Molecular Cloning of the cDNA Encoding pp36, a Tyrosine-phosphorylated Adaptor Protein Selectively Expressed by T Cells and Natural Killer Cells

Activation of T and natural killer (NK) cells leads to the tyrosine phosphorylation of pp36 and to its association with several signaling molecules, including phospholipase Cγ-1 and Grb2. Microsequencing of peptides derived from purified rat pp36 protein led to the cloning, in rat and man, of cDNA encoding a T- and NK cell–specific protein with several putative Src homology 2 domain–binding motifs. A rabbit antiserum directed against a peptide sequence from the cloned rat molecule recognized tyrosine phosphorylated pp36 from pervanadate-treated rat thymocytes. When expressed in 293T human fibroblast cells and tyrosine-phosphorylated, pp36 associated with phospholipase Cγ-1 and Grb2. Studies with GST–Grb2 fusion proteins demonstrated that the association was specific for the Src homology 2 domain of Grb-2. Molecular cloning of the gene encoding pp36 should facilitate studies examining the role of this adaptor protein in proximal signaling events during T and NK cell activation.

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The importance of Src homology 2 domain-containing leukocyte phosphoprotein of 76 kilodaltons sterile-α motif domain in thymic selection and T-cell activation

Blood ◽

10.1182/blood-2008-09-177832 ◽

2009 ◽

Vol 114 (1) ◽

pp. 74-84 ◽

Cited By ~ 10

Author(s):

Shudan Shen ◽

Jasmine Lau ◽

Minghua Zhu ◽

Jianwei Zou ◽

Deirdre Fuller ◽

...

Keyword(s):

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Adaptor Protein ◽

Thymic Selection ◽

Thymocyte Development ◽

Sam Domain ◽

Src Homology 2 ◽

Src Homology ◽

Src Homology 2 Domain

Abstract The Src homology 2 domain–containing leukocyte phosphoprotein of 76 kilodaltons (SLP-76) is a cytosolic adaptor protein essential for thymocyte development and T-cell activation. It contains a sterile-α motif (SAM) domain, 3 phosphotyrosine motifs, a proline-rich region, and a Src homology 2 domain. Whereas the other domains have been extensively studied, the role of the SAM domain in SLP-76 function is not known. To understand the function of this domain, we generated SLP-76 knockin mice with the SAM domain deleted. Analysis of these mice showed that thymocyte development was partially blocked at the double-positive to single-positive transition. Positive and negative thymic selection was also impaired. In addition, we analyzed T-cell receptor (TCR)–mediated signaling in T cells from these mutant mice. TCR-mediated inositol 1,4,5-triphosphate production, calcium flux, and extracellular signal-regulated kinase activation were decreased, leading to defective interleukin-2 production and proliferation. Moreover, despite normal association between Gads and SLP-76, TCR-mediated formation of SLP-76 microclusters was impaired by the deletion of the SAM domain. Altogether, our data demonstrated that the SAM domain is indispensable for optimal SLP-76 signaling.

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Aberrant overexpression of microRNAs activate AKT signaling via down-regulation of tumor suppressors in natural killer–cell lymphoma/leukemia

Blood ◽

10.1182/blood-2009-06-222794 ◽

2009 ◽

Vol 114 (15) ◽

pp. 3265-3275 ◽

Cited By ~ 139

Author(s):

Yasuo Yamanaka ◽

Hiroyuki Tagawa ◽

Naoto Takahashi ◽

Atsushi Watanabe ◽

Yong-Mei Guo ◽

...

Keyword(s):

Natural Killer ◽

Nk Cell ◽

Cell Lymphoma ◽

Killer Cell ◽

Primary Lymphoma ◽

Down Regulation ◽

Endogenous Expression ◽

Programmed Cell Death 4 ◽

Src Homology ◽

Src Homology 2 Domain

Abstract The gene(s) responsible for natural killer (NK)–cell lymphoma/leukemia have not been identified. In the present study, we found that in NK-cell lymphoma lines (n = 10) and specimens of primary lymphoma (n = 10), levels of miR-21 and miR-155 expression were inversely related and were significantly greater than those found in normal natural killer (CD3−CD56+) cells (n = 8). To determine the functions of these microRNAs in lymphomagenesis, we examined the effects of antisense oligonucleotides (ASOs) targeting miR-21 (ASO-21) and/or miR-155 (ASO-155) in NK-cell lymphoma lines overexpressing one or both of these miRNAs. Conversely, cells showing little endogenous expression of miR-21 or miR-155 were transduced by the use of lentiviral vectors, leading to their overexpression. Reducing expression of miR-21 or miR-155 led to up-regulation of phosphatase and tensin homologue (PTEN), programmed cell death 4 (PDCD4), or Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP1). ASO-21– and ASO-155–treated cell lines all showed down-regulation of phosphorylated AKTser473. Moreover, transduction with either miR-21 or miR-155 led to down-regulation of PTEN and PDCD4 or SHIP1 with up-regulation of phosphorylated AKTser473. Collectively, these results provide important new insight into the pathogenesis of NK-cell lymphoma/leukemia and suggest targeting miR-21 and/or miR-155 may represent a useful approach to treating NK-cell lymphoma/leukemia.

