Interaction of the cytoplasmic tail of CTLA-4 (CD152) with a clathrin- associated protein is negatively regulated by tyrosine phosphorylation

Jeffrey D. Bradshaw, Pin Lu, Gina Leytze, Julie Rodgers, Gary L. Schieven, Kelly L. Bennett, Peter S. Linsley, Stephen E. Kurtz

Research output: Contribution to journalArticle

99 Citations (Scopus)

Abstract

CTLA-4 (CD152), high-avidity receptor for CD80 and CD86, is a powerful regulator of T cell activation. While CTLA-4 functions at the cell surface, it is primarily localized in intracellular vesicles and cycles to the cell surface. The CTLA-4 cytoplasmic domain contains sequences that direct its intracellular localization and regulate its signaling. Here we demonstrate that effector molecules involved in receptor trafficking and signaling interact with distinct, but overlapping, sequences in the CTLA-4 cytoplasmic domain. Using the yeast two-hybrid method, we demonstrate association of the μ2 subunit of AP-2, the clathrin-associated complex found in plasma membrane-associated coated pits, with the cytoplasmic tail of CTLA-4, but not CD28. The μl subunit of AP-1, found in Golgi-associated coated pits, associated with neither CTLA-4 nor CD28. Sequences required for interaction of μ2 and CTLA-4 were localized to residues, 161TTGVY in CTLA-4; this sequence is N-terminal to, but overlaps with, a previously identified SH2 binding motif, 165YVKM, involved in CTLA-4 signaling. μ2 interacted preferentially with CTLA-4 when residue 165Y was nonphosphorylated, whereas a PI3 kinase SH2 domain interacted preferentially when 165Y was phosphorylated. In co-transfection experiments, both tyrosine residues in the cytoplasmic tail of CTLA-4 ( 165Y and 182Y) were phosphorylated by the T lymphocyte-associated tyrosine kinase, p561ck. Thus, phosphorylation of CTLA- 4 residue 165Y may reciprocally regulate signaling and trafficking of CTLA-4 by determining which effector molecules bind to its cytoplasmic tail.

Original languageEnglish (US)
Pages (from-to)15975-15982
Number of pages8
JournalBiochemistry
Volume36
Issue number50
DOIs
StatePublished - Dec 16 1997
Externally publishedYes

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Vesicular Transport Adaptor Proteins
Phosphorylation
Tyrosine
T-Lymphocytes
Clathrin
Two-Hybrid System Techniques
src Homology Domains
T-cells
Transcription Factor AP-1
Phosphatidylinositol 3-Kinases
Protein-Tyrosine Kinases
Transfection
Cell Cycle
Yeasts
Cell Membrane
Molecules
Cell membranes
Yeast
Chemical activation
Association reactions

ASJC Scopus subject areas

  • Biochemistry

Cite this

Interaction of the cytoplasmic tail of CTLA-4 (CD152) with a clathrin- associated protein is negatively regulated by tyrosine phosphorylation. / Bradshaw, Jeffrey D.; Lu, Pin; Leytze, Gina; Rodgers, Julie; Schieven, Gary L.; Bennett, Kelly L.; Linsley, Peter S.; Kurtz, Stephen E.

In: Biochemistry, Vol. 36, No. 50, 16.12.1997, p. 15975-15982.

Research output: Contribution to journalArticle

Bradshaw, Jeffrey D. ; Lu, Pin ; Leytze, Gina ; Rodgers, Julie ; Schieven, Gary L. ; Bennett, Kelly L. ; Linsley, Peter S. ; Kurtz, Stephen E. / Interaction of the cytoplasmic tail of CTLA-4 (CD152) with a clathrin- associated protein is negatively regulated by tyrosine phosphorylation. In: Biochemistry. 1997 ; Vol. 36, No. 50. pp. 15975-15982.
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T1 - Interaction of the cytoplasmic tail of CTLA-4 (CD152) with a clathrin- associated protein is negatively regulated by tyrosine phosphorylation

AU - Bradshaw, Jeffrey D.

AU - Lu, Pin

AU - Leytze, Gina

AU - Rodgers, Julie

AU - Schieven, Gary L.

AU - Bennett, Kelly L.

AU - Linsley, Peter S.

AU - Kurtz, Stephen E.

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N2 - CTLA-4 (CD152), high-avidity receptor for CD80 and CD86, is a powerful regulator of T cell activation. While CTLA-4 functions at the cell surface, it is primarily localized in intracellular vesicles and cycles to the cell surface. The CTLA-4 cytoplasmic domain contains sequences that direct its intracellular localization and regulate its signaling. Here we demonstrate that effector molecules involved in receptor trafficking and signaling interact with distinct, but overlapping, sequences in the CTLA-4 cytoplasmic domain. Using the yeast two-hybrid method, we demonstrate association of the μ2 subunit of AP-2, the clathrin-associated complex found in plasma membrane-associated coated pits, with the cytoplasmic tail of CTLA-4, but not CD28. The μl subunit of AP-1, found in Golgi-associated coated pits, associated with neither CTLA-4 nor CD28. Sequences required for interaction of μ2 and CTLA-4 were localized to residues, 161TTGVY in CTLA-4; this sequence is N-terminal to, but overlaps with, a previously identified SH2 binding motif, 165YVKM, involved in CTLA-4 signaling. μ2 interacted preferentially with CTLA-4 when residue 165Y was nonphosphorylated, whereas a PI3 kinase SH2 domain interacted preferentially when 165Y was phosphorylated. In co-transfection experiments, both tyrosine residues in the cytoplasmic tail of CTLA-4 ( 165Y and 182Y) were phosphorylated by the T lymphocyte-associated tyrosine kinase, p561ck. Thus, phosphorylation of CTLA- 4 residue 165Y may reciprocally regulate signaling and trafficking of CTLA-4 by determining which effector molecules bind to its cytoplasmic tail.

AB - CTLA-4 (CD152), high-avidity receptor for CD80 and CD86, is a powerful regulator of T cell activation. While CTLA-4 functions at the cell surface, it is primarily localized in intracellular vesicles and cycles to the cell surface. The CTLA-4 cytoplasmic domain contains sequences that direct its intracellular localization and regulate its signaling. Here we demonstrate that effector molecules involved in receptor trafficking and signaling interact with distinct, but overlapping, sequences in the CTLA-4 cytoplasmic domain. Using the yeast two-hybrid method, we demonstrate association of the μ2 subunit of AP-2, the clathrin-associated complex found in plasma membrane-associated coated pits, with the cytoplasmic tail of CTLA-4, but not CD28. The μl subunit of AP-1, found in Golgi-associated coated pits, associated with neither CTLA-4 nor CD28. Sequences required for interaction of μ2 and CTLA-4 were localized to residues, 161TTGVY in CTLA-4; this sequence is N-terminal to, but overlaps with, a previously identified SH2 binding motif, 165YVKM, involved in CTLA-4 signaling. μ2 interacted preferentially with CTLA-4 when residue 165Y was nonphosphorylated, whereas a PI3 kinase SH2 domain interacted preferentially when 165Y was phosphorylated. In co-transfection experiments, both tyrosine residues in the cytoplasmic tail of CTLA-4 ( 165Y and 182Y) were phosphorylated by the T lymphocyte-associated tyrosine kinase, p561ck. Thus, phosphorylation of CTLA- 4 residue 165Y may reciprocally regulate signaling and trafficking of CTLA-4 by determining which effector molecules bind to its cytoplasmic tail.

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