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Parallel phosphatidylinositol 3-kinase (PI3K)-dependent and Src-dependent pathways lead to CXCL8-mediated Rac2 activation and chemotaxis.

ABSTRACT: The requirement for phosphatidylinositol 3-kinase (PI3K) in the establishment of cell polarity and motility in a number of cell types has recently come into question. In this study, we demonstrate that inhibition of PI3K by wortmannin in neutrophil-like differentiated HL60 cells expressing CXCR2 resulted in reduced cell motility but normal chemotaxis in response to a gradient of CXCL8. However, wortmannin inhibition of PI3K did impair the ability of cells to re-orient their polarity and respond quickly to a change in the direction of the CXCL8 gradient. We hypothesized that Src-regulated ELMO-Dock2-Rac2 activation mediates chemotaxis in the absence of PI3K activity. Inhibition of Src with the small molecule inhibitor, PP2, or inhibition of Dock2 by shRNA knockdown confirmed the functional role of Src and Dock2 in regulating chemotaxis when PI3K was inhibited. Moreover, neutrophils isolated from bone marrow of hck(-/-)fgr(-/-)lyn(-/-) mice exhibited much more severe inhibition of chemotaxis when PI3K was blocked with wortmannin as compared with neutrophils isolated from bone marrow of wild-type mice. Thus, PI3K and Src-ELMO-Dock2 pathways work in parallel to activate Rac2 and modulate chemotaxis in response to a CXCL8 gradient in neutrophils.

SUBMITTER: Sai J

PROVIDER: S-EPMC2546539 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Parallel phosphatidylinositol 3-kinase (PI3K)-dependent and Src-dependent pathways lead to CXCL8-mediated Rac2 activation and chemotaxis.

Sai Jiqing J Raman Dayanidhi D Liu Yuxin Y Wikswo John J Richmond Ann A

The Journal of biological chemistry 20080728 39

The requirement for phosphatidylinositol 3-kinase (PI3K) in the establishment of cell polarity and motility in a number of cell types has recently come into question. In this study, we demonstrate that inhibition of PI3K by wortmannin in neutrophil-like differentiated HL60 cells expressing CXCR2 resulted in reduced cell motility but normal chemotaxis in response to a gradient of CXCL8. However, wortmannin inhibition of PI3K did impair the ability of cells to re-orient their polarity and respond ...[more]

PMID: 18662984

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Project description:BACKGROUND: BMP-induced chemotaxis of mesenchymal progenitors is fundamental for vertebrate development, disease and tissue repair. BMP2 induces Smad and non-Smad signalling. Whereas signal transduction via Smads lead to transcriptional responses, non-Smad signalling induces both, transcriptional and immediate/early non-transcriptional responses. However, the molecular mechanisms by which BMP2 facilitates planar cell polarity, cortical actin rearrangements, lamellipodia formation and chemotaxis of mesenchymal progenitors are poorly understood. Our aim was to uncover the molecular mechanism by which BMP2 facilitates chemotaxis via the BMP2-dependent activation of PI3K and spatiotemporal control of PIP3 production important for actin rearrangements at the mesenchymal cell cytocortex. RESULTS: We unveiled the molecular mechanism by which BMP2 induces non-Smad signalling by PI3K and the role of the second messenger PIP3 in BMP2-induced planar cell polarity, cortical actin reorganisation and lamellipodia formation. By using protein interaction studies, we identified the class Ia PI3K regulatory subunit p55? to act as a specific and non-redundant binding partner for BMP receptor type II (BMPRII) in concert with the catalytic subunit p110?. We mapped the PI3K interaction to a region within the BMPRII kinase. Either BMP2 stimulation or increasing amounts of BMPRI facilitated p55? association with BMPRII, but BMPRII kinase activity was not required for the interaction. We visualised BMP2-dependent PIP3 production via PI3K p55?/p110? and were able to localise PIP3 to the leading edge of intact cells during the process of BMP2-induced planar cell polarity and actin dependent lamellipodia formation. Using mass spectrometry, we found the highly PIP3-sensitive PH-domain protein LL5? to act as a novel BMP2 effector in orchestrating cortical actin rearrangements. By use of live cell imaging we found that knock-down of p55? or LL5? or pharmacological inhibition of PI3K impaired BMP2-induced migratory responses. CONCLUSIONS: Our results provide evidence for an important contribution of the BMP2-PI3K (p55?/p110?)- PIP3-LL5? signalling axis in mesenchymal progenitor cell chemotaxis. We demonstrate molecular insights into BMP2-induced PI3K signalling on the level of actin reorganisation at the leading edge cytocortex. These findings are important to better understand BMP2-induced cytoskeletal reorganisation and chemotaxis of mesenchymal progenitors in different physiological or pathophysiological contexts.

Dataset Information

Parallel phosphatidylinositol 3-kinase (PI3K)-dependent and Src-dependent pathways lead to CXCL8-mediated Rac2 activation and chemotaxis.

Publications

Parallel phosphatidylinositol 3-kinase (PI3K)-dependent and Src-dependent pathways lead to CXCL8-mediated Rac2 activation and chemotaxis.

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