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X-Linked Lymphoproliferative Disease

Journal of Experimental Medicine ◽

10.1084/jem.192.3.337 ◽

2000 ◽

Vol 192 (3) ◽

pp. 337-346 ◽

Cited By ~ 351

Author(s):

Silvia Parolini ◽

Cristina Bottino ◽

Michela Falco ◽

Raffaella Augugliaro ◽

Silvia Giliani ◽

...

Keyword(s):

B Cell ◽

Nk Cells ◽

Cell Lines ◽

Nk Cell ◽

Hla Class I ◽

Lymphoproliferative Disease ◽

Class I ◽

Src Homology 2 ◽

Src Homology ◽

Src Homology 2 Domain

2B4 is a surface molecule involved in activation of the natural killer (NK) cell–mediated cytotoxicity. It binds a protein termed Src homology 2 domain–containing protein (SH2D1A) or signaling lymphocyte activation molecule (SLAM)-associated protein (SAP), which in turn has been proposed to function as a regulator of the 2B4-associated signal transduction pathway. In this study, we analyzed patients with X-linked lymphoproliferative disease (XLP), a severe inherited immunodeficiency characterized by critical mutations in the SH2D1A gene and by the inability to control Epstein-Barr virus (EBV) infections. We show that, in these patients, 2B4 not only fails to transduce triggering signals, but also mediates a sharp inhibition of the NK-mediated cytolysis. Other receptors involved in NK cell triggering, including CD16, NKp46, NKp44, and NKp30, displayed a normal functional capability. However, their activating function was inhibited upon engagement of 2B4 molecules. CD48, the natural ligand of 2B4, is highly expressed on the surface of EBV+ B cell lines. Remarkably, NK cells from XLP patients could not kill EBV+ B cell lines. This failure was found to be the consequence of inhibitory signals generated by the interaction between 2B4 and CD48, as the antibody-mediated disruption of the 2B4–CD48 interaction restored lysis of EBV+ target cells lacking human histocompatibility leukocyte antigen (HLA) class I molecules. In the case of autologous or allogeneic (HLA class I+) EBV+ lymphoblastoid cell lines, restoration of lysis was achieved only by the simultaneous disruption of 2B4–CD48 and NK receptor–HLA class I interactions. Molecular analysis revealed that 2B4 molecules isolated from either XLP or normal NK cells were identical. As expected, in XLP-NK cells, 2B4 did not associate with SH2D1A, whereas similar to 2B4 molecules isolated from normal NK cells, it did associate with Src homology 2 domain–containing phosphatase 1.

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Lymphocyte Antigen Receptor Signal Integration and Regulation by the SHC Adaptor

Biological Chemistry ◽

10.1515/bc.1999.020 ◽

1999 ◽

Vol 380 (2) ◽

Cited By ~ 13

Author(s):

C.T. Baldari ◽

J.L. Telford.

Keyword(s):

Cell Activation ◽

Control Point ◽

Adaptor Protein ◽

Membrane Receptors ◽

Nucleotide Exchange ◽

Transmembrane Receptors ◽

Ras Pathway ◽

Receptor Signal ◽

Src Homology ◽

Src Homology 2 Domain

AbstractThe Shc adaptor protein transduces signals from transmembrane receptors to the Ras pathway of cell activation by providing binding sites for the recruitment to the submembrane compartment of the Grb2/Sos G-nucleotide exchange complex. The need for Shc in this process is however unclear since Grb2 can be recruited directly to phosphotyrosine containing membrane receptors through its src-homology-2 domain. Evidence from studies in lymphocytes indicates that Shc is multifunctional and is involved in the integration of independent signals to the Ras pathway. Furthermore, Shc may be a key control point at which signaling can be modulated both by interfering signals and by feedback mechanisms. Here we review recent literature to support these functions for Shc.

