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Proteomic analysis of secretagogue-stimulated neutrophils implicates a role for actin and actin-interacting proteins in Rac2-mediated granule exocytosis.

Eitzen G, Lo AN, Mitchell T, Kim JD, Chao DV, Lacy P - Proteome Sci (2011)

Bottom Line: Protein abundance was analyzed by 2-dimensional SDS-PAGE of fluorescently labelled samples which allowed the detection ~3500 proteins.We also found changes in abundance of many actin remodelling proteins including coronin-1A, β-actin and the F-actin capping protein, (CapZ-β).We found coronin-1A to be a novel downstream effector protein of this pathway that is threonine phosphorylated in response to secretagogue stimulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, University of Alberta, Edmonton, AB, Canada. gary.eitzen@ualberta.ca.

ABSTRACT

Background: Neutrophils are abundant leukocytes that play a primary role in defence against pathogens. Neutrophils enter sites of infection where they eliminate pathogens via phagocytosis and the release of antimicrobial mediators via degranulation. Rho GTPases, particularly Rac2, play a key role in neutrophil degranulation. The purpose of this study was to identify Rac2-dependent changes in protein abundance in stimulated neutrophils.

Methods: We performed a proteomic analysis on secretagogue-stimulated bone marrow neutrophils that were isolated from wild-type and Rac2-/- mice. Protein abundance was analyzed by 2-dimensional SDS-PAGE of fluorescently labelled samples which allowed the detection ~3500 proteins.

Results: We identified 22 proteins that showed significant changes in abundance after secretagogue-stimulation of wild-type neutrophils, which did not occur in neutrophils isolated from Rac2-/- mice. As expected, the abundance of several granule proteins was reduced in wild-type cells; this did not occur in Rac2-/- neutrophils which confirms the requirement for Rac2 in degranulation. We also found changes in abundance of many actin remodelling proteins including coronin-1A, β-actin and the F-actin capping protein, (CapZ-β). Coronin-1A showed elevated levels of several isoforms after stimulation of neutrophils from wild-type, but not from Rac2-/- mice. These isoforms were immunoreactive with anti-phospho-threonine antibodies, suggesting that neutrophil stimulation triggers a Rac2-dependent kinase cascade that results in the phosphorylation of coronin-1A.

Conclusion: The control of Rac2-mediated degranulation in neutrophils likely functions through actin remodelling via activation of several actin-binding proteins. We found coronin-1A to be a novel downstream effector protein of this pathway that is threonine phosphorylated in response to secretagogue stimulation.

No MeSH data available.


Related in: MedlinePlus

Immunoblot for actin and coronin in BMN samples. Cell lysates were prepared from BMN isolated from wild-type and Rac2-/- mice. Cells were either unstimulated or stimulated for 15 min with CB/fMLF. A. Lysates were probed for coronin-1A, β-actin and β-tubulin. B and C. Quantification of coronin-1A (B) and β-actin (C) band intensities measured from three samples, normalized to β-tubulin for each sample (n = 3). *, p < 0.05; **, p < 0.01; ns, p > 0.05 (not significant).
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Figure 6: Immunoblot for actin and coronin in BMN samples. Cell lysates were prepared from BMN isolated from wild-type and Rac2-/- mice. Cells were either unstimulated or stimulated for 15 min with CB/fMLF. A. Lysates were probed for coronin-1A, β-actin and β-tubulin. B and C. Quantification of coronin-1A (B) and β-actin (C) band intensities measured from three samples, normalized to β-tubulin for each sample (n = 3). *, p < 0.05; **, p < 0.01; ns, p > 0.05 (not significant).

Mentions: We next examined BMN lysates by immunoblot analysis of 1D gels to further examine changes in actin and the actin-interacting protein, coronin-1A [16]. Actin levels showed a significant reduction after stimulation of wild-type BMNs, which is consistent with the abundance changes identified in the proteomic analysis (Figure 6A, and 6C). Although coronin-1A showed a significant increase in abundance in stimulated wild-type BMNs via 2D-DiGE proteomic analysis, immunoblot analysis of 1D gels showed a less significant increase (Figure 6A, and 6B). However, immunoblot analysis of lysates run on 2D-gels revealed several coronin-1A isoforms which did not resolve by 1D electrophoresis (Figure 7A). Several coronin-1A spots in wild-type samples migrated to more acidic pI values, suggesting the appearance of various isoforms of phosphorylated coronin-1A (Figure 7, A and B, compare wild-type unstim to wild-type stim). The more acidic spots (Figure 7, spots 4 and 5) increased in abundance after stimulation, suggesting rapid multiple phosphorylation events. To test this, we immunoprecipitated coronin-1A from lysates, ran these on 1D gels, and carried out immunoblotting with anti-phospho-specific antibodies. Coronin-1A was efficiently immunoprecipitated from all lysates (Figure 7C, left panel), and an anti-phospho-threonine immunoreactive protein species was detected in wild-type stimulated samples (Figure 7C, right panel). Samples were also probed with anti-phospho-tyrosine and anti-phospho-serine antibodies, but no immunoreactive species were observed (data not shown).


