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DOCK8 functions as an adaptor that links TLR-MyD88 signaling to B cell activation.

Jabara HH, McDonald DR, Janssen E, Massaad MJ, Ramesh N, Borzutzky A, Rauter I, Benson H, Schneider L, Baxi S, Recher M, Notarangelo LD, Wakim R, Dbaibo G, Dasouki M, Al-Herz W, Barlan I, Baris S, Kutukculer N, Ochs HD, Plebani A, Kanariou M, Lefranc G, Reisli I, Fitzgerald KA, Golenbock D, Manis J, Keles S, Ceja R, Chatila TA, Geha RS - Nat. Immunol. (2012)

Bottom Line: DOCK8 associated constitutively with MyD88 and the tyrosine kinase Pyk2 in normal B cells.After ligation of TLR9, DOCK8 became tyrosine-phosphorylated by Pyk2, bound the Src-family kinase Lyn and linked TLR9 to a Src-kinase Syk-transcription factor STAT3 cascade essential for TLR9-driven B cell proliferation and differentiation.Thus, DOCK8 functions as an adaptor in a TLR9-MyD88 signaling pathway in B cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, Children's Hospital and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
The adaptors DOCK8 and MyD88 have been linked to serological memory. Here we report that DOCK8-deficient patients had impaired antibody responses and considerably fewer CD27(+) memory B cells. B cell proliferation and immunoglobulin production driven by Toll-like receptor 9 (TLR9) were considerably lower in DOCK8-deficient B cells, but those driven by the costimulatory molecule CD40 were not. In contrast, TLR9-driven expression of AICDA (which encodes the cytidine deaminase AID), the immunoglobulin receptor CD23 and the costimulatory molecule CD86 and activation of the transcription factor NF-κB, the kinase p38 and the GTPase Rac1 were intact. DOCK8 associated constitutively with MyD88 and the tyrosine kinase Pyk2 in normal B cells. After ligation of TLR9, DOCK8 became tyrosine-phosphorylated by Pyk2, bound the Src-family kinase Lyn and linked TLR9 to a Src-kinase Syk-transcription factor STAT3 cascade essential for TLR9-driven B cell proliferation and differentiation. Thus, DOCK8 functions as an adaptor in a TLR9-MyD88 signaling pathway in B cells.

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DOCK8 mediates CpG activation of a Pyk2-Src-Syk-STAT3 cascade essential for B cell proliferation and differentiation(a,b) Representative immunoblot analysis of lysates from CpG-stimulated PBMCs from a DOCK8 deficient patient and a normal control for tyrosine phophorylated proteins (a) and for phosphorylation of Pyk2 at Y402 and Src at Y416 (b). BLNK was used as loading control. The higher amounts of BLNK in PBMCs from the patients reflect the higher percentage of CD19+ B cells in the patients compared to controls (23% versus 7 % in a and 19% versus 8% in b respectively). (c) Immunoblot analysis of lysates from CpG-stimulated splenic B cells from Myd88−/− mice and BALB/c WT controls for pY402Pyk2 and pY416Src. (d–g) Effect of the Pyk2 inhibitor tyrphostin A9 (Tyr A9) and the Src kinase inhibitor PP2 on CpG-driven phosphorylation of Pyk2 at Y402 (d), Src at Y416 (e), Syk Y352 (f), and STAT3 at Y705 (g) in normal PBMCs. (h) Effect of Tyr A9 and PP2 on CpG-driven IgG secretion by normal PBMCs. Stimulation with anti-CD40+IL-21 was used as control. Results in (a–g) are representative of three independent experiments. Data in (h) represent mean and s.e. of three experiments. *P<0.05 and **P<0.01 (Student’s t-test).
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Figure 6: DOCK8 mediates CpG activation of a Pyk2-Src-Syk-STAT3 cascade essential for B cell proliferation and differentiation(a,b) Representative immunoblot analysis of lysates from CpG-stimulated PBMCs from a DOCK8 deficient patient and a normal control for tyrosine phophorylated proteins (a) and for phosphorylation of Pyk2 at Y402 and Src at Y416 (b). BLNK was used as loading control. The higher amounts of BLNK in PBMCs from the patients reflect the higher percentage of CD19+ B cells in the patients compared to controls (23% versus 7 % in a and 19% versus 8% in b respectively). (c) Immunoblot analysis of lysates from CpG-stimulated splenic B cells from Myd88−/− mice and BALB/c WT controls for pY402Pyk2 and pY416Src. (d–g) Effect of the Pyk2 inhibitor tyrphostin A9 (Tyr A9) and the Src kinase inhibitor PP2 on CpG-driven phosphorylation of Pyk2 at Y402 (d), Src at Y416 (e), Syk Y352 (f), and STAT3 at Y705 (g) in normal PBMCs. (h) Effect of Tyr A9 and PP2 on CpG-driven IgG secretion by normal PBMCs. Stimulation with anti-CD40+IL-21 was used as control. Results in (a–g) are representative of three independent experiments. Data in (h) represent mean and s.e. of three experiments. *P<0.05 and **P<0.01 (Student’s t-test).

Mentions: Stimulation of normal PBMCs with CpG resulted in tyrosine phosphorylation of proteins with molecular weights that correspond to those of Src family kinases (54–56 kDa), their target Syk (72 kDa), and Pyk2 (110 kDa), a tyrosine kinase that functionally interacts with Src kinases (Fig. 6a). In contrast, it caused minimal protein tyrosine phosphorylation in PBMCs from DOCK8-deficient patients. This defect was selective to CpG stimulation, because BCR crosslinking caused comparable protein tyrosine phosphorylation in PBMCs from DOCK8-deficient patients and controls (data not shown), consistent with the normal activation of B cells from DOCK8 mutant mice following BCR crosslinking22.


