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GxcC connects Rap and Rac signaling during Dictyostelium development.

Plak K, Veltman D, Fusetti F, Beeksma J, Rivero F, Van Haastert PJ, Kortholt A - BMC Cell Biol. (2013)

Bottom Line: RapA is also important in late development, however so far little is known about the downstream effectors of RapA that play a role in this process.GxcC binds directly and specifically to active RapA and binds to a subset of Dictyostelium Rac proteins.Deletion studies revealed that this pathway is involved in regulating Dictyostelium development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biochemistry, University of Groningen, Nijenborgh 7, Groningen, AG, 9747, The Netherlands.

ABSTRACT

Background: Rap proteins belong to the Ras family of small G-proteins. Dictyostelium RapA is essential and implicated in processes throughout the life cycle. In early development and chemotaxis competent cells RapA induces pseudopod formation by activating PI3K and it regulates substrate attachment and myosin disassembly via the serine/threonine kinase Phg2. RapA is also important in late development, however so far little is known about the downstream effectors of RapA that play a role in this process.

Results: Here we show that cells expressing constitutively active RapA exhibit a high level of Rac activation. With a pull-down screen coupled to mass spectrometry, we identified the Rac specific guanine nucleotide exchange factor, GxcC, as Rap binding partner. GxcC binds directly and specifically to active RapA and binds to a subset of Dictyostelium Rac proteins. Deletion studies revealed that this pathway is involved in regulating Dictyostelium development.

Conclusions: GxcC provides a novel link between Rap and Rac signalling and is one of the Rap effectors regulating the progression of multicellular development.

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Related in: MedlinePlus

GxcC is a specific RapA interaction partner. (A) Dynamics of Rac activation visualized with CRIB-GFP. Cells were developed in shaken suspension for 3 hours and pulsed with 50 nM cAMP for another 2 hours. Developed cells were transferred to a glass bottom dish and overlayed with PB + 0.4% agarose. Images were collected using TIRF microscopy (original experiments are shown in Additional file 1: Movie 1 and Additional file 2: Movie 2). Scale bar is 10 μM. (B) Domain composition of GxcC. ARM – Armadillo repeats, CC – Coiled Coil region, RacGEF – Guanine Nucleotide Exchange factor for Rac, PH – Pleckstrin Homology domain. Arrows indicate the boundaries of truncated constructs. (C) Pull down in Dictyostelium lysate with GST-GxcC as bait and GFP-RapACA as prey. (D) Pull down with GST-ARM or GST-DH-PH as bait and GFP-RapACA as prey. The amount of prey was detected by western blotting using antibody specific for GFP (E) SDS PAGE analysis of recombinant GST-ARM (95 kDa) purified from E coli. (F) GDI assay in which the dissociation rate of mGppNHP from RapA was measured in the presence of varying concentrations of GST-ARM. The addition of increasing concentrations of the effector results in a concentration-dependent decrease of the observed rate constant (kobs).
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Figure 1: GxcC is a specific RapA interaction partner. (A) Dynamics of Rac activation visualized with CRIB-GFP. Cells were developed in shaken suspension for 3 hours and pulsed with 50 nM cAMP for another 2 hours. Developed cells were transferred to a glass bottom dish and overlayed with PB + 0.4% agarose. Images were collected using TIRF microscopy (original experiments are shown in Additional file 1: Movie 1 and Additional file 2: Movie 2). Scale bar is 10 μM. (B) Domain composition of GxcC. ARM – Armadillo repeats, CC – Coiled Coil region, RacGEF – Guanine Nucleotide Exchange factor for Rac, PH – Pleckstrin Homology domain. Arrows indicate the boundaries of truncated constructs. (C) Pull down in Dictyostelium lysate with GST-GxcC as bait and GFP-RapACA as prey. (D) Pull down with GST-ARM or GST-DH-PH as bait and GFP-RapACA as prey. The amount of prey was detected by western blotting using antibody specific for GFP (E) SDS PAGE analysis of recombinant GST-ARM (95 kDa) purified from E coli. (F) GDI assay in which the dissociation rate of mGppNHP from RapA was measured in the presence of varying concentrations of GST-ARM. The addition of increasing concentrations of the effector results in a concentration-dependent decrease of the observed rate constant (kobs).

Mentions: Rap proteins are involved in the regulation of the actin cytoskeleton. In mammals Rap promotes cell spreading via the RacGEFs Vav2 and Tiam1 [16]. In Dictyostelium expression of constitutive active RapA (RapACA) results in an increased actin response and F-actin polymerization is induced at the sites were RapA is activated in response to cAMP [17]. However the pathways and mechanism by which Rap regulates cytoskeleton reorganization are not completely understood. The dynamics of Rac activation in Dictyostelium cells can be monitored using the fluorescent probe CRIB-GFP that consists of the CRIB domain of Dictyostelium PakB, that specifically binds to the active form of Rac [18,19]. To test if RapA can indeed activate the Rac pathway we coexpressed RapACA and CRIB-GFP and visualised active Rac using total internal reflection microscopy. In random moving wild type (AX3) cells there is a broad patch of active Rac in pseudopods. In unstimulated developed cells, there is usually a single (1.3 ±0.5, n = 20) stable patch that slowly migrates along with the pseudopod (Figure 1A, Additional file 1: Movie 1). Cells expressing RapACA are flattened and unpolarized in appearance [13]. In these cells, active Rac patches are highly motile (Figure 1A, Additional file 2: Movie 2). Patches grow to unusually large size and often break up into multiple (2.4 ± 0.9, n = 20) patches, which leads to multiple simultaneous protrusions.


