CD44 Selectively Associates With Active Src Family Protein Tyrosine Kinases Lck and Fyn in Glycosphingolipid-Rich Plasma Membrane Domains of Human Peripheral Blood Lymphocytes

Abstract CD44 is the major cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan and is implicated in a variety of biological events that include embryonic morphogenesis, lymphocyte recirculation, inflammation, and tumor metastasis. CD44 delivers activation signals to T lymphocytes, B lymphocytes, natural killer cells, polymorphonuclear leukocytes, and macrophages by stimulating protein tyrosine phosphorylation and calcium influx. The mechanism of signal transduction via CD44 remains undefined, although CD44 was shown to physically associate with intracellular protein tyrosine kinase Lck in T lymphocytes. In the present report, we show that a significant proportion of CD44 in human peripheral blood T lymphocytes and endothelial cells is associated with low-density plasma membrane fractions that represent specialized plasma membrane domains enriched in glycosphingolipids and glycosylphosphatidylinositol (GPI)-anchored proteins. CD44 and the GPI-anchored CD59 do not appear to directly interact in the low-density membrane fractions. In human peripheral blood T lymphocytes, 20% to 30% of the Src family protein tyrosine kinases, Lck and Fyn, are recovered from these fractions. CD44-associated protein kinase activity was selectively recovered from the low-density membrane fractions, corresponding to glycosphingolipid-rich plasma membrane microdomains. Reprecipitation of the in vitro phosphorylated proteins showed that CD44 associates not only with Lck but also with Fyn kinase in these membrane domains. Our results suggest that cellular stimulation via CD44 may proceed through the signaling machinery of glycosphingolipid-enriched plasma membrane microdomains and, hence, depend on the functional integrity of such domains.

Download Full-text

CD44 Selectively Associates With Active Src Family Protein Tyrosine Kinases Lck and Fyn in Glycosphingolipid-Rich Plasma Membrane Domains of Human Peripheral Blood Lymphocytes

Blood ◽

10.1182/blood.v91.10.3901.3901_3901_3908 ◽

1998 ◽

Vol 91 (10) ◽

pp. 3901-3908 ◽

Cited By ~ 3

Author(s):

Subburaj Ilangumaran ◽

Anne Briol ◽

Daniel C. Hoessli

Keyword(s):

Plasma Membrane ◽

T Lymphocytes ◽

Peripheral Blood ◽

Tyrosine Kinases ◽

Human Peripheral Blood ◽

Membrane Microdomains ◽

Low Density ◽

Membrane Domains ◽

Protein Tyrosine ◽

Plasma Membrane Microdomains

CD44 is the major cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan and is implicated in a variety of biological events that include embryonic morphogenesis, lymphocyte recirculation, inflammation, and tumor metastasis. CD44 delivers activation signals to T lymphocytes, B lymphocytes, natural killer cells, polymorphonuclear leukocytes, and macrophages by stimulating protein tyrosine phosphorylation and calcium influx. The mechanism of signal transduction via CD44 remains undefined, although CD44 was shown to physically associate with intracellular protein tyrosine kinase Lck in T lymphocytes. In the present report, we show that a significant proportion of CD44 in human peripheral blood T lymphocytes and endothelial cells is associated with low-density plasma membrane fractions that represent specialized plasma membrane domains enriched in glycosphingolipids and glycosylphosphatidylinositol (GPI)-anchored proteins. CD44 and the GPI-anchored CD59 do not appear to directly interact in the low-density membrane fractions. In human peripheral blood T lymphocytes, 20% to 30% of the Src family protein tyrosine kinases, Lck and Fyn, are recovered from these fractions. CD44-associated protein kinase activity was selectively recovered from the low-density membrane fractions, corresponding to glycosphingolipid-rich plasma membrane microdomains. Reprecipitation of the in vitro phosphorylated proteins showed that CD44 associates not only with Lck but also with Fyn kinase in these membrane domains. Our results suggest that cellular stimulation via CD44 may proceed through the signaling machinery of glycosphingolipid-enriched plasma membrane microdomains and, hence, depend on the functional integrity of such domains.

