Fcγ receptor type IIb induced recruitment of inositol and protein phosphatases to the signal transductory complex of human B-cell

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Abstract

Co-clustering of FcγRIIb and B-cell receptor (BCR) inhibits cell activation by interrupting BCR stimulated signal transduction. The immunoreceptor tyrosine-based inhibitory motif (ITIM) of FcγRIIb becomes tyrosyl phosphorylated (P-ITIM) upon co-clustering with BCR then P-ITIM interacts with several signalling molecules, some of which negatively regulate the cell activation process. The molecules recruited by the P-ITIM of human FcγRIIb have not been characterised yet. In order to affinity isolate the potential functional partner molecules of human FcγRIIb, synthetic peptides were designed to cover almost the entire intracellular FcγRIIb domain, including FcγRIIb1 and FcγRIIb2 specific sequences and stretches containing the phosphorylated and non-phosphorylated ITIM. We report here that several tyrosyl phosphorylated proteins bind to the P-ITIM peptide from both resting and activated B-cell lysates, the 53-56 kDa being the most prominent one. A fraction of the 53-56 kDa bands were identified as the protein tyrosine kinase (PTK), Lyn which also bound to ITIM peptide, pointing to its role in initiating FcγRIIb-mediated negative regulation. Among the P-ITIM associated tyr phosphorylated components, the 145 kDa one was identified as the inositol polyphosphate 5-phosphatase, SHIP and the 72 kDa protein as the protein tyrosine phosphatase (PTP) SHP2, whereas SHP1 was not detected. Phosphatase activity assays showed that P-ITIM bound about five times higher SHIP and four times higher PTP activity than the ITIM containing peptide. Furthermore, we detected PKC and MAPK in both ITIM and P-ITIM peptides precipitated samples. Since human B-cells express both FcγRIIb1 and FcγRIIb2, differing in a 19 amino acid insert in the cytoplasmic tail of the former, we investigated the components binding to FcγRIIb1 and FcγRIIb2 specific sequences. Synthetic peptide representing FcγRIIb1 and FcγRIIb2 specific sequences weakly bound unidentified tyr phosphorylated proteins at 50-56 kDa, while the insert itself did not bind a detectable amount of protein. Neither of the ITIM or P-ITIM bound molecules were observed in samples precipitated with peptides corresponding to FcγRIIb1 or FcγRIIb2 specific sequences. These observations suggest that protein kinases associate with both ITIM and P-ITIM of human FcγRIIb, Lyn being responsible for the tyrosyl phosphorylation of ITIM. SH1P and SHP2 phosphatases selectively bind to the phosphorylated ITIM. Based on these data we assume that SHIP and SHP2 recruited in vivo to the FcγRIIb co-clustered BCR are responsible for the FcγRIIb mediated negative regulation of human B-cell activation.

Original languageEnglish
Pages (from-to)159-164
Number of pages6
JournalImmunology Letters
Volume57
Issue number1-3
DOIs
Publication statusPublished - 1997

Fingerprint

Fc Receptors
Phosphoprotein Phosphatases
Tyrosine
B-Lymphocytes
Peptides
myo-inositol-1 (or 4)-monophosphatase
Protein Tyrosine Phosphatases
Cluster Analysis
Proteins
Non-Receptor Type 11 Protein Tyrosine Phosphatase

Keywords

  • FcγRIIb
  • Human B-lymphocytes
  • Phosphatases
  • Regulation
  • Signal transduction

