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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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(A) TCR-LAT assembly formation requires palmitoylation of LAT. LAT-deficient ANJ3 cells and ANJ3 cells reconstituted with WT or palmitoylation-deficient C26/29A LAT were used for α-CD3 immunoisolation. Immunoisolates were analyzed by Western blot using antibodies against the indicated antigens. A, B, and C show relative amounts of proteins in 1/10 of pelleted homogenates of ANJ3, ANJ3 wt LAT, and ANJ3 C26/29A derivatives, respectively (exposure times were 1/3 of the immunoisolates). (B) LAT with a Lck raft membrane anchor accumulates in TCR immunoisolates. LAT-deficient JCaM2 cells, JCaM2 expressing wt LAT, and Lck-LAT were subjected to α-CD3 immunoisolation. A, B, and C show relative amounts of proteins in 1/10 of pelleted homogenates of JCaM2, JCaM2 wt LAT, and Lck-LAT, respectively. Asterisks mark the position of Ab heavy chain; closed circles mark the position of the Ab light chain. The positions of molecular mass markers (in kilodaltons) are shown.
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Figure 5: (A) TCR-LAT assembly formation requires palmitoylation of LAT. LAT-deficient ANJ3 cells and ANJ3 cells reconstituted with WT or palmitoylation-deficient C26/29A LAT were used for α-CD3 immunoisolation. Immunoisolates were analyzed by Western blot using antibodies against the indicated antigens. A, B, and C show relative amounts of proteins in 1/10 of pelleted homogenates of ANJ3, ANJ3 wt LAT, and ANJ3 C26/29A derivatives, respectively (exposure times were 1/3 of the immunoisolates). (B) LAT with a Lck raft membrane anchor accumulates in TCR immunoisolates. LAT-deficient JCaM2 cells, JCaM2 expressing wt LAT, and Lck-LAT were subjected to α-CD3 immunoisolation. A, B, and C show relative amounts of proteins in 1/10 of pelleted homogenates of JCaM2, JCaM2 wt LAT, and Lck-LAT, respectively. Asterisks mark the position of Ab heavy chain; closed circles mark the position of the Ab light chain. The positions of molecular mass markers (in kilodaltons) are shown.

Mentions: Next, we tested whether LAT requires raft association for its accumulation in the TCR microenvironment. We used the LAT-deficient Jurkat derivative ANJ3 cells either reconstituted with wild-type LAT or with LAT lacking palmitoylation sites, i.e., a non-raft LAT mutant (Fig. 5 A; Zhang et al. 1998b; Lin et al. 1999). α-CD3 immunoisolates from ANJ3 contained TCR ζ-chain, albeit at a reduced immunoisolation efficiency, but lacked LAT and associated PLC-γ and Grb2. Importantly, LAT, PLC-γ, and Grb2 were efficiently coisolated in α-CD3–immunoisolated PM from wt LAT-reconstituted ANJ3 cells. In contrast, a LAT variant mutated in the S-palmitoylation sites Cys 26 and Cys 29 was not recruited into the α-CD3 isolates and failed to reconstitute PLC-γ and Grb2 recruitment into α-CD3 immunoisolates. Palmitoylation-deficient LAT mutant as well as wild-type LAT are both located in the PM (Zhang et al. 1998b). Thus, in addition to Lck/Fyn tyrosine kinase activity, LAT requires palmitoylation for its accumulation in the vicinity of triggered TCR.


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

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

(A) TCR-LAT assembly formation requires palmitoylation of LAT. LAT-deficient ANJ3 cells and ANJ3 cells reconstituted with WT or palmitoylation-deficient C26/29A LAT were used for α-CD3 immunoisolation. Immunoisolates were analyzed by Western blot using antibodies against the indicated antigens. A, B, and C show relative amounts of proteins in 1/10 of pelleted homogenates of ANJ3, ANJ3 wt LAT, and ANJ3 C26/29A derivatives, respectively (exposure times were 1/3 of the immunoisolates). (B) LAT with a Lck raft membrane anchor accumulates in TCR immunoisolates. LAT-deficient JCaM2 cells, JCaM2 expressing wt LAT, and Lck-LAT were subjected to α-CD3 immunoisolation. A, B, and C show relative amounts of proteins in 1/10 of pelleted homogenates of JCaM2, JCaM2 wt LAT, and Lck-LAT, respectively. Asterisks mark the position of Ab heavy chain; closed circles mark the position of the Ab light chain. The positions of molecular mass markers (in kilodaltons) are shown.
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Related In: Results  -  Collection

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Figure 5: (A) TCR-LAT assembly formation requires palmitoylation of LAT. LAT-deficient ANJ3 cells and ANJ3 cells reconstituted with WT or palmitoylation-deficient C26/29A LAT were used for α-CD3 immunoisolation. Immunoisolates were analyzed by Western blot using antibodies against the indicated antigens. A, B, and C show relative amounts of proteins in 1/10 of pelleted homogenates of ANJ3, ANJ3 wt LAT, and ANJ3 C26/29A derivatives, respectively (exposure times were 1/3 of the immunoisolates). (B) LAT with a Lck raft membrane anchor accumulates in TCR immunoisolates. LAT-deficient JCaM2 cells, JCaM2 expressing wt LAT, and Lck-LAT were subjected to α-CD3 immunoisolation. A, B, and C show relative amounts of proteins in 1/10 of pelleted homogenates of JCaM2, JCaM2 wt LAT, and Lck-LAT, respectively. Asterisks mark the position of Ab heavy chain; closed circles mark the position of the Ab light chain. The positions of molecular mass markers (in kilodaltons) are shown.
Mentions: Next, we tested whether LAT requires raft association for its accumulation in the TCR microenvironment. We used the LAT-deficient Jurkat derivative ANJ3 cells either reconstituted with wild-type LAT or with LAT lacking palmitoylation sites, i.e., a non-raft LAT mutant (Fig. 5 A; Zhang et al. 1998b; Lin et al. 1999). α-CD3 immunoisolates from ANJ3 contained TCR ζ-chain, albeit at a reduced immunoisolation efficiency, but lacked LAT and associated PLC-γ and Grb2. Importantly, LAT, PLC-γ, and Grb2 were efficiently coisolated in α-CD3–immunoisolated PM from wt LAT-reconstituted ANJ3 cells. In contrast, a LAT variant mutated in the S-palmitoylation sites Cys 26 and Cys 29 was not recruited into the α-CD3 isolates and failed to reconstitute PLC-γ and Grb2 recruitment into α-CD3 immunoisolates. Palmitoylation-deficient LAT mutant as well as wild-type LAT are both located in the PM (Zhang et al. 1998b). Thus, in addition to Lck/Fyn tyrosine kinase activity, LAT requires palmitoylation for its accumulation in the vicinity of triggered TCR.

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