Download Full-text

Diacylglycerol activates the influx of extracellular cations in T-lymphocytes independently of intracellular calcium-store depletion and possibly involving endogenous TRP6 gene products

Biochemical Journal ◽

10.1042/bj3640245 ◽

2002 ◽

Vol 364 (1) ◽

pp. 245-254 ◽

Cited By ~ 60

Author(s):

Alessandra GAMBERUCCI ◽

Emanuele GIURISATO ◽

Paola PIZZO ◽

Maristella TASSI ◽

Roberta GIUNTI ◽

...

Keyword(s):

Plasma Membrane ◽

T Lymphocytes ◽

Peripheral Blood ◽

Transient Receptor Potential ◽

Human Peripheral Blood ◽

Receptor Potential ◽

Animal Cells ◽

Bivalent Cation ◽

Intracellular Calcium Store ◽

Transient Receptor

In Jurkat and human peripheral blood T-lymphocytes, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analogue of diacylglycerol, activated the influx of Ca2+, Ba2+ and Sr2+. OAG also caused plasma-membrane depolarization in Ca2+-free media that was recovered by the addition of bivalent cation, indicating the activation of Na+ influx. OAG-induced cation influx was (i) mimicked by the natural dacylglycerol 1-stearoyl-2-arachidonyl-sn-glycerol, (ii) not blocked by inhibiting protein kinase C or in the absence of phopholipase C activity and (iii) blocked by La3+ and Gd3+. Differently from OAG, both thapsigargin and phytohaemagglutinin activated a potent influx of Ca2+, but little influx of Ba2+ and Sr2+. Moreover, the influx of Ca2+ activated by thapsigargin and that activated by OAG were additive. Furthermore, several drugs (i.e. econazole, SKF96365, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, 2-aminoethoxy diphenylborate and calyculin-A), while inhibiting the influx of Ca2+ induced by both thapsigargin and phytohaemagglutinin, did not affect OAG-stimulated cation influx. Transient receptor potential (TRP) 3 and TRP6 proteins have been shown previously to be activated by diacylglycerol when expressed heterologously in animal cells [Hofmann, Obukhov, Schaefer, Harteneck, Gudermann and Schultz (1999) Nature (London) 397, 259–263]. In both Jurkat and peripheral blood T-lymphocytes, mRNA encoding TRP proteins 1, 3, 4 and 6 was detected by reverse transcriptase PCR, and the TRP6 protein was detected by Western blotting in a purified plasma-membrane fraction. We conclude that T-cells express a diacylglycerol-activated cation channel, unrelated to the channel involved in capacitative Ca2+ entry, and associated with the expression of TRP6 protein.

Download Full-text

Identification of low-density Triton X-100-insoluble plasma membrane microdomains in higher plants

European Journal of Biochemistry ◽

10.1046/j.1432-1327.2000.01776.x ◽

2000 ◽

Vol 267 (24) ◽

pp. 6989-6995 ◽

Cited By ~ 93

Author(s):

Tatjana Peskan ◽

Martin Westermann ◽

Ralf Oelmüller

Keyword(s):

Plasma Membrane ◽

Higher Plants ◽

Membrane Microdomains ◽

Low Density ◽

Triton X ◽

Plasma Membrane Microdomains

Download Full-text

Distinct intracellular localization of Lck and Fyn protein tyrosine kinases in human T lymphocytes.

