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A balance between positive and negative signals in cytotoxic lymphocytes regulates the polarization of lipid rafts during the development of cell-mediated killing.

Lou Z, Jevremovic D, Billadeau DD, Leibson PJ - J. Exp. Med. (2000)

Bottom Line: The cross-linking of certain types of cell surface receptors initiates the redistribution of these lipid rafts, resulting in the formation of signaling complexes.This inhibition is dependent on the catalytic activity of KIR-associated SHP-1, a Src homology 2 (SH2) domain containing tyrosine phosphatase.These results suggest that the influence of integrated positive and negative signals on raft redistribution critically influences the development of cell-mediated cytotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Mayo Graduate and Medical Schools, Mayo Clinic, Rochester, Minnesota 55905, USA.

ABSTRACT
Plasma membrane microdomains containing sphingolipids and cholesterol (lipid rafts) are enriched in signaling molecules. The cross-linking of certain types of cell surface receptors initiates the redistribution of these lipid rafts, resulting in the formation of signaling complexes. However, little is known about the regulation of the initial raft redistribution and whether negative regulatory signaling pathways target this phase of cellular activation. We used natural killer (NK) cells as a model to investigate the regulation of raft redistribution, as both positive and negative signals have been implicated in the development of their cellular function. Here we show that after NK cells form conjugates with sensitive tumor cells, rafts become polarized to the site of target recognition. This redistribution of lipid rafts requires the activation of both Src and Syk family protein tyrosine kinases. In contrast, engagement of major histocompatibility complex (MHC)-recognizing killer cell inhibitory receptors (KIRs) on NK cells by resistant, MHC-bearing tumor targets blocks raft redistribution. This inhibition is dependent on the catalytic activity of KIR-associated SHP-1, a Src homology 2 (SH2) domain containing tyrosine phosphatase. These results suggest that the influence of integrated positive and negative signals on raft redistribution critically influences the development of cell-mediated cytotoxicity.

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Expression of dominant negative, catalytically inactive Syk inhibits raft redistribution. Left panels: NK cells were infected for 4 h at a multiplicity of infection of 20 with control recombinant vaccinia viruses (PSC-65) or recombinant vaccinia encoding either wild-type Syk or catalytically inactive, truncated Syk (Syk T). Infected NK cells were stained with FITC–CTx and incubated with hydroethidine-labeled K562. NK–target conjugates were scored for raft redistribution. Right panels: infected NK cells were incubated with 51Cr-labeled K562 cells for 4 h. The two results shown are representative of five total experiments.
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Figure 5: Expression of dominant negative, catalytically inactive Syk inhibits raft redistribution. Left panels: NK cells were infected for 4 h at a multiplicity of infection of 20 with control recombinant vaccinia viruses (PSC-65) or recombinant vaccinia encoding either wild-type Syk or catalytically inactive, truncated Syk (Syk T). Infected NK cells were stained with FITC–CTx and incubated with hydroethidine-labeled K562. NK–target conjugates were scored for raft redistribution. Right panels: infected NK cells were incubated with 51Cr-labeled K562 cells for 4 h. The two results shown are representative of five total experiments.

Mentions: SykT is a catalytically inactive truncated mutant of Syk kinase that can inhibit cytotoxicity when expressed in NK cells 14. We transiently expressed SykT in NK cells using recombinant vaccinia virus. The expression levels of the virus-encoded Syk T and wild-type Syk were equivalent to the levels of endogenous Syk (data not shown). As shown in Fig. 5, expression of SykT inhibits raft polarization and NK cell–mediated cytotoxicity when compared with expression of wild-type Syk or vector alone (PSC-65). Therefore, experiments using either pharmacologic or genetic approaches suggest that raft polarization requires the activity of proximal PTKs.


A balance between positive and negative signals in cytotoxic lymphocytes regulates the polarization of lipid rafts during the development of cell-mediated killing.

Lou Z, Jevremovic D, Billadeau DD, Leibson PJ - J. Exp. Med. (2000)

Expression of dominant negative, catalytically inactive Syk inhibits raft redistribution. Left panels: NK cells were infected for 4 h at a multiplicity of infection of 20 with control recombinant vaccinia viruses (PSC-65) or recombinant vaccinia encoding either wild-type Syk or catalytically inactive, truncated Syk (Syk T). Infected NK cells were stained with FITC–CTx and incubated with hydroethidine-labeled K562. NK–target conjugates were scored for raft redistribution. Right panels: infected NK cells were incubated with 51Cr-labeled K562 cells for 4 h. The two results shown are representative of five total experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195747&req=5

Figure 5: Expression of dominant negative, catalytically inactive Syk inhibits raft redistribution. Left panels: NK cells were infected for 4 h at a multiplicity of infection of 20 with control recombinant vaccinia viruses (PSC-65) or recombinant vaccinia encoding either wild-type Syk or catalytically inactive, truncated Syk (Syk T). Infected NK cells were stained with FITC–CTx and incubated with hydroethidine-labeled K562. NK–target conjugates were scored for raft redistribution. Right panels: infected NK cells were incubated with 51Cr-labeled K562 cells for 4 h. The two results shown are representative of five total experiments.
Mentions: SykT is a catalytically inactive truncated mutant of Syk kinase that can inhibit cytotoxicity when expressed in NK cells 14. We transiently expressed SykT in NK cells using recombinant vaccinia virus. The expression levels of the virus-encoded Syk T and wild-type Syk were equivalent to the levels of endogenous Syk (data not shown). As shown in Fig. 5, expression of SykT inhibits raft polarization and NK cell–mediated cytotoxicity when compared with expression of wild-type Syk or vector alone (PSC-65). Therefore, experiments using either pharmacologic or genetic approaches suggest that raft polarization requires the activity of proximal PTKs.

Bottom Line: The cross-linking of certain types of cell surface receptors initiates the redistribution of these lipid rafts, resulting in the formation of signaling complexes.This inhibition is dependent on the catalytic activity of KIR-associated SHP-1, a Src homology 2 (SH2) domain containing tyrosine phosphatase.These results suggest that the influence of integrated positive and negative signals on raft redistribution critically influences the development of cell-mediated cytotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Mayo Graduate and Medical Schools, Mayo Clinic, Rochester, Minnesota 55905, USA.

ABSTRACT
Plasma membrane microdomains containing sphingolipids and cholesterol (lipid rafts) are enriched in signaling molecules. The cross-linking of certain types of cell surface receptors initiates the redistribution of these lipid rafts, resulting in the formation of signaling complexes. However, little is known about the regulation of the initial raft redistribution and whether negative regulatory signaling pathways target this phase of cellular activation. We used natural killer (NK) cells as a model to investigate the regulation of raft redistribution, as both positive and negative signals have been implicated in the development of their cellular function. Here we show that after NK cells form conjugates with sensitive tumor cells, rafts become polarized to the site of target recognition. This redistribution of lipid rafts requires the activation of both Src and Syk family protein tyrosine kinases. In contrast, engagement of major histocompatibility complex (MHC)-recognizing killer cell inhibitory receptors (KIRs) on NK cells by resistant, MHC-bearing tumor targets blocks raft redistribution. This inhibition is dependent on the catalytic activity of KIR-associated SHP-1, a Src homology 2 (SH2) domain containing tyrosine phosphatase. These results suggest that the influence of integrated positive and negative signals on raft redistribution critically influences the development of cell-mediated cytotoxicity.

Show MeSH
Related in: MedlinePlus