DOCK8 functions as an adaptor that links TLR-MyD88 signaling to B cell activation.

Jabara HH, McDonald DR, Janssen E, Massaad MJ, Ramesh N, Borzutzky A, Rauter I, Benson H, Schneider L, Baxi S, Recher M, Notarangelo LD, Wakim R, Dbaibo G, Dasouki M, Al-Herz W, Barlan I, Baris S, Kutukculer N, Ochs HD, Plebani A, Kanariou M, Lefranc G, Reisli I, Fitzgerald KA, Golenbock D, Manis J, Keles S, Ceja R, Chatila TA, Geha RS - Nat. Immunol. (2012)

DOCK8 mediates CpG activation of a Pyk2-Src-Syk-STAT3 cascade essential for B cell proliferation and differentiation(a,b) Representative immunoblot analysis of lysates from CpG-stimulated PBMCs from a DOCK8 deficient patient and a normal control for tyrosine phophorylated proteins (a) and for phosphorylation of Pyk2 at Y402 and Src at Y416 (b). BLNK was used as loading control. The higher amounts of BLNK in PBMCs from the patients reflect the higher percentage of CD19+ B cells in the patients compared to controls (23% versus 7 % in a and 19% versus 8% in b respectively). (c) Immunoblot analysis of lysates from CpG-stimulated splenic B cells from Myd88−/− mice and BALB/c WT controls for pY402Pyk2 and pY416Src. (d–g) Effect of the Pyk2 inhibitor tyrphostin A9 (Tyr A9) and the Src kinase inhibitor PP2 on CpG-driven phosphorylation of Pyk2 at Y402 (d), Src at Y416 (e), Syk Y352 (f), and STAT3 at Y705 (g) in normal PBMCs. (h) Effect of Tyr A9 and PP2 on CpG-driven IgG secretion by normal PBMCs. Stimulation with anti-CD40+IL-21 was used as control. Results in (a–g) are representative of three independent experiments. Data in (h) represent mean and s.e. of three experiments. *P<0.05 and **P<0.01 (Student’s t-test).
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Figure 6: DOCK8 mediates CpG activation of a Pyk2-Src-Syk-STAT3 cascade essential for B cell proliferation and differentiation(a,b) Representative immunoblot analysis of lysates from CpG-stimulated PBMCs from a DOCK8 deficient patient and a normal control for tyrosine phophorylated proteins (a) and for phosphorylation of Pyk2 at Y402 and Src at Y416 (b). BLNK was used as loading control. The higher amounts of BLNK in PBMCs from the patients reflect the higher percentage of CD19+ B cells in the patients compared to controls (23% versus 7 % in a and 19% versus 8% in b respectively). (c) Immunoblot analysis of lysates from CpG-stimulated splenic B cells from Myd88−/− mice and BALB/c WT controls for pY402Pyk2 and pY416Src. (d–g) Effect of the Pyk2 inhibitor tyrphostin A9 (Tyr A9) and the Src kinase inhibitor PP2 on CpG-driven phosphorylation of Pyk2 at Y402 (d), Src at Y416 (e), Syk Y352 (f), and STAT3 at Y705 (g) in normal PBMCs. (h) Effect of Tyr A9 and PP2 on CpG-driven IgG secretion by normal PBMCs. Stimulation with anti-CD40+IL-21 was used as control. Results in (a–g) are representative of three independent experiments. Data in (h) represent mean and s.e. of three experiments. *P<0.05 and **P<0.01 (Student’s t-test).
Mentions: Stimulation of normal PBMCs with CpG resulted in tyrosine phosphorylation of proteins with molecular weights that correspond to those of Src family kinases (54–56 kDa), their target Syk (72 kDa), and Pyk2 (110 kDa), a tyrosine kinase that functionally interacts with Src kinases (Fig. 6a). In contrast, it caused minimal protein tyrosine phosphorylation in PBMCs from DOCK8-deficient patients. This defect was selective to CpG stimulation, because BCR crosslinking caused comparable protein tyrosine phosphorylation in PBMCs from DOCK8-deficient patients and controls (data not shown), consistent with the normal activation of B cells from DOCK8 mutant mice following BCR crosslinking22.

Bottom Line: DOCK8 associated constitutively with MyD88 and the tyrosine kinase Pyk2 in normal B cells.After ligation of TLR9, DOCK8 became tyrosine-phosphorylated by Pyk2, bound the Src-family kinase Lyn and linked TLR9 to a Src-kinase Syk-transcription factor STAT3 cascade essential for TLR9-driven B cell proliferation and differentiation.Thus, DOCK8 functions as an adaptor in a TLR9-MyD88 signaling pathway in B cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, Children's Hospital and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
The adaptors DOCK8 and MyD88 have been linked to serological memory. Here we report that DOCK8-deficient patients had impaired antibody responses and considerably fewer CD27(+) memory B cells. B cell proliferation and immunoglobulin production driven by Toll-like receptor 9 (TLR9) were considerably lower in DOCK8-deficient B cells, but those driven by the costimulatory molecule CD40 were not. In contrast, TLR9-driven expression of AICDA (which encodes the cytidine deaminase AID), the immunoglobulin receptor CD23 and the costimulatory molecule CD86 and activation of the transcription factor NF-κB, the kinase p38 and the GTPase Rac1 were intact. DOCK8 associated constitutively with MyD88 and the tyrosine kinase Pyk2 in normal B cells. After ligation of TLR9, DOCK8 became tyrosine-phosphorylated by Pyk2, bound the Src-family kinase Lyn and linked TLR9 to a Src-kinase Syk-transcription factor STAT3 cascade essential for TLR9-driven B cell proliferation and differentiation. Thus, DOCK8 functions as an adaptor in a TLR9-MyD88 signaling pathway in B cells.

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