DOCK2 is a Rac activator that regulates motility and polarity during neutrophil chemotaxis

Kunisaki, Yuya; Nishikimi, Akihiko; Tanaka, Yoshihiko; Takii, Ryosuke; Noda, Masahiro; Inayoshi, Ayumi; Watanabe, Kenji; Sanematsu, Fumiyuki; Sasazuki, Takehiko; Sasaki, Takehiko; Fukui, Yoshihiro

doi:10.1084/jem2039oia23

Cited by 47 publications

(80 citation statements)

References 17 publications

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“…5 and 6), both activation of phosphatidylinositol-specific PLC and PI3K␥ are normal in Hck/Fgrdeficient neutrophils. Consistent with the evidence that NADPH oxidase activation requires the synergistic action of Vav1, P-Rex1, and DOCK2 GEFs (55,63,64,66), in assay conditions revealing defective fMLP-induced superoxide anion generation by hck Ϫ/Ϫ fgr Ϫ/Ϫ neutrophils, a PI3K␥ inhibitor markedly reduced this response in WT neutrophils and totally suppressed it in KO cells (Fig. 7).…”

Section: Discussionsupporting

confidence: 86%

“…13). Interestingly, a very recent study showed that fMLP-induced activation of both Rac1 and Rac2 is reduced by 70% in PMNs deficient of DOCK2, a CDM family member Rac GEF, and DOCK2 deficiency results in a marked inhibition of cell polarity and translocation speed (66). Additionally, similarly to vav1 Ϫ/Ϫ or P-rex1 Ϫ/Ϫ PMNs, dock2 Ϫ/Ϫ PMNs are markedly defective in NADPH oxidase activation.…”

Section: Discussionmentioning

confidence: 99%

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The Src Family Kinases Hck and Fgr Regulate Neutrophil Responses to N-Formyl-Methionyl-Leucyl-Phenylalanine

Fumagalli

Zhang

Baruzzi

et al. 2007

The Journal of Immunology

106

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The chemotactic peptide formyl-methionyl-leucyl-phenilalanine (fMLP) triggers intracellular protein tyrosine phosphorylation leading to neutrophil activation. Deficiency of the Src family kinases Hck and Fgr have previously been found to regulate fMLP-induced degranulation. In this study, we further investigate fMLP signaling in hck−/−fgr−/− neutrophils and find that they fail to activate a respiratory burst and display reduced F-actin polymerization in response to fMLP. Additionally, albeit migration of both hck−/−fgr−/− mouse neutrophils and human neutrophils incubated with the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) through 3-μm pore size Transwells was normal, deficiency, or inhibition, of Src kinases resulted in a failure of neutrophils to migrate through 1-μm pore size Transwells. Among MAPKs, phosphorylation of ERK1/2 was not different, phosphorylation of p38 was only partially affected, and phosphorylation of JNK was markedly decreased in fMLP-stimulated hck−/−fgr−/− neutrophils and in human neutrophils incubated with PP2. An increase in intracellular Ca2+ concentration and phosphorylation of Akt/PKB occurred normally in fMLP-stimulated hck−/−fgr−/− neutrophils, indicating that activation of both phosphoinositide-specific phospholipase C and PI3K is independent of Hck and Fgr. In contrast, phosphorylation of the Rho/Rac guanine nucleotide exchange factor Vav1 and the Rac target p21-activated kinases were markedly reduced in both hck−/−fgr−/− neutrophils and human neutrophils incubated with a PP2. Consistent with these findings, PP2 inhibited Rac2 activation in human neutrophils. We suggest that Hck and Fgr act within a signaling pathway triggered by fMLP receptors that involves Vav1 and p21-activated kinases, leading to respiratory burst and F-actin polymerization.

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Section: Discussionsupporting

confidence: 86%

Section: Discussionmentioning

confidence: 99%

The Src Family Kinases Hck and Fgr Regulate Neutrophil Responses to N-Formyl-Methionyl-Leucyl-Phenylalanine

Fumagalli

Zhang

Baruzzi

et al. 2007

The Journal of Immunology

106

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“…We also found that B-Raf interacted with Rictor, a known binding partner of mTor (68). Further, we identified Dock2, a guanine nucleotide exchange factor that activates the small G protein Rac (69), and GM130, a Golgi matrix protein, as binding partners for B-Raf and Raf-1. Erk signaling from different locations in a T cell can have different outcomes (70,71), and we contemplate that GM130 might be involved in targeting Raf-1 and B-Raf to the Golgi.…”