Proteomic analysis of secretagogue-stimulated neutrophils implicates a role for actin and actin-interacting proteins in Rac2-mediated granule exocytosis.

Eitzen G, Lo AN, Mitchell T, Kim JD, Chao DV, Lacy P - Proteome Sci (2011)

Immunoblot for actin and coronin in BMN samples. Cell lysates were prepared from BMN isolated from wild-type and Rac2-/- mice. Cells were either unstimulated or stimulated for 15 min with CB/fMLF. A. Lysates were probed for coronin-1A, β-actin and β-tubulin. B and C. Quantification of coronin-1A (B) and β-actin (C) band intensities measured from three samples, normalized to β-tubulin for each sample (n = 3). *, p < 0.05; **, p < 0.01; ns, p > 0.05 (not significant).
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Figure 6: Immunoblot for actin and coronin in BMN samples. Cell lysates were prepared from BMN isolated from wild-type and Rac2-/- mice. Cells were either unstimulated or stimulated for 15 min with CB/fMLF. A. Lysates were probed for coronin-1A, β-actin and β-tubulin. B and C. Quantification of coronin-1A (B) and β-actin (C) band intensities measured from three samples, normalized to β-tubulin for each sample (n = 3). *, p < 0.05; **, p < 0.01; ns, p > 0.05 (not significant).
Mentions: We next examined BMN lysates by immunoblot analysis of 1D gels to further examine changes in actin and the actin-interacting protein, coronin-1A [16]. Actin levels showed a significant reduction after stimulation of wild-type BMNs, which is consistent with the abundance changes identified in the proteomic analysis (Figure 6A, and 6C). Although coronin-1A showed a significant increase in abundance in stimulated wild-type BMNs via 2D-DiGE proteomic analysis, immunoblot analysis of 1D gels showed a less significant increase (Figure 6A, and 6B). However, immunoblot analysis of lysates run on 2D-gels revealed several coronin-1A isoforms which did not resolve by 1D electrophoresis (Figure 7A). Several coronin-1A spots in wild-type samples migrated to more acidic pI values, suggesting the appearance of various isoforms of phosphorylated coronin-1A (Figure 7, A and B, compare wild-type unstim to wild-type stim). The more acidic spots (Figure 7, spots 4 and 5) increased in abundance after stimulation, suggesting rapid multiple phosphorylation events. To test this, we immunoprecipitated coronin-1A from lysates, ran these on 1D gels, and carried out immunoblotting with anti-phospho-specific antibodies. Coronin-1A was efficiently immunoprecipitated from all lysates (Figure 7C, left panel), and an anti-phospho-threonine immunoreactive protein species was detected in wild-type stimulated samples (Figure 7C, right panel). Samples were also probed with anti-phospho-tyrosine and anti-phospho-serine antibodies, but no immunoreactive species were observed (data not shown).

Bottom Line: Protein abundance was analyzed by 2-dimensional SDS-PAGE of fluorescently labelled samples which allowed the detection ~3500 proteins.We also found changes in abundance of many actin remodelling proteins including coronin-1A, β-actin and the F-actin capping protein, (CapZ-β).We found coronin-1A to be a novel downstream effector protein of this pathway that is threonine phosphorylated in response to secretagogue stimulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, University of Alberta, Edmonton, AB, Canada. gary.eitzen@ualberta.ca.

ABSTRACT

Background: Neutrophils are abundant leukocytes that play a primary role in defence against pathogens. Neutrophils enter sites of infection where they eliminate pathogens via phagocytosis and the release of antimicrobial mediators via degranulation. Rho GTPases, particularly Rac2, play a key role in neutrophil degranulation. The purpose of this study was to identify Rac2-dependent changes in protein abundance in stimulated neutrophils.

Methods: We performed a proteomic analysis on secretagogue-stimulated bone marrow neutrophils that were isolated from wild-type and Rac2-/- mice. Protein abundance was analyzed by 2-dimensional SDS-PAGE of fluorescently labelled samples which allowed the detection ~3500 proteins.

Results: We identified 22 proteins that showed significant changes in abundance after secretagogue-stimulation of wild-type neutrophils, which did not occur in neutrophils isolated from Rac2-/- mice. As expected, the abundance of several granule proteins was reduced in wild-type cells; this did not occur in Rac2-/- neutrophils which confirms the requirement for Rac2 in degranulation. We also found changes in abundance of many actin remodelling proteins including coronin-1A, β-actin and the F-actin capping protein, (CapZ-β). Coronin-1A showed elevated levels of several isoforms after stimulation of neutrophils from wild-type, but not from Rac2-/- mice. These isoforms were immunoreactive with anti-phospho-threonine antibodies, suggesting that neutrophil stimulation triggers a Rac2-dependent kinase cascade that results in the phosphorylation of coronin-1A.

Conclusion: The control of Rac2-mediated degranulation in neutrophils likely functions through actin remodelling via activation of several actin-binding proteins. We found coronin-1A to be a novel downstream effector protein of this pathway that is threonine phosphorylated in response to secretagogue stimulation.

No MeSH data available.


Related in: MedlinePlus