ASJC Scopus subject areas

  • Immunology
  • Immunology and Allergy

Cite this

@article{400d0e23e17049b49199a8dd817ecbb8,
title = "Fcγ receptor type IIb induced recruitment of inositol and protein phosphatases to the signal transductory complex of human B-cell",
abstract = "Co-clustering of FcγRIIb and B-cell receptor (BCR) inhibits cell activation by interrupting BCR stimulated signal transduction. The immunoreceptor tyrosine-based inhibitory motif (ITIM) of FcγRIIb becomes tyrosyl phosphorylated (P-ITIM) upon co-clustering with BCR then P-ITIM interacts with several signalling molecules, some of which negatively regulate the cell activation process. The molecules recruited by the P-ITIM of human FcγRIIb have not been characterised yet. In order to affinity isolate the potential functional partner molecules of human FcγRIIb, synthetic peptides were designed to cover almost the entire intracellular FcγRIIb domain, including FcγRIIb1 and FcγRIIb2 specific sequences and stretches containing the phosphorylated and non-phosphorylated ITIM. We report here that several tyrosyl phosphorylated proteins bind to the P-ITIM peptide from both resting and activated B-cell lysates, the 53-56 kDa being the most prominent one. A fraction of the 53-56 kDa bands were identified as the protein tyrosine kinase (PTK), Lyn which also bound to ITIM peptide, pointing to its role in initiating FcγRIIb-mediated negative regulation. Among the P-ITIM associated tyr phosphorylated components, the 145 kDa one was identified as the inositol polyphosphate 5-phosphatase, SHIP and the 72 kDa protein as the protein tyrosine phosphatase (PTP) SHP2, whereas SHP1 was not detected. Phosphatase activity assays showed that P-ITIM bound about five times higher SHIP and four times higher PTP activity than the ITIM containing peptide. Furthermore, we detected PKC and MAPK in both ITIM and P-ITIM peptides precipitated samples. Since human B-cells express both FcγRIIb1 and FcγRIIb2, differing in a 19 amino acid insert in the cytoplasmic tail of the former, we investigated the components binding to FcγRIIb1 and FcγRIIb2 specific sequences. Synthetic peptide representing FcγRIIb1 and FcγRIIb2 specific sequences weakly bound unidentified tyr phosphorylated proteins at 50-56 kDa, while the insert itself did not bind a detectable amount of protein. Neither of the ITIM or P-ITIM bound molecules were observed in samples precipitated with peptides corresponding to FcγRIIb1 or FcγRIIb2 specific sequences. These observations suggest that protein kinases associate with both ITIM and P-ITIM of human FcγRIIb, Lyn being responsible for the tyrosyl phosphorylation of ITIM. SH1P and SHP2 phosphatases selectively bind to the phosphorylated ITIM. Based on these data we assume that SHIP and SHP2 recruited in vivo to the FcγRIIb co-clustered BCR are responsible for the FcγRIIb mediated negative regulation of human B-cell activation.",
keywords = "FcγRIIb, Human B-lymphocytes, Phosphatases, Regulation, Signal transduction",
author = "G. S{\'a}rmay and G. Koncz and I. Pecht and J. Gergely",
year = "1997",
doi = "10.1016/S0165-2478(97)00055-2",
language = "English",
volume = "57",
pages = "159--164",
journal = "Immunology Letters",
issn = "0165-2478",
publisher = "Elsevier",
number = "1-3",

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TY - JOUR

T1 - Fcγ receptor type IIb induced recruitment of inositol and protein phosphatases to the signal transductory complex of human B-cell

AU - Sármay, G.

AU - Koncz, G.

AU - Pecht, I.

AU - Gergely, J.

PY - 1997

Y1 - 1997

N2 - Co-clustering of FcγRIIb and B-cell receptor (BCR) inhibits cell activation by interrupting BCR stimulated signal transduction. The immunoreceptor tyrosine-based inhibitory motif (ITIM) of FcγRIIb becomes tyrosyl phosphorylated (P-ITIM) upon co-clustering with BCR then P-ITIM interacts with several signalling molecules, some of which negatively regulate the cell activation process. The molecules recruited by the P-ITIM of human FcγRIIb have not been characterised yet. In order to affinity isolate the potential functional partner molecules of human FcγRIIb, synthetic peptides were designed to cover almost the entire intracellular FcγRIIb domain, including FcγRIIb1 and FcγRIIb2 specific sequences and stretches containing the phosphorylated and non-phosphorylated ITIM. We report here that several tyrosyl phosphorylated proteins bind to the P-ITIM peptide from both resting and activated B-cell lysates, the 53-56 kDa being the most prominent one. A fraction of the 53-56 kDa bands were identified as the protein tyrosine kinase (PTK), Lyn which also bound to ITIM peptide, pointing to its role in initiating FcγRIIb-mediated negative regulation. Among the P-ITIM associated tyr phosphorylated components, the 145 kDa one was identified as the inositol polyphosphate 5-phosphatase, SHIP and the 72 kDa protein as the protein tyrosine phosphatase (PTP) SHP2, whereas SHP1 was not detected. Phosphatase activity assays showed that P-ITIM bound about five times higher SHIP and four times higher PTP activity than the ITIM containing peptide. Furthermore, we detected PKC and MAPK in both ITIM and P-ITIM peptides precipitated samples. Since human B-cells express both FcγRIIb1 and FcγRIIb2, differing in a 19 amino acid insert in the cytoplasmic tail of the former, we investigated the components binding to FcγRIIb1 and FcγRIIb2 specific sequences. Synthetic peptide representing FcγRIIb1 and FcγRIIb2 specific sequences weakly bound unidentified tyr phosphorylated proteins at 50-56 kDa, while the insert itself did not bind a detectable amount of protein. Neither of the ITIM or P-ITIM bound molecules were observed in samples precipitated with peptides corresponding to FcγRIIb1 or FcγRIIb2 specific sequences. These observations suggest that protein kinases associate with both ITIM and P-ITIM of human FcγRIIb, Lyn being responsible for the tyrosyl phosphorylation of ITIM. SH1P and SHP2 phosphatases selectively bind to the phosphorylated ITIM. Based on these data we assume that SHIP and SHP2 recruited in vivo to the FcγRIIb co-clustered BCR are responsible for the FcγRIIb mediated negative regulation of human B-cell activation.

