Phosphorylated Tyr317, and to a lesser extent, Tyr239, bound the Grb2 adaptor and SHIP. Furthermore, a pTyr317 Shc phosphopeptide selectively recognized Grb2, Sos1, SHIP, and the p85 subunit of phosphatidylinositol 3′ kinase from mast cells, as characterized by mass spectrometry. These results indicate that Shc undergoes an interdependent series of pTyr-mediated interactions in IL-3–stimulated mast cells, resulting in the recruitment of proteins that regulate the Ras pathway and phospholipid metabolism.

The Shc adaptor protein forms interdependent phosphotyrosine-mediated protein complexes in mast cells stimulated with interleukin 3

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 132-138 ◽  
Author(s):  
Laura Velazquez ◽  
Gerald D. Gish ◽  
Peter van der Geer ◽  
Lorne Taylor ◽  
Johanna Shulman ◽  
...  
Keyword(s):  
Mast Cells ◽  
Protein Complexes ◽  
Adaptor Protein ◽  
Phospholipid Metabolism ◽  
Sh2 Domain ◽  
Interleukin 3 ◽  
Inositol Phosphatase ◽  
Ptb Domain

The Shc adaptor protein possesses 2 distinct phosphotyrosine (pTyr) recognition modules—the pTyr binding (PTB) domain and the Src homology 2 (SH2) domain—and multiple potential sites for tyrosine (Tyr) phosphorylation (Tyr residues 239, 240, and 317). On stimulation of hematopoietic cells with interleukin 3 (IL-3), Shc becomes phosphorylated and may therefore contribute to IL-3 signaling. We investigated the interactions mediated by the Shc modular domains and pTyr sites in IL-3–dependent IC2 premast cells. The Shc PTB domain, rather than the SH2 domain, associated both in vitro and in vivo with the Tyr-phosphorylated β subunit of the IL-3 receptor and with the SH2-containing 5′ inositol phosphatase (SHIP), and it recognized specific NXXpY phosphopeptides from these binding partners. In IL-3–stimulated mast cells, Shc phosphorylation occurred primarily on Tyr239 and 317 and was dependent on a functional PTB domain. Phosphorylated Tyr317, and to a lesser extent, Tyr239, bound the Grb2 adaptor and SHIP. Furthermore, a pTyr317 Shc phosphopeptide selectively recognized Grb2, Sos1, SHIP, and the p85 subunit of phosphatidylinositol 3′ kinase from mast cells, as characterized by mass spectrometry. These results indicate that Shc undergoes an interdependent series of pTyr-mediated interactions in IL-3–stimulated mast cells, resulting in the recruitment of proteins that regulate the Ras pathway and phospholipid metabolism.

scholarly journals Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins.

1997 ◽  
Vol 17 (3) ◽  
pp. 1702-1713 ◽  
Author(s):  
D D Schlaepfer ◽  
M A Broome ◽  
T Hunter
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Intracellular Signaling ◽  
Protein Tyrosine Kinase ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Sh2 Domain ◽  
Mitogen Activated Protein Kinase