Section: Discussionmentioning

confidence: 61%

Kidins220/ARMS Associates with B-Raf and the TCR, Promoting Sustained Erk Signaling in T Cells

Deswal

Meyer

Fiala

et al. 2013

The Journal of Immunology

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The activation kinetics of MAPK Erk are critical for T cell development and activation. In particular, sustained Erk signaling is required for T cell activation and effector functions, such as IL-2 production. Although Raf-1 triggers transient Erk activation, B-Raf is implicated in sustained Erk signaling after TCR stimulation. In this study, we show that B-Raf is dephosphorylated on its inhibitory serine 365 upon TCR triggering. However, it is unknown how B-Raf activation is coupled to the TCR. Using mass spectrometry, we identified protein kinase D–interacting substrate of 220 kDa (Kidins220)/ankyrin repeat-rich membrane spanning protein, mammalian target of rapamycin, Rictor, Dock2, and GM130 as novel B-Raf interaction partners. We focused on Kidins220, a protein that has been studied in neuronal cells and found that it associated with the pre-TCR, αβTCR, and γδTCR. Upon prolonged TCR stimulation, the Kidins220–TCR interaction was reduced, as demonstrated by immunoprecipitation and proximity ligation assays. We show that Kidins220 is required for TCR-induced sustained, but not transient, Erk activation. Consequently, induction of the immediate early gene products and transcription factors c-Fos and Erg-1 was blocked, and upregulation of the activation markers CD69, IL-2, and IFN-γ was reduced. Further, Kidins220 was required for optimal calcium signaling. In conclusion, we describe Kidins220 as a novel TCR-interacting protein that couples B-Raf to the TCR. Kidins220 is mandatory for sustained Erk signaling; thus, it is crucial for TCR-mediated T cell activation.

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“…Cells were resuspended in PBS/HSA/hep and held at 4°C until use. Murine bone marrow-derived neutrophils were nucleofected as previously described (Kunisaki et al, 2006) and maintained at 37°C at 5% CO 2 in the presence of 25 ng/ml recombinant murine GM-CSF (R&D Systems, Minneapolis, MN) for 24 h. The transfection efficiency was generally between 10 and 15% at 24 h, with good viability after 16 -24 h. Primary human neutrophils used in RT-PCR and immunoblot analysis were obtained from healthy donors as described previously (Lokuta et al, 2003).…”

Section: Isolation Of Primary Neutrophils and Nucleofectionmentioning

confidence: 99%

Type Iγ PIP Kinase Is a Novel Uropod Component that Regulates Rear Retraction during Neutrophil Chemotaxis

Lokuta

Senetar²,

Bennin

et al. 2007

MBoC

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Cell polarization is necessary for directed migration and leukocyte recruitment to inflamed tissues. Recent progress has been made in defining the molecular mechanisms that regulate chemoattractant-induced cell polarity during chemotaxis, including the contribution of phosphoinositide 3-kinase (PI3K)-dependent phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P 3 ] synthesis at the leading edge. However, less is known about the molecular composition of the cell rear and how the uropod functions during cell motility. Here, we demonstrate that phosphatidylinositol phosphate kinase type I␥ (PIPKI␥661), which generates PtdIns(4,5)P 2 , is enriched in the uropod during chemotaxis of primary neutrophils and differentiated HL-60 cells (dHL-60). Using time-lapse microscopy, we show that enrichment of PIPKI␥661 at the cell rear occurs early upon chemoattractant stimulation and is persistent during chemotaxis. Accordingly, we were able to detect enrichment of PtdIns(4,5)P 2 at the uropod during chemotaxis. Overexpression of kinase-dead PIPKI␥661 compromised uropod formation and rear retraction similar to inhibition of ROCK signaling, suggesting that PtdIns(4,5)P 2 synthesis is important to elicit the backness response during chemotaxis. Together, our findings identify a previously unknown function for PIPKI␥661 as a novel component of the backness signal that regulates rear retraction during chemotaxis.

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DOCK2 is a Rac activator that regulates motility and polarity during neutrophil chemotaxis

Cited by 47 publications

References 17 publications

The Src Family Kinases Hck and Fgr Regulate Neutrophil Responses to N-Formyl-Methionyl-Leucyl-Phenylalanine

The Src Family Kinases Hck and Fgr Regulate Neutrophil Responses to N-Formyl-Methionyl-Leucyl-Phenylalanine

Kidins220/ARMS Associates with B-Raf and the TCR, Promoting Sustained Erk Signaling in T Cells

Type Iγ PIP Kinase Is a Novel Uropod Component that Regulates Rear Retraction during Neutrophil Chemotaxis

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