The Journal of Cell Biology ◽

10.1083/jcb.125.3.639 ◽

1994 ◽

Vol 125 (3) ◽

pp. 639-649 ◽

Cited By ~ 119

Author(s):

S C Ley ◽

M Marsh ◽

C R Bebbington ◽

K Proudfoot ◽

P Jordan

Keyword(s):

T Cells ◽

Plasma Membrane ◽

T Cell ◽

T Lymphocytes ◽

Tyrosine Kinases ◽

Intracellular Localization ◽

Protein Tyrosine Kinases ◽

Jurkat T Cells ◽

Protein Tyrosine ◽

Mitotic Cells

Two src family kinases, lck and fyn, participate in the activation of T lymphocytes. Both of these protein tyrosine kinases are thought to function via their interaction with cell surface receptors. Thus, lck is associated with CD4, CD8, and Thy-1, whereas fyn is associated with the T cell antigen receptor and Thy-1. In this study, the intracellular localization of these two protein tyrosine kinases in T cells was analyzed by immunofluorescence and confocal microscopy. Lck was present at the plasma membrane, consistent with its proposed role in transmembrane signalling, and was also associated with pericentrosomal vesicles which co-localized with the cation-independent mannose 6-phosphate receptor. Surprisingly, fyn was not detected at the plasma membrane in either Jurkat T cells or T lymphoblasts but was closely associated with the centrosome and to microtubule bundles radiating from the centrosome. In mitotic cells, fyn co-localized with the mitotic spindle and poles. The essentially non-overlapping intracellular distributions of lck and fyn suggest that these kinases may be accessible to distinct regulatory proteins and substrates and, therefore, may regulate different aspects of T cell activation. Anti-phosphotyrosine antibody staining at the plasma membrane increases dramatically after CD3 cross-linking of Jurkat T cells. The localization of lck to the plasma membrane suggests that it may participate in mediating this increase in tyrosine phosphorylation, rather than fyn. Furthermore, the distribution of fyn in mitotic cells raises the possibility that it functions at the M phase of the cell cycle.

Download Full-text

Activation of human peripheral blood T lymphocytes by pharmacological induction of protein-tyrosine phosphorylation.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.89.21.10306 ◽

1992 ◽

Vol 89 (21) ◽

pp. 10306-10310 ◽

Cited By ~ 110

Author(s):

J. J. O'Shea ◽

D. W. McVicar ◽

T. L. Bailey ◽

C. Burns ◽

M. J. Smyth

Keyword(s):

T Lymphocytes ◽

Tyrosine Phosphorylation ◽

Peripheral Blood ◽

Human Peripheral Blood ◽

Protein Tyrosine Phosphorylation ◽

Protein Tyrosine

Download Full-text

Membrane microdomains: lessons from microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140257 ◽

1995 ◽

Vol 53 ◽

pp. 776-777

Author(s):

J.M. Robinson ◽

J.M Oliver

Keyword(s):

Spatial Distribution ◽

Plasma Membrane ◽

Epithelial Cells ◽

Plasma Membranes ◽

Cell Types ◽

Imaging Modalities ◽

Membrane Microdomains ◽

Membrane Domains ◽

Lateral Heterogeneity ◽

Functional Importance

Specialized regions of plasma membranes displaying lateral heterogeneity are the focus of this Symposium. Specialized membrane domains are known for certain cell types such as differentiated epithelial cells where lateral heterogeneity in lipids and proteins exists between the apical and basolateral portions of the plasma membrane. Lateral heterogeneity and the presence of microdomains in membranes that are uniform in appearance have been more difficult to establish. Nonetheless a number of studies have provided evidence for membrane microdomains and indicated a functional importance for these structures.This symposium will focus on the use of various imaging modalities and related approaches to define membrane microdomains in a number of cell types. The importance of existing as well as emerging imaging technologies for use in the elucidation of membrane microdomains will be highlighted. The organization of membrane microdomains in terms of dimensions and spatial distribution is of considerable interest and will be addressed in this Symposium.