The focal adhesion kinase (FAK), a protein-tyrosine kinase (PTK), associates with integrin receptors and is activated by cell binding to extracellular matrix proteins, such as fibronectin (FN). FAK autophosphorylation at Tyr-397 promotes Src homology 2 (SH2) domain binding of Src family PTKs, and c-Src phosphorylation of FAK at Tyr-925 creates an SH2 binding site for the Grb2 SH2-SH3 adaptor protein. FN-stimulated Grb2 binding to FAK may facilitate intracellular signaling to targets such as ERK2-mitogen-activated protein kinase. We examined FN-stimulated signaling to ERK2 and found that ERK2 activation was reduced 10-fold in Src- fibroblasts, compared to that of Src- fibroblasts stably reexpressing wild-type c-Src. FN-stimulated FAK phosphotyrosine (P.Tyr) and Grb2 binding to FAK were reduced, whereas the tyrosine phosphorylation of another signaling protein, p130cas, was not detected in the Src- cells. Stable expression of residues 1 to 298 of Src (Src 1-298, which encompass the SH3 and SH2 domains of c-Src) in the Src- cells blocked Grb2 binding to FAK; but surprisingly, Src 1-298 expression also resulted in elevated p130cas P.Tyr levels and a two- to threefold increase in FN-stimulated ERK2 activity compared to levels in Src- cells. Src 1-298 bound to both FAK and p130cas and promoted FAK association with p130cas in vivo. FAK was observed to phosphorylate p130cas in vitro and could thus phosphorylate p130cas upon FN stimulation of the Src 1-298-expressing cells. FAK-induced phosphorylation of p130cas in the Src 1-298 cells promoted the SH2 domain-dependent binding of the Nck adaptor protein to p130cas, which may facilitate signaling to ERK2. These results show that there are additional FN-stimulated pathways to ERK2 that do not involve Grb2 binding to FAK.

scholarly journals Physical and functional interactions between Stat5 and the tyrosine- phosphorylated receptors for erythropoietin and interleukin-3

Blood ◽  
1996 ◽  
Vol 88 (12) ◽  
pp. 4415-4425 ◽  
Author(s):  
H Chin ◽  
N Nakamura ◽  
R Kamiyama ◽  
N Miyasaka ◽  
JN Ihle ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Sh2 Domain ◽  
Binding Studies ◽  
Interleukin 3 ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
Docking Sites ◽  
Carboxy Terminal

Erythropoietin (Epo) and interleukin-3 (IL-3) stimulate activation of the Jak2 tyrosine kinase and induce tyrosine phosphorylation and activation of Stat5. In the present study, we have shown that Epo or IL-3 stimulation induces binding of Stat5 to the tyrosine-phosphorylated Epo receptor (EpoR) or IL-3 receptor beta subunit (betaIL3), respectively, in IL-3-dependent 32D cells expressing the EpoR. The binding of Stat5 to these cytokine receptors was shown to be rapid and transient, occurring within 1 minute of stimulation of cells and significantly decreasing after 5 minutes of cell treatment. In vivo binding experiments in COS cells showed that binding of Stat5 to the EpoR was mediated through the Stat5 Src homology 2 (SH2) domain. In vitro binding studies further showed that Stat5, but not other Stats examined, bound specifically to tyrosine-phosphorylated recombinant EpoR fusion proteins. In these in vivo and in vitro binding studies, Stat5 bound, albeit to a lesser degree, to truncated EpoR mutants in which all the intracellular tyrosines except Y-343 were removed. Furthermore, EpoR-derived synthetic phosphotyrosine peptides corresponding to Y-343, Y-401, Y-431, and Y-479 inhibited the in vitro binding of Stat5. When expressed in 32D cells, a mutant EpoR in which all the intracellular tyrosines were removed by carboxy-terminal truncation showed a significantly impaired ability to induce tyrosine phosphorylation of Stat5, particularly at low concentrations of Epo, but exhibited an increased sensitivity to Epo for growth signaling as compared with the wild-type EpoR. These results indicate that Stat5 specifically and transiently binds to the EpoR through the interaction between the Stat5 SH2 domain and specific phosphorylated tyrosines, including Y-343, in the EpoR cytoplasmic domain. It was implied that betaIL3 may also have similar Stat5 docking sites. The Stat5 docking sites in the EpoR were shown to facilitate specific activation of Stat5, which, however, may not be required for the EpoR-mediated growth signaling.

