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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

Kinetic analysis of neutrophil exocytosis. Neutrophils were isolated from bone marrow of wild-type and Rac2-/- mice. 1 × 106 cells were incubated with 5 μM CB for 5 min followed by 5 μM fMLF. At the indicated times samples were taken and cell-free supernatants were analyzed for MPO, an enzyme contained in primary granules. Exocytosis was measure as the % MPO release, calculated as a ratio of total cellular MPO. A. Shown is a representative result from a typical degranulation assays. B. Pre-incubation of BMN with 10 μM cycloheximide for 1 hr at 37°C prior to CB/fMLF stimulation does not affect degranulation (n = 3).
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Figure 1: Kinetic analysis of neutrophil exocytosis. Neutrophils were isolated from bone marrow of wild-type and Rac2-/- mice. 1 × 106 cells were incubated with 5 μM CB for 5 min followed by 5 μM fMLF. At the indicated times samples were taken and cell-free supernatants were analyzed for MPO, an enzyme contained in primary granules. Exocytosis was measure as the % MPO release, calculated as a ratio of total cellular MPO. A. Shown is a representative result from a typical degranulation assays. B. Pre-incubation of BMN with 10 μM cycloheximide for 1 hr at 37°C prior to CB/fMLF stimulation does not affect degranulation (n = 3).

Mentions: Rac2 is a hematopoietic-specific GTPase which is required for the activation of neutrophil immune cell functions, including granule exocytosis and activation of the NADPH oxidase complex [7,10]. Here, we performed a proteomic screen to characterize the Rac2-dependent neutrophil response to secretagogue stimulation, which activates exocytosis (degranulation). Our goal was to identify proteins which may be post-translationally modified in response to stimulation. Rac2-dependent changes can be identified by comparing samples prepared from wild-type versus Rac2-/- mice. Three independent experiments were performed, whereby bone marrow neutrophils (BMN) were isolated from two wild-type and two Rac2-/- mice. Cells were incubated with vehicle (unstimulated) or the secretagogue CB/fMLF (stimulated) for 15 min at 37°C. A 15 min stimulation was selected as the ideal time-point for comparative analyses because this is the time required to obtain maximal degranulation in mouse neutrophils (Figure 1A). We have previously shown that Rac2 activation (the formation of Rac2-GTP) is sustained for 15 min when using CB/fMLF as the secretagogue [10]. As well, this would be insufficient time to allow de novo protein synthesis, which focuses our proteomic screen on Rac2-dependent post-translational modifications, proteolysis or secretion. Pre-incubation of neutrophils with the translation inhibitor cycloheximide did not affect degranulation, confirming that de novo protein synthesis is not required for degranulation (Figure 1B). Examination of BMN by microscopy revealed no obvious morphological differences in cell shape and structure other than more intense cortical actin in unstimulated Rac2-/- cells (Figure 2, unstim). Stimulation resulted in the redistribution of azurophilic CD63+ granule staining which accumulated at the cell periphery in wild-type cells but not in Rac2-/- cells (Figure 2, stim).


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)

Kinetic analysis of neutrophil exocytosis. Neutrophils were isolated from bone marrow of wild-type and Rac2-/- mice. 1 × 106 cells were incubated with 5 μM CB for 5 min followed by 5 μM fMLF. At the indicated times samples were taken and cell-free supernatants were analyzed for MPO, an enzyme contained in primary granules. Exocytosis was measure as the % MPO release, calculated as a ratio of total cellular MPO. A. Shown is a representative result from a typical degranulation assays. B. Pre-incubation of BMN with 10 μM cycloheximide for 1 hr at 37°C prior to CB/fMLF stimulation does not affect degranulation (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Kinetic analysis of neutrophil exocytosis. Neutrophils were isolated from bone marrow of wild-type and Rac2-/- mice. 1 × 106 cells were incubated with 5 μM CB for 5 min followed by 5 μM fMLF. At the indicated times samples were taken and cell-free supernatants were analyzed for MPO, an enzyme contained in primary granules. Exocytosis was measure as the % MPO release, calculated as a ratio of total cellular MPO. A. Shown is a representative result from a typical degranulation assays. B. Pre-incubation of BMN with 10 μM cycloheximide for 1 hr at 37°C prior to CB/fMLF stimulation does not affect degranulation (n = 3).
Mentions: Rac2 is a hematopoietic-specific GTPase which is required for the activation of neutrophil immune cell functions, including granule exocytosis and activation of the NADPH oxidase complex [7,10]. Here, we performed a proteomic screen to characterize the Rac2-dependent neutrophil response to secretagogue stimulation, which activates exocytosis (degranulation). Our goal was to identify proteins which may be post-translationally modified in response to stimulation. Rac2-dependent changes can be identified by comparing samples prepared from wild-type versus Rac2-/- mice. Three independent experiments were performed, whereby bone marrow neutrophils (BMN) were isolated from two wild-type and two Rac2-/- mice. Cells were incubated with vehicle (unstimulated) or the secretagogue CB/fMLF (stimulated) for 15 min at 37°C. A 15 min stimulation was selected as the ideal time-point for comparative analyses because this is the time required to obtain maximal degranulation in mouse neutrophils (Figure 1A). We have previously shown that Rac2 activation (the formation of Rac2-GTP) is sustained for 15 min when using CB/fMLF as the secretagogue [10]. As well, this would be insufficient time to allow de novo protein synthesis, which focuses our proteomic screen on Rac2-dependent post-translational modifications, proteolysis or secretion. Pre-incubation of neutrophils with the translation inhibitor cycloheximide did not affect degranulation, confirming that de novo protein synthesis is not required for degranulation (Figure 1B). Examination of BMN by microscopy revealed no obvious morphological differences in cell shape and structure other than more intense cortical actin in unstimulated Rac2-/- cells (Figure 2, unstim). Stimulation resulted in the redistribution of azurophilic CD63+ granule staining which accumulated at the cell periphery in wild-type cells but not in Rac2-/- cells (Figure 2, stim).

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