Phosphatase CD45 Both Positively and Negatively Regulates T Cell Receptor Phosphorylation in Reconstituted Membrane Protein Clusters

Furlan, Gabriela; Minowa, Takashi; Hanagata, Nobutaka; Kataoka-Hamai, Chiho; Kaizuka, Yoshihisa

doi:10.1074/jbc.m114.574319

Cited by 30 publications

(33 citation statements)

References 39 publications

(40 reference statements)

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“…In this work, we presented an unconventional approach to quantify the role of protein-membrane interactions in regulating the accessibility of ITAM tyrosines, and thereby their phosphorylation, on the cytoplasmic domain of the T-cell receptor, a process that is at the root of signal transduction in T-cell signaling. Although the impact of cyt phosphorylation and its variation though membrane interactions have been extensively studied, mostly in the cellular context (25,27), consensus on these processes has not been established due to contradicting results from distinct experiments (19,26,51,52). In vitro experiments that reconstruct significant aspects of the T-cell receptor signaling cascade may help to elucidate this problem (51,52).…”

Section: Discussionmentioning

confidence: 99%

“…Although the impact of cyt phosphorylation and its variation though membrane interactions have been extensively studied, mostly in the cellular context (25,27), consensus on these processes has not been established due to contradicting results from distinct experiments (19,26,51,52). In vitro experiments that reconstruct significant aspects of the T-cell receptor signaling cascade may help to elucidate this problem (51,52). Here we took the radical approach to study the soluble TCR cyt domain in the extremely simple but well-controlled experimental environment of tethered synthetic bilayers probed by SPR and NR.…”

Section: Discussionmentioning

confidence: 99%

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The cytosolic domain of T-cell receptor ζ associates with membranes in a dynamic equilibrium and deeply penetrates the bilayer

Zimmermann¹,

Eells²,

Heinrich

et al. 2017

Journal of Biological Chemistry

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Interactions between lipid bilayers and the membrane-proximal regions of membrane-associated proteins play important roles in regulating membrane protein structure and function. The T-cell antigen receptor is an assembly of eight single-pass membrane-spanning subunits on the surface of T lymphocytes that initiates cytosolic signaling cascades upon binding antigens presented by MHC-family proteins on antigen-presenting cells. Its ζ-subunit contains multiple cytosolic immunoreceptor tyrosine-based activation motifs involved in signal transduction, and this subunit by itself is sufficient to couple extracellular stimuli to intracellular signaling events. Interactions of the cytosolic domain of ζ (ζ) with acidic lipids have been implicated in the initiation and regulation of transmembrane signaling. ζ is unstructured in solution. Interaction with acidic phospholipids induces structure, but its disposition when bound to lipid bilayers is controversial. Here, using surface plasmon resonance and neutron reflection, we characterized the interaction of ζ with planar lipid bilayers containing mixtures of acidic and neutral lipids. We observed two binding modes of ζ to the bilayers in dynamic equilibrium: one in which ζ is peripherally associated with lipid headgroups and one in which it penetrates deeply into the bilayer. Such an equilibrium between the peripherally bound and embedded forms of ζ apparently controls accessibility of the immunoreceptor tyrosine-based activation signal transduction pathway. Our results reconcile conflicting findings of the ζ structure reported in previous studies and provide a framework for understanding how lipid interactions regulate motifs to tyrosine kinases and may regulate the T-cell antigen receptor biological activities for this cell-surface receptor system.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

The cytosolic domain of T-cell receptor ζ associates with membranes in a dynamic equilibrium and deeply penetrates the bilayer

Zimmermann¹,

Eells²,

Heinrich

et al. 2017

Journal of Biological Chemistry

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“…The importance of this spatio-temporal organization is illustrated by studies in the immunological synapse (IS) in which the dynamic inclusion and exclusion of CD45 is known to be important for balancing the opposing roles of CD45 in T cell signaling. As a result, CD45 can function as both a positive and a negative factor in T cell activation, depending on its spatio-temporal organization and local concentration 7, 44 . In the past, numerous mechanisms have been proposed to regulate the activity of CD45, these include localization and dimerization 36 .…”