MHC Class II and T Cell Receptor Signals Are Dispensable for IL-2-Induced Regulatory T Cell Proliferation In Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2181-2181
Author(s):  
Tao Zou ◽  
Atsushi Satake ◽  
Jonathan Maltzman ◽  
Taku Kambayashi
Keyword(s):  
T Cell ◽  
Conflicts Of Interest ◽  
Interleukin 2 ◽  
Adaptor Protein ◽  
Class Ii ◽  
Sh2 Domain ◽  
Tcr Signaling ◽  
Mhc Ii

Abstract Abstract 2181 Regulatory T cells (Tregs) protect the host from autoimmunity and inappropriate immune activation. Thus, to ensure immune tolerance in the steady state, an adequate number of peripheral Tregs must be constantly maintained. Prior work has suggested that major histocompatibility class II (MHC II) and interleukin-2 (IL-2) are both necessary to maintain peripheral Treg homeostasis and proliferation in vivo. However, we have recently reported that Treg proliferation may not strictly depend on MHC II, as the provision of IL-2 was sufficient to drive proliferation of Tregs in an MHC II-independent manner in vitro, as long as the Tregs interacted with dendritic cells (DC)s. Here, extending our previous in vitro observations, we tested the dependence of Treg proliferation on IL-2, DCs, and TCR signaling in vivo. Proliferation of adoptively transferred Tregs was detected in wildtype (WT) mice. This proliferation was markedly enhanced when the mice were injected with IL-2 immune complexes (IC)s but not when the IL-2 IC-injected mice lacked DCs, suggesting that IL-2-induced Treg proliferation was dependent on DCs in vivo. As previously reported, adoptively transferred Tregs did not proliferate in MHC II-deficient hosts. However, the injection of IL-2 ICs into these mice induced Treg proliferation comparable to those transferred into IL-2 IC-injected WT mice, suggesting that IL-2 signaling by Tregs obviated the need of MHC II for their proliferation. Furthermore, while the ablation of TCR signaling by conditional deletion of the adaptor protein SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) rendered Tregs unable to proliferate by themselves, IL-2 IC treatment partially rescued this deficiency. We next examined the signaling pathways involved in Treg proliferation downstream of the IL-2 receptor. Despite the importance of the Stat5 pathway in IL-2 receptor signaling during Treg development in the thymus, activation of Stat5b alone was insufficient to rescue proliferation of SLP-76-deficient Tregs, indicating that alternative pathways must also be activated for Treg proliferation. Additional studies investigating the role of other signaling molecules downstream of the IL-2 receptor are currently underway. In summary, we have demonstrated for the first time that Tregs do not require TCR signaling through interaction with MHC II for their proliferation in vivo. We propose that this MHC II-independent mode of Treg proliferation allows Tregs with multiple antigen specificities to proliferate, which ensures that a diverse TCR repertoire is continuously maintained in the Treg pool. Furthermore, we believe that exploitation of these pathways may be therapeutically beneficial in autoimmunity and in transplantation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Enhancement of the biologic effects of interleukin-3 in vivo by anti- interleukin-3 antibodies

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1133-1141
Author(s):  
AT Jones ◽  
HJ Ziltener
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Half Life ◽  
Low Dose ◽  
Interleukin 3 ◽  
Concurrent Use ◽  
Biologic Effects ◽  
Total Body

Interleukin-3 (IL-3) has been shown to be a promising agent in the stimulation of bone marrow regeneration following myeloablative therapy. The biologic half-life of this agent is very short (5 to 15 minutes), which limits the effectiveness of low-dose therapy. Here we show that the biologic effects of low-dose IL-3 in mice may be enhanced by concurrent use of polyclonal anti-IL-3 antibodies. The biologic effects of IL-3 in vivo were enhanced dramatically by the combination of the cytokine and polyclonal rabbit anti-IL-3 antibodies, which recognized a peptide comprising the first 29 amino acids of the IL-3 molecule. Enhancing effects were not apparent in vitro, where weak neutralizing properties were observed for these antibodies. The mechanism of this enhancement by the antibody appears to be via a ninefold reduction in the total-body clearance of the cytokine in vivo. The apparent volumes of distribution for IL-3 and for the IL-3/antibody complex were surprisingly similar and exceeded the expected intravascular volume. The prolonged biologic half-life of IL-3 was reproducibly associated with a threefold to fivefold increase in splenic mast-cell precursors over levels observed in mice treated with IL-3 alone; increases in the numbers of mature mucosal-type mast cells in the spleen, but not in the jejunum or lung; increases in IL-3- dependent colony-forming unit-cell in the spleen; and an apparent redistribution of mast cells away from the bone marrow. These experiments demonstrate that antibodies to a cytokine can enhance the biologic activity of that cytokine in vivo.

