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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

Scatter plots of standardized log abundance of identified proteins. Abundance of spots was determined from fluorescently labelled samples of BMN run on 2D gels. Standardization of spots was to a pooled sample run on all six gels. The statistical significance is shown for paired comparisons (n = 6 for each condition). **, p < 0.01; ***, p < 0.001; ns, p > 0.05 (not significant).
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Figure 5: Scatter plots of standardized log abundance of identified proteins. Abundance of spots was determined from fluorescently labelled samples of BMN run on 2D gels. Standardization of spots was to a pooled sample run on all six gels. The statistical significance is shown for paired comparisons (n = 6 for each condition). **, p < 0.01; ***, p < 0.001; ns, p > 0.05 (not significant).

Mentions: Abundance data was derived from spot mapping and densitometry using the DeCyder software DIA module. The accuracy of the mapping procedure was examined which showed that spot maps were uniform and included only the primary area of the spot for densitometry (Figure 4). Table 1 shows the changes in abundance between wild-type unstimulated and stimulated samples of the identified proteins with negative ratios indicating reduced protein abundance (e.g. due to protein secretion), while positive ratios indicate an increase in protein abundance (e.g. due to protein modification). Likewise, Table 2 shows changes in abundance between Rac2-/- unstimulated and stimulated samples. Table 3 shows the changes in abundance between wild-type stimulated and Rac2-/- stimulated neutrophils. Abundance data of six identified proteins is graphically represented by scatter plot in Figure 5, which highlights the changes in protein abundance that occurred in wild-type neutrophils, and that did not occur to the same extent in Rac2-/- neutrophils.


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)

Scatter plots of standardized log abundance of identified proteins. Abundance of spots was determined from fluorescently labelled samples of BMN run on 2D gels. Standardization of spots was to a pooled sample run on all six gels. The statistical significance is shown for paired comparisons (n = 6 for each condition). **, p < 0.01; ***, p < 0.001; ns, p > 0.05 (not significant).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3379032&req=5

Figure 5: Scatter plots of standardized log abundance of identified proteins. Abundance of spots was determined from fluorescently labelled samples of BMN run on 2D gels. Standardization of spots was to a pooled sample run on all six gels. The statistical significance is shown for paired comparisons (n = 6 for each condition). **, p < 0.01; ***, p < 0.001; ns, p > 0.05 (not significant).
Mentions: Abundance data was derived from spot mapping and densitometry using the DeCyder software DIA module. The accuracy of the mapping procedure was examined which showed that spot maps were uniform and included only the primary area of the spot for densitometry (Figure 4). Table 1 shows the changes in abundance between wild-type unstimulated and stimulated samples of the identified proteins with negative ratios indicating reduced protein abundance (e.g. due to protein secretion), while positive ratios indicate an increase in protein abundance (e.g. due to protein modification). Likewise, Table 2 shows changes in abundance between Rac2-/- unstimulated and stimulated samples. Table 3 shows the changes in abundance between wild-type stimulated and Rac2-/- stimulated neutrophils. Abundance data of six identified proteins is graphically represented by scatter plot in Figure 5, which highlights the changes in protein abundance that occurred in wild-type neutrophils, and that did not occur to the same extent in Rac2-/- neutrophils.

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