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Selective accumulation of raft-associated membrane protein LAT in T cell receptor signaling assemblies.

Harder T, Kuhn M - J. Cell Biol. (2000)

Bottom Line: We found that the raft transmembrane protein linker for activation of T cells (LAT), but not a palmitoylation-deficient non-raft LAT mutant, strongly accumulated in TCR-enriched immunoisolates in a tyrosine phosphorylation-dependent manner.In contrast, other raft-associated molecules, including protein tyrosine kinases Lck and Fyn, GM1, and cholesterol, were not highly concentrated in TCR-enriched plasma membrane immunoisolates.Our results indicate that TCR signaling assemblies in plasma membrane subdomains, rather than generally concentrating raft-associated membrane proteins and lipids, form by a selective protein-mediated anchoring of the raft membrane protein LAT in vicinity of TCR.

View Article: PubMed Central - PubMed

Affiliation: Basel Institute for Immunology, CH-4005, Basel, Switzerland.

ABSTRACT
Activation of T cell antigen receptor (TCR) induces tyrosine phosphorylations that mediate the assembly of signaling protein complexes. Moreover, cholesterol-sphingolipid raft membrane domains have been implicated to play a role in TCR signal transduction. Here, we studied the assembly of TCR with signal transduction proteins and raft markers in plasma membrane subdomains of Jurkat T leukemic cells. We employed a novel method to immunoisolate plasma membrane subfragments that were highly concentrated in activated TCR-CD3 complexes and associated signaling proteins. We found that the raft transmembrane protein linker for activation of T cells (LAT), but not a palmitoylation-deficient non-raft LAT mutant, strongly accumulated in TCR-enriched immunoisolates in a tyrosine phosphorylation-dependent manner. In contrast, other raft-associated molecules, including protein tyrosine kinases Lck and Fyn, GM1, and cholesterol, were not highly concentrated in TCR-enriched plasma membrane immunoisolates. Many downstream signaling proteins coisolated with the TCR/LAT-enriched plasma membrane fragments, suggesting that LAT/TCR assemblies form a structural scaffold for TCR signal transduction proteins. Our results indicate that TCR signaling assemblies in plasma membrane subdomains, rather than generally concentrating raft-associated membrane proteins and lipids, form by a selective protein-mediated anchoring of the raft membrane protein LAT in vicinity of TCR.

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α-CD3 immunoisolation from cholesterol-depleted Jurkat cells. The conditions of MβCD depletion depicted as 0 mM, mock depletion; 4 mM, 4 mM MβCD for 40 min; and 10 mM, 10 mM MβCD for 20 min. Western blots were probed with Abs against the indicated proteins. (A) Association with Triton X-100–insoluble membranes: DRMs were recovered in fraction 2, whereas fractions 6 and 7 contained Triton X-100–soluble proteins. (B) Effect of cholesterol depletions on recovery of signaling protein in α-CD3 immunoisolates. α-CD3 immunoisolates from MβCD-extracted cells were analyzed by Western blot using antibodies against the indicated antigens. Asterisks mark the position of heavy chain; closed circles mark the position of the light chain of the antibody used for the immunoisolation. The positions of the p21 and phosphorylated p23 form of TCR ζ-chain are shown. Positions of molecular mass markers (in kilodaltons) are indicated.
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Figure 6: α-CD3 immunoisolation from cholesterol-depleted Jurkat cells. The conditions of MβCD depletion depicted as 0 mM, mock depletion; 4 mM, 4 mM MβCD for 40 min; and 10 mM, 10 mM MβCD for 20 min. Western blots were probed with Abs against the indicated proteins. (A) Association with Triton X-100–insoluble membranes: DRMs were recovered in fraction 2, whereas fractions 6 and 7 contained Triton X-100–soluble proteins. (B) Effect of cholesterol depletions on recovery of signaling protein in α-CD3 immunoisolates. α-CD3 immunoisolates from MβCD-extracted cells were analyzed by Western blot using antibodies against the indicated antigens. Asterisks mark the position of heavy chain; closed circles mark the position of the light chain of the antibody used for the immunoisolation. The positions of the p21 and phosphorylated p23 form of TCR ζ-chain are shown. Positions of molecular mass markers (in kilodaltons) are indicated.

Mentions: Disruption of raft domains using cholesterol depletion has been used to study the role of raft domains in TCR signaling (Xavier et al. 1998; Kabouridis et al. 2000). Importantly, Kabouridis et al. 2000 showed that cholesterol depletion of Jurkat cells with 10 mM MβCD abrogated DRM association of Lck and LAT within the first 20 min of extraction and, in parallel, stimulated tyrosine phosphorylations and the ras pathway (Kabouridis et al. 2000). Therefore, we studied the effects of these cholesterol depletion conditions on the recovery of TCR signaling proteins in our immunoisolation procedure. Extraction with 10 mM MβCD for 20 min reduced the amount of cholesterol in Jurkat cells to 35 ± 2% SD of the control value. Moreover, it strongly reduced DRM association of Lck and LAT in Triton X-100 Jurkat lysates, suggesting a reduction of raft association (Fig. 6 A). Interestingly, the α-CD3 immunoisolates of Jurkat cells treated for 20 min with 10 mM MβCD contained the same set of tyrosine-phosphorylated signaling proteins as the isolates from the control cells. Indeed, the recovery of TCR-LAT signaling assemblies from these cells was slightly enhanced (Fig. 6 B). This showed that TCR signaling protein assemblies form after MβCD extraction when raft domains are disrupted but tyrosine phosphorylations are increased and deregulated. This strongly supports the view that LAT-TCR assemblies are stably connected via tyrosine phosphorylation–dependent protein–protein interactions and are not coupled by cholesterol-dependent raft lipid domains.