scholarly journals CAS/Crk Coupling Serves as a “Molecular Switch” for Induction of Cell Migration

1998 ◽  
Vol 140 (4) ◽  
pp. 961-972 ◽  
Author(s):  
Richard L. Klemke ◽  
Jie Leng ◽  
Rachel Molander ◽  
Peter C. Brooks ◽  
Kristiina Vuori ◽  
...  
Keyword(s):  
Cell Migration ◽  
Adaptor Protein ◽  
Molecular Switch ◽  
Sh2 Domain ◽  
Carcinoma Cells ◽  
Dominant Negative ◽  
Promote Cell Migration ◽  
Invasive Property

Abstract. Carcinoma cells selected for their ability to migrate in vitro showed enhanced invasive properties in vivo. Associated with this induction of migration was the anchorage-dependent phosphorylation of p130CAS (Crk-associated substrate), leading to its coupling to the adaptor protein c-CrkII (Crk). In fact, expression of CAS or its adaptor protein partner Crk was sufficient to promote cell migration, and this depended on CAS tyrosine phosphorylation facilitating an SH2-mediated complex with Crk. Cytokine-stimulated cell migration was blocked by CAS lacking the Crk binding site or Crk containing a mutant SH2 domain. This migration response was characterized by CAS/Crk localization to membrane ruffles and blocked by the dominant-negative GTPase, Rac, but not Ras. Thus, CAS/Crk assembly serves as a “molecular switch” for the induction of cell migration and appears to contribute to the invasive property of tumors.

scholarly journals Loss of Src-like Adaptor Protein 2 Expression Increases the Transforming Potential of Oncogenic FLT3-ITD

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5106-5106
Author(s):  
Sausan A. Moharram ◽  
Julhash U. Kazi ◽  
Lars Rönnstrand
Keyword(s):  
Conflicts Of Interest ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Tumor Formation ◽  
Signaling Proteins ◽  
Short Term Treatment ◽  
Tyrosine Residues ◽  
Alternative Approach

Abstract The receptor tyrosine kinase FLT3 is found to be a mutated oncogene in hematological malignancies including acute myeloid leukemia (AML). FLT3 inhibitors in combination with chemotherapy display promising results in a clinical setting, but patients relapse after short-term treatment due to the development of resistant disease. Therefore, targeting signaling proteins downstream of FLT3 can be an alternative approach for the treatment of patients carrying mutant FLT3. Activated FLT3 is constitutively phosphorylated on several tyrosine residues. These tyrosine residues facilitate association of SH2 domain-containing signaling proteins. By using a panel of SH2 domain-containing proteins we identified SLAP2 as a potent interaction partner of FLT3. The interaction in between FLT3 and SLAP2 occurs when FLT3 is activated and an intact SH2 domain of SLAP2 is required for the interaction. SLAP2 associates with FLT3 mainly through its SRC binding sites and expression of SLAP2 inhibited oncogenic FLT3-ITD-mediated cell proliferation and colony formation in vitro, and tumor formation in vivo. By analysis of patient expression data, we found that loss of SLAP2 expression correlates with poor prognosis of AML patients carrying FLT3-ITD. SLAP2 inhibits FLT3-mediated downstream signaling such as activation of AKT, ERK, p38 and STAT5. Inhibition is partially mediated through ubiquitination-mediated degradation of FLT3. Taken together our current study demonstrates that SLAP2 is an important regulator of FLT3-mediated oncogenic signaling and thus modulation of the SLAP2 expression levels can be an alternative approach for the treatment of FLT3-ITD positive malignancies. Disclosures No relevant conflicts of interest to declare.

