The mechanism of CSF-1-induced Wiskott-Aldrich syndrome protein activation in vivo. A role for phosphatidylinositol 3-kinase and Cdc42

Michael Cammer, Jean Claude Gevrey, Mike Lorenz, Athanassios Dovas, John S. Condeelis, Dianne Cox

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

A role for Wiskott-Aldrich syndrome protein (WASP) in chemotaxis to various agents has been demonstrated in monocyte-derived cell types. Although WASP has been shown to be activated by multiple mechanisms in vitro, it is unclear how WASP is regulated in vivo. A WASP biosensor (WASPbs), which uses intramolecular fluorescence resonance energy transfer to report WASP activation in vivo, was constructed, and following transfection of macrophages, activation of WASPbs upon treatment with colony-stimulating factor-1 (CSF-1) was detected globally as early as 30 s and remained localized to protrusive regions at later time points. Similar results were obtained when endogenous WASP activation was determined using conformation-sensitive antibodies. In vivo CSF-1-induced WASP activation was fully Cdc42-dependent. Activation of WASP in response to treatment with CSF-1 was also shown to be phosphatidylinositol 3-kinase-dependent. However, treatment with the Src family kinase inhibitors PP2 or SU6656 or disruption of the major tyrosine phosphorylation site of WASPbs (Y291F mutation) did not reduce the level of CSF-1-inducedWASPactivation. Our results indicate that WASP activation downstream of CSF-1R is phosphatidylinositol 3-kinase- and Cdc42-dependent consistent with an involvement of these molecules in macrophage migration. However, although tyrosine phosphorylation of WASP has been proposed to stimulate WASP activity, we found no evidence to indicate that this occurs in vivo.

Original languageEnglish (US)
Pages (from-to)23302-23311
Number of pages10
JournalJournal of Biological Chemistry
Volume284
Issue number35
DOIs
StatePublished - Aug 28 2009

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Wiskott-Aldrich Syndrome Protein
Phosphatidylinositol 3-Kinase
Macrophage Colony-Stimulating Factor
Chemical activation
Biosensing Techniques
Biosensors
Phosphorylation
Macrophages
Tyrosine
Fluorescence Resonance Energy Transfer
Macrophage Activation
Chemotaxis
Transfection
Conformations
Monocytes

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

The mechanism of CSF-1-induced Wiskott-Aldrich syndrome protein activation in vivo. A role for phosphatidylinositol 3-kinase and Cdc42. / Cammer, Michael; Gevrey, Jean Claude; Lorenz, Mike; Dovas, Athanassios; Condeelis, John S.; Cox, Dianne.

In: Journal of Biological Chemistry, Vol. 284, No. 35, 28.08.2009, p. 23302-23311.

Research output: Contribution to journalArticle

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abstract = "A role for Wiskott-Aldrich syndrome protein (WASP) in chemotaxis to various agents has been demonstrated in monocyte-derived cell types. Although WASP has been shown to be activated by multiple mechanisms in vitro, it is unclear how WASP is regulated in vivo. A WASP biosensor (WASPbs), which uses intramolecular fluorescence resonance energy transfer to report WASP activation in vivo, was constructed, and following transfection of macrophages, activation of WASPbs upon treatment with colony-stimulating factor-1 (CSF-1) was detected globally as early as 30 s and remained localized to protrusive regions at later time points. Similar results were obtained when endogenous WASP activation was determined using conformation-sensitive antibodies. In vivo CSF-1-induced WASP activation was fully Cdc42-dependent. Activation of WASP in response to treatment with CSF-1 was also shown to be phosphatidylinositol 3-kinase-dependent. However, treatment with the Src family kinase inhibitors PP2 or SU6656 or disruption of the major tyrosine phosphorylation site of WASPbs (Y291F mutation) did not reduce the level of CSF-1-inducedWASPactivation. Our results indicate that WASP activation downstream of CSF-1R is phosphatidylinositol 3-kinase- and Cdc42-dependent consistent with an involvement of these molecules in macrophage migration. However, although tyrosine phosphorylation of WASP has been proposed to stimulate WASP activity, we found no evidence to indicate that this occurs in vivo.",
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