Section: Discussionmentioning

confidence: 99%

“…CD45 is a large single pass transmembrane glycoprotein (180-220 kDa) responsible for dephosphorylating the tyrosine kinase Lck at position Y505 in T cells, thereby relieving the auto-inhibition of Lck 7 . Activated Lck is then able to directly phosphorylate the TCR and ZAP70, and thereby initiates the T cell signaling cascade.…”

Section: Introductionmentioning

confidence: 99%

Dynamic regulation of CD45 by tetraspanin CD53

Dunlock

Arp

Jansen

et al. 2019

Preprint

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T cells are central to the adaptive immune response, playing a role in both the direct and indirect killing of pathogens and transformed cells. The activation of T cells is the result of a complex signaling cascade, initiated at the T cell receptor (TCR), and ending with the induction of proliferation. CD45, a member of the protein tyrosine phosphatase family, is one of the most abundant membrane proteins on T cells and functions by regulating activation directly downstream of the TCR. As a result of alternative splicing, CD45 can be expressed in multiple isoforms, naive T cells express the CD45RA isoform, while activated T cells gain expression of CD45RO, which has been proposed to increase signaling. Though the importance of CD45 in TCR signaling, proliferation and cytokine production is well established, little is known about the regulation of CD45 activity. We discovered that the immune-specific tetraspanin CD53 directly affects the stability and function of CD45RO in T cells. We have identified CD53 as a T cell co-stimulatory molecule in primary human and murine cells. Furthermore, we have shown that the absence of CD53 leads to an altered CD45 isoform expression as a result of decreased CD45RO stability on the cell surface. This instability was accompanied by increased mobility as measured by FRAP. Together, this indicates that CD53 functions as a stabilizer of CD45RO, and therefore as a positive regulator of TCR signaling at the T cell surface. Our data provides novel insight into the role of tetraspanins in the regulation of immune signaling and may provide a new avenue for the regulation of T cell signaling.

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“…Typically, in such experiments, α-CD3 is monobiotinylated and ICAM-1 is poly-His tagged (Kaizuka et al, 2007; Ilani et al, 2009; Hsu et al, 2012; Yi et al, 2012; Dillard et al, 2014; Furlan et al, 2014). …”

Section: Introductionmentioning

confidence: 99%

Supported Lipid Bilayer Technology for the Study of Cellular Interfaces

et al. 2015

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Glass‐supported lipid bilayers presenting freely diffusing proteins have served as a powerful tool for studying cell‐cell interfaces, in particular, T cell–antigen presenting cell (APC) interactions, using optical microscopy. Here we expand upon existing protocols and describe the preparation of liposomes by an extrusion method, and describe how this system can be used to study immune synapse formation by Jurkat cells. We also present a method for forming such lipid bilayers on silica beads for the study of signaling responses by population methods, such as western blotting, flow cytometry, and gene‐expression analysis. Finally, we describe how to design and prepare transmembrane‐anchored protein‐laden liposomes, following expression in suspension CHO (CHOs) cells, a mammalian expression system alternative to insect and bacterial cell lines, which do not produce mammalian glycosylation patterns. Such transmembrane‐anchored proteins may have many novel applications in cell biology and immunology. © 2015 by John Wiley & Sons, Inc.

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Phosphatase CD45 Both Positively and Negatively Regulates T Cell Receptor Phosphorylation in Reconstituted Membrane Protein Clusters

Cited by 30 publications

References 39 publications

The cytosolic domain of T-cell receptor ζ associates with membranes in a dynamic equilibrium and deeply penetrates the bilayer

The cytosolic domain of T-cell receptor ζ associates with membranes in a dynamic equilibrium and deeply penetrates the bilayer

Dynamic regulation of CD45 by tetraspanin CD53

Supported Lipid Bilayer Technology for the Study of Cellular Interfaces

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