GxcC connects Rap and Rac signaling during Dictyostelium development.

Plak K, Veltman D, Fusetti F, Beeksma J, Rivero F, Van Haastert PJ, Kortholt A - BMC Cell Biol. (2013)

GxcC is a specific RapA interaction partner. (A) Dynamics of Rac activation visualized with CRIB-GFP. Cells were developed in shaken suspension for 3 hours and pulsed with 50 nM cAMP for another 2 hours. Developed cells were transferred to a glass bottom dish and overlayed with PB + 0.4% agarose. Images were collected using TIRF microscopy (original experiments are shown in Additional file 1: Movie 1 and Additional file 2: Movie 2). Scale bar is 10 μM. (B) Domain composition of GxcC. ARM – Armadillo repeats, CC – Coiled Coil region, RacGEF – Guanine Nucleotide Exchange factor for Rac, PH – Pleckstrin Homology domain. Arrows indicate the boundaries of truncated constructs. (C) Pull down in Dictyostelium lysate with GST-GxcC as bait and GFP-RapACA as prey. (D) Pull down with GST-ARM or GST-DH-PH as bait and GFP-RapACA as prey. The amount of prey was detected by western blotting using antibody specific for GFP (E) SDS PAGE analysis of recombinant GST-ARM (95 kDa) purified from E coli. (F) GDI assay in which the dissociation rate of mGppNHP from RapA was measured in the presence of varying concentrations of GST-ARM. The addition of increasing concentrations of the effector results in a concentration-dependent decrease of the observed rate constant (kobs).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675359&req=5

Figure 1: GxcC is a specific RapA interaction partner. (A) Dynamics of Rac activation visualized with CRIB-GFP. Cells were developed in shaken suspension for 3 hours and pulsed with 50 nM cAMP for another 2 hours. Developed cells were transferred to a glass bottom dish and overlayed with PB + 0.4% agarose. Images were collected using TIRF microscopy (original experiments are shown in Additional file 1: Movie 1 and Additional file 2: Movie 2). Scale bar is 10 μM. (B) Domain composition of GxcC. ARM – Armadillo repeats, CC – Coiled Coil region, RacGEF – Guanine Nucleotide Exchange factor for Rac, PH – Pleckstrin Homology domain. Arrows indicate the boundaries of truncated constructs. (C) Pull down in Dictyostelium lysate with GST-GxcC as bait and GFP-RapACA as prey. (D) Pull down with GST-ARM or GST-DH-PH as bait and GFP-RapACA as prey. The amount of prey was detected by western blotting using antibody specific for GFP (E) SDS PAGE analysis of recombinant GST-ARM (95 kDa) purified from E coli. (F) GDI assay in which the dissociation rate of mGppNHP from RapA was measured in the presence of varying concentrations of GST-ARM. The addition of increasing concentrations of the effector results in a concentration-dependent decrease of the observed rate constant (kobs).
Mentions: Rap proteins are involved in the regulation of the actin cytoskeleton. In mammals Rap promotes cell spreading via the RacGEFs Vav2 and Tiam1 [16]. In Dictyostelium expression of constitutive active RapA (RapACA) results in an increased actin response and F-actin polymerization is induced at the sites were RapA is activated in response to cAMP [17]. However the pathways and mechanism by which Rap regulates cytoskeleton reorganization are not completely understood. The dynamics of Rac activation in Dictyostelium cells can be monitored using the fluorescent probe CRIB-GFP that consists of the CRIB domain of Dictyostelium PakB, that specifically binds to the active form of Rac [18,19]. To test if RapA can indeed activate the Rac pathway we coexpressed RapACA and CRIB-GFP and visualised active Rac using total internal reflection microscopy. In random moving wild type (AX3) cells there is a broad patch of active Rac in pseudopods. In unstimulated developed cells, there is usually a single (1.3 ±0.5, n = 20) stable patch that slowly migrates along with the pseudopod (Figure 1A, Additional file 1: Movie 1). Cells expressing RapACA are flattened and unpolarized in appearance [13]. In these cells, active Rac patches are highly motile (Figure 1A, Additional file 2: Movie 2). Patches grow to unusually large size and often break up into multiple (2.4 ± 0.9, n = 20) patches, which leads to multiple simultaneous protrusions.

Bottom Line: RapA is also important in late development, however so far little is known about the downstream effectors of RapA that play a role in this process.GxcC binds directly and specifically to active RapA and binds to a subset of Dictyostelium Rac proteins.Deletion studies revealed that this pathway is involved in regulating Dictyostelium development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biochemistry, University of Groningen, Nijenborgh 7, Groningen, AG, 9747, The Netherlands.

ABSTRACT

Background: Rap proteins belong to the Ras family of small G-proteins. Dictyostelium RapA is essential and implicated in processes throughout the life cycle. In early development and chemotaxis competent cells RapA induces pseudopod formation by activating PI3K and it regulates substrate attachment and myosin disassembly via the serine/threonine kinase Phg2. RapA is also important in late development, however so far little is known about the downstream effectors of RapA that play a role in this process.

Results: Here we show that cells expressing constitutively active RapA exhibit a high level of Rac activation. With a pull-down screen coupled to mass spectrometry, we identified the Rac specific guanine nucleotide exchange factor, GxcC, as Rap binding partner. GxcC binds directly and specifically to active RapA and binds to a subset of Dictyostelium Rac proteins. Deletion studies revealed that this pathway is involved in regulating Dictyostelium development.

Conclusions: GxcC provides a novel link between Rap and Rac signalling and is one of the Rap effectors regulating the progression of multicellular development.

Show MeSH
Related in: MedlinePlus