AB - Co-clustering of FcγRIIb and B-cell receptor (BCR) inhibits cell activation by interrupting BCR stimulated signal transduction. The immunoreceptor tyrosine-based inhibitory motif (ITIM) of FcγRIIb becomes tyrosyl phosphorylated (P-ITIM) upon co-clustering with BCR then P-ITIM interacts with several signalling molecules, some of which negatively regulate the cell activation process. The molecules recruited by the P-ITIM of human FcγRIIb have not been characterised yet. In order to affinity isolate the potential functional partner molecules of human FcγRIIb, synthetic peptides were designed to cover almost the entire intracellular FcγRIIb domain, including FcγRIIb1 and FcγRIIb2 specific sequences and stretches containing the phosphorylated and non-phosphorylated ITIM. We report here that several tyrosyl phosphorylated proteins bind to the P-ITIM peptide from both resting and activated B-cell lysates, the 53-56 kDa being the most prominent one. A fraction of the 53-56 kDa bands were identified as the protein tyrosine kinase (PTK), Lyn which also bound to ITIM peptide, pointing to its role in initiating FcγRIIb-mediated negative regulation. Among the P-ITIM associated tyr phosphorylated components, the 145 kDa one was identified as the inositol polyphosphate 5-phosphatase, SHIP and the 72 kDa protein as the protein tyrosine phosphatase (PTP) SHP2, whereas SHP1 was not detected. Phosphatase activity assays showed that P-ITIM bound about five times higher SHIP and four times higher PTP activity than the ITIM containing peptide. Furthermore, we detected PKC and MAPK in both ITIM and P-ITIM peptides precipitated samples. Since human B-cells express both FcγRIIb1 and FcγRIIb2, differing in a 19 amino acid insert in the cytoplasmic tail of the former, we investigated the components binding to FcγRIIb1 and FcγRIIb2 specific sequences. Synthetic peptide representing FcγRIIb1 and FcγRIIb2 specific sequences weakly bound unidentified tyr phosphorylated proteins at 50-56 kDa, while the insert itself did not bind a detectable amount of protein. Neither of the ITIM or P-ITIM bound molecules were observed in samples precipitated with peptides corresponding to FcγRIIb1 or FcγRIIb2 specific sequences. These observations suggest that protein kinases associate with both ITIM and P-ITIM of human FcγRIIb, Lyn being responsible for the tyrosyl phosphorylation of ITIM. SH1P and SHP2 phosphatases selectively bind to the phosphorylated ITIM. Based on these data we assume that SHIP and SHP2 recruited in vivo to the FcγRIIb co-clustered BCR are responsible for the FcγRIIb mediated negative regulation of human B-cell activation.

KW - FcγRIIb

KW - Human B-lymphocytes

KW - Phosphatases

KW - Regulation

KW - Signal transduction

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U2 - 10.1016/S0165-2478(97)00055-2

DO - 10.1016/S0165-2478(97)00055-2

M3 - Article

VL - 57

SP - 159

EP - 164

JO - Immunology Letters

JF - Immunology Letters

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