Selective accumulation of raft-associated membrane protein LAT in T cell receptor signaling assemblies.

Harder T, Kuhn M - J. Cell Biol. (2000)

α-CD3 immunoisolation from cholesterol-depleted Jurkat cells. The conditions of MβCD depletion depicted as 0 mM, mock depletion; 4 mM, 4 mM MβCD for 40 min; and 10 mM, 10 mM MβCD for 20 min. Western blots were probed with Abs against the indicated proteins. (A) Association with Triton X-100–insoluble membranes: DRMs were recovered in fraction 2, whereas fractions 6 and 7 contained Triton X-100–soluble proteins. (B) Effect of cholesterol depletions on recovery of signaling protein in α-CD3 immunoisolates. α-CD3 immunoisolates from MβCD-extracted cells were analyzed by Western blot using antibodies against the indicated antigens. Asterisks mark the position of heavy chain; closed circles mark the position of the light chain of the antibody used for the immunoisolation. The positions of the p21 and phosphorylated p23 form of TCR ζ-chain are shown. Positions of molecular mass markers (in kilodaltons) are indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2192654&req=5

Figure 6: α-CD3 immunoisolation from cholesterol-depleted Jurkat cells. The conditions of MβCD depletion depicted as 0 mM, mock depletion; 4 mM, 4 mM MβCD for 40 min; and 10 mM, 10 mM MβCD for 20 min. Western blots were probed with Abs against the indicated proteins. (A) Association with Triton X-100–insoluble membranes: DRMs were recovered in fraction 2, whereas fractions 6 and 7 contained Triton X-100–soluble proteins. (B) Effect of cholesterol depletions on recovery of signaling protein in α-CD3 immunoisolates. α-CD3 immunoisolates from MβCD-extracted cells were analyzed by Western blot using antibodies against the indicated antigens. Asterisks mark the position of heavy chain; closed circles mark the position of the light chain of the antibody used for the immunoisolation. The positions of the p21 and phosphorylated p23 form of TCR ζ-chain are shown. Positions of molecular mass markers (in kilodaltons) are indicated.
Mentions: Disruption of raft domains using cholesterol depletion has been used to study the role of raft domains in TCR signaling (Xavier et al. 1998; Kabouridis et al. 2000). Importantly, Kabouridis et al. 2000 showed that cholesterol depletion of Jurkat cells with 10 mM MβCD abrogated DRM association of Lck and LAT within the first 20 min of extraction and, in parallel, stimulated tyrosine phosphorylations and the ras pathway (Kabouridis et al. 2000). Therefore, we studied the effects of these cholesterol depletion conditions on the recovery of TCR signaling proteins in our immunoisolation procedure. Extraction with 10 mM MβCD for 20 min reduced the amount of cholesterol in Jurkat cells to 35 ± 2% SD of the control value. Moreover, it strongly reduced DRM association of Lck and LAT in Triton X-100 Jurkat lysates, suggesting a reduction of raft association (Fig. 6 A). Interestingly, the α-CD3 immunoisolates of Jurkat cells treated for 20 min with 10 mM MβCD contained the same set of tyrosine-phosphorylated signaling proteins as the isolates from the control cells. Indeed, the recovery of TCR-LAT signaling assemblies from these cells was slightly enhanced (Fig. 6 B). This showed that TCR signaling protein assemblies form after MβCD extraction when raft domains are disrupted but tyrosine phosphorylations are increased and deregulated. This strongly supports the view that LAT-TCR assemblies are stably connected via tyrosine phosphorylation–dependent protein–protein interactions and are not coupled by cholesterol-dependent raft lipid domains.

Bottom Line: We found that the raft transmembrane protein linker for activation of T cells (LAT), but not a palmitoylation-deficient non-raft LAT mutant, strongly accumulated in TCR-enriched immunoisolates in a tyrosine phosphorylation-dependent manner.In contrast, other raft-associated molecules, including protein tyrosine kinases Lck and Fyn, GM1, and cholesterol, were not highly concentrated in TCR-enriched plasma membrane immunoisolates.Our results indicate that TCR signaling assemblies in plasma membrane subdomains, rather than generally concentrating raft-associated membrane proteins and lipids, form by a selective protein-mediated anchoring of the raft membrane protein LAT in vicinity of TCR.

View Article: PubMed Central - PubMed

Affiliation: Basel Institute for Immunology, CH-4005, Basel, Switzerland.

ABSTRACT
Activation of T cell antigen receptor (TCR) induces tyrosine phosphorylations that mediate the assembly of signaling protein complexes. Moreover, cholesterol-sphingolipid raft membrane domains have been implicated to play a role in TCR signal transduction. Here, we studied the assembly of TCR with signal transduction proteins and raft markers in plasma membrane subdomains of Jurkat T leukemic cells. We employed a novel method to immunoisolate plasma membrane subfragments that were highly concentrated in activated TCR-CD3 complexes and associated signaling proteins. We found that the raft transmembrane protein linker for activation of T cells (LAT), but not a palmitoylation-deficient non-raft LAT mutant, strongly accumulated in TCR-enriched immunoisolates in a tyrosine phosphorylation-dependent manner. In contrast, other raft-associated molecules, including protein tyrosine kinases Lck and Fyn, GM1, and cholesterol, were not highly concentrated in TCR-enriched plasma membrane immunoisolates. Many downstream signaling proteins coisolated with the TCR/LAT-enriched plasma membrane fragments, suggesting that LAT/TCR assemblies form a structural scaffold for TCR signal transduction proteins. Our results indicate that TCR signaling assemblies in plasma membrane subdomains, rather than generally concentrating raft-associated membrane proteins and lipids, form by a selective protein-mediated anchoring of the raft membrane protein LAT in vicinity of TCR.

Show MeSH
Related in: MedlinePlus