Download Full-text

Faculty Opinions recommendation of Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signaling molecules in T cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1026541.328582 ◽

2005 ◽

Author(s):

Ken Jacobson

Keyword(s):

T Cells ◽

Plasma Membrane ◽

Single Molecule ◽

Signaling Molecules ◽

Membrane Microdomains ◽

Protein Networks ◽

Single Molecule Microscopy ◽

Plasma Membrane Microdomains

Download Full-text

Differential chemokine expression profiles in human peripheral blood T lymphocytes: dependence on T-cell coreceptor and calcineurin signaling

Blood ◽

10.1182/blood-2002-03-0697 ◽

2003 ◽

Vol 101 (1) ◽

pp. 216-225 ◽

Cited By ~ 18

Author(s):

Anthony D. Cristillo ◽

Mirtha J. Macri ◽

Barbara E. Bierer

Keyword(s):

T Cell ◽

T Lymphocytes ◽

Signaling Pathways ◽

Peripheral Blood ◽

Human Peripheral Blood ◽

Expression Profiles ◽

Lymphoid Tissues ◽

Rnase Protection ◽

Chemokine Expression ◽

Calcineurin Activity

Abstract The chemokine superfamily consists of small (8-10 kDa) molecules that function to attract, selectively, different subsets of leukocytes. Binding of chemokines to their appropriate G-protein–coupled receptors is necessary for primary immune responses and for homing of leukocytes to lymphoid tissues. Here, we have characterized the signaling pathways in primary T lymphocytes that regulate chemokine gene induction using an RNase protection assay. Dependence on stimulation through the coreceptor CD28 and sensitivity to the calcineurin inhibitors cyclosporine and tacrolimus were studied using purified human peripheral blood lymphocytes. Lymphotactin (Ltn), macrophage inflammatory protein (MIP)–1α, and MIP-1β were all rapidly induced and sensitive to cyclosporine treatment. At later time points, the expression of MIP-1α and MIP-1β, but not of Ltn, was restored despite the inhibition of calcineurin activity. By contrast, the induction of interleukin-8 was delayed and was found to be cyclosporine insensitive. Calcineurin activity of IP-10 mRNA induction was contingent on the specific T-cell stimulation conditions, suggesting that IP-10 expression is modulated by calcineurin-dependent and -independent signaling pathways. Differential chemokine expression profiles result from the engagement of T-cell coreceptors and the requirement for, and the dependence on, calcineurin phosphatase activity.

Download Full-text

Flotillin microdomains interact with the cortical cytoskeleton to control uropod formation and neutrophil recruitment

The Journal of Cell Biology ◽

10.1083/jcb.201005140 ◽

2010 ◽

Vol 191 (4) ◽

pp. 771-781 ◽

Cited By ~ 76

Author(s):

Alexander Ludwig ◽

Grant P. Otto ◽

Kirsi Riento ◽

Emily Hams ◽

Padraic G. Fallon ◽

...

Keyword(s):

Plasma Membrane ◽

Ex Vivo ◽

Neutrophil Migration ◽

Neutrophil Recruitment ◽

Membrane Microdomains ◽

Neutrophil Chemotaxis ◽

Myosin Iia ◽

Plasma Membrane Microdomains ◽

Cortical Cytoskeleton

We studied the function of plasma membrane microdomains defined by the proteins flotillin 1 and flotillin 2 in uropod formation and neutrophil chemotaxis. Flotillins become concentrated in the uropod of neutrophils after exposure to chemoattractants such as N-formyl-Met-Leu-Phe (fMLP). Here, we show that mice lacking flotillin 1 do not have flotillin microdomains, and that recruitment of neutrophils toward fMLP in vivo is reduced in these mice. Ex vivo, migration of neutrophils through a resistive matrix is reduced in the absence of flotillin microdomains, but the machinery required for sensing chemoattractant functions normally. Flotillin microdomains specifically associate with myosin IIa, and spectrins. Both uropod formation and myosin IIa activity are compromised in flotillin 1 knockout neutrophils. We conclude that the association between flotillin microdomains and cortical cytoskeleton has important functions during neutrophil migration, in uropod formation, and in the regulation of myosin IIa.

Download Full-text