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Functional Requirements for Interactions Between CD84 and Src Homology 2 Domain-Containing Proteins and Their Contribution to Human T Cell Activation

The Journal of Immunology ◽

10.4049/jimmunol.171.5.2485 ◽

2003 ◽

Vol 171 (5) ◽

pp. 2485-2495 ◽

Cited By ~ 46

Author(s):

Stuart G. Tangye ◽

Kim E. Nichols ◽

Nathan J. Hare ◽

Barbara C. M. van de Weerdt

Keyword(s):

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Functional Requirements ◽

Src Homology 2 ◽

Human T Cell ◽

Src Homology ◽

Src Homology 2 Domain

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Cloning and Characterization of Human Lnk, an Adaptor Protein with Pleckstrin Homology and Src Homology 2 Domains that Can Inhibit T Cell Activation

The Journal of Immunology ◽

10.4049/jimmunol.164.10.5199 ◽

2000 ◽

Vol 164 (10) ◽

pp. 5199-5206 ◽

Cited By ~ 75

Author(s):

Yijin Li ◽

Xiaoqing He ◽

Josephine Schembri-King ◽

Scott Jakes ◽

Jun Hayashi

Keyword(s):

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Adaptor Protein ◽

Pleckstrin Homology ◽

Src Homology 2 ◽

Src Homology

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c-Abl phosphorylates Dok1 to promote filopodia during cell spreading

The Journal of Cell Biology ◽

10.1083/jcb.200312171 ◽

2004 ◽

Vol 165 (4) ◽

pp. 493-503 ◽

Cited By ~ 56

Author(s):

Pamela J. Woodring ◽

Jill Meisenhelder ◽

Sam A. Johnson ◽

Guo-Lei Zhou ◽

Jeffrey Field ◽

...

Keyword(s):

Cell Adhesion ◽

Tyrosine Kinase ◽

Actin Cytoskeleton ◽

Adaptor Protein ◽

Cell Spreading ◽

Unknown Mechanism ◽

Abl Tyrosine Kinase ◽

Src Homology 2 ◽

Src Homology ◽

Src Homology 2 Domain

Filopodia are dynamic F-actin structures that cells use to explore their environment. c-Abl tyrosine kinase promotes filopodia during cell spreading through an unknown mechanism that does not require Cdc42 activity. Using an unbiased approach, we identified Dok1 as a specific c-Abl substrate in spreading fibroblasts. When activated by cell adhesion, c-Abl phosphorylates Y361 of Dok1, promoting its association with the Src homology 2 domain (SH2)/SH3 adaptor protein Nck. Each signaling component was critical for filopodia formation during cell spreading, as evidenced by the finding that mouse fibroblasts lacking c-Abl, Dok1, or Nck had fewer filopodia than cells reexpressing the product of the disrupted gene. Dok1 and c-Abl stimulated filopodia in a mutually interdependent manner, indicating that they function in the same signaling pathway. Dok1 and c-Abl were both detected in filopodia of spreading cells, and therefore may act locally to modulate actin. Our data suggest a novel pathway by which c-Abl transduces signals to the actin cytoskeleton through phosphorylating Dok1 Y361 and recruiting Nck.

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EAT-2, a SAP-like adaptor, controls NK cell activation through phospholipase Cγ, Ca++, and Erk, leading to granule polarization

Journal of Experimental Medicine ◽

10.1084/jem.20132038 ◽

2014 ◽

Vol 211 (4) ◽

pp. 727-742 ◽

Cited By ~ 44

Author(s):

Luis-Alberto Pérez-Quintero ◽

Romain Roncagalli ◽

Huaijian Guo ◽

Sylvain Latour ◽

Dominique Davidson ◽

...

Keyword(s):

Nk Cells ◽

Protein Tyrosine Kinase ◽

Cell Activation ◽

Nk Cell ◽

Target Cells ◽

Cellular Mechanisms ◽

Src Homology ◽

Slam Family ◽

Src Homology 2 Domain ◽

Signaling Lymphocytic Activation Molecule

Ewing’s sarcoma-associated transcript 2 (EAT-2) is an Src homology 2 domain-containing intracellular adaptor related to signaling lymphocytic activation molecule (SLAM)–associated protein (SAP), the X-linked lymphoproliferative gene product. Both EAT-2 and SAP are expressed in natural killer (NK) cells, and their combined expression is essential for NK cells to kill abnormal hematopoietic cells. SAP mediates this function by coupling SLAM family receptors to the protein tyrosine kinase Fyn and the exchange factor Vav, thereby promoting conjugate formation between NK cells and target cells. We used a variety of genetic, biochemical, and imaging approaches to define the molecular and cellular mechanisms by which EAT-2 controls NK cell activation. We found that EAT-2 mediates its effects in NK cells by linking SLAM family receptors to phospholipase Cγ, calcium fluxes, and Erk kinase. These signals are triggered by one or two tyrosines located in the carboxyl-terminal tail of EAT-2 but not found in SAP. Unlike SAP, EAT-2 does not enhance conjugate formation. Rather, it accelerates polarization and exocytosis of cytotoxic granules toward hematopoietic target cells. Hence, EAT-2 promotes NK cell activation by molecular and cellular mechanisms distinct from those of SAP. These findings explain the cooperative and essential function of these two adaptors in NK cell activation.

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