Hematopoietic cell phosphatase associates with the interleukin-3 (IL-3) receptor beta chain and down-regulates IL-3-induced tyrosine phosphorylation and mitogenesis

1993 ◽  
Vol 13 (12) ◽  
pp. 7577-7586
Author(s):  
T Yi ◽  
A L Mui ◽  
G Krystal ◽  
J N Ihle
Keyword(s):  
Cell Growth ◽  
Tyrosine Phosphorylation ◽  
Growth Regulation ◽  
Sh2 Domain ◽  
Hematopoietic Cell ◽  
Beta Chain ◽  
Interleukin 3 ◽  
Amino Terminal

Hematopoietic cell phosphatase (HCP) is a tyrosine phosphatase with two Src homology 2 (SH2) domains that is predominantly expressed in hematopoietic cells, including cells whose growth is regulated by interleukin-3 (IL-3). The potential effects of HCP on IL-3-induced protein tyrosine phosphorylation and growth regulation were examined to assess the role of HCP in hematopoiesis. Our studies demonstrate that, following ligand binding, HCP specifically associates with the beta chain of the IL-3 receptor through the amino-terminal SH2 domain of HCP, both in vivo and in vitro, and can dephosphorylate the receptor chain in vitro. The effects of increasing or decreasing HCP levels in IL-3-dependent cells were assessed with dexamethasone-inducible constructs containing an HCP cDNA in sense and antisense orientations. Increased HCP levels were found to reduce the levels of IL-3-induced tyrosine phosphorylation of the receptor and to dramatically suppress cell growth. Conversely, decreasing the levels of HCP increased IL-3-induced tyrosine phosphorylation of the receptor and marginally increased growth rate. These results support a role for HCP in the regulation of hematopoietic cell growth and begin to provide a mechanistic explanation for the dramatic effects that the genetic loss of HCP, which occurs in motheaten (me) and viable motheaten (mev) mice, has on hematopoiesis.

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 Enhancement of the biologic effects of interleukin-3 in vivo by anti- interleukin-3 antibodies

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1133-1141 ◽  
Author(s):  
AT Jones ◽  
HJ Ziltener
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Half Life ◽  
Low Dose ◽  
Interleukin 3 ◽  
Concurrent Use ◽  
Biologic Effects ◽  
Total Body

Abstract Interleukin-3 (IL-3) has been shown to be a promising agent in the stimulation of bone marrow regeneration following myeloablative therapy. The biologic half-life of this agent is very short (5 to 15 minutes), which limits the effectiveness of low-dose therapy. Here we show that the biologic effects of low-dose IL-3 in mice may be enhanced by concurrent use of polyclonal anti-IL-3 antibodies. The biologic effects of IL-3 in vivo were enhanced dramatically by the combination of the cytokine and polyclonal rabbit anti-IL-3 antibodies, which recognized a peptide comprising the first 29 amino acids of the IL-3 molecule. Enhancing effects were not apparent in vitro, where weak neutralizing properties were observed for these antibodies. The mechanism of this enhancement by the antibody appears to be via a ninefold reduction in the total-body clearance of the cytokine in vivo. The apparent volumes of distribution for IL-3 and for the IL-3/antibody complex were surprisingly similar and exceeded the expected intravascular volume. The prolonged biologic half-life of IL-3 was reproducibly associated with a threefold to fivefold increase in splenic mast-cell precursors over levels observed in mice treated with IL-3 alone; increases in the numbers of mature mucosal-type mast cells in the spleen, but not in the jejunum or lung; increases in IL-3- dependent colony-forming unit-cell in the spleen; and an apparent redistribution of mast cells away from the bone marrow. These experiments demonstrate that antibodies to a cytokine can enhance the biologic activity of that cytokine in vivo.

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