Limits...
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.

Show MeSH

Related in: MedlinePlus

GxcC binds to a subset of Dictyostelium Rac proteins. (A) GST and various purified GST-Rac proteins were incubated with GFP-GxcC cell lysate. Beads were precipitated and the amount of GFP-GxcC and bait was detected by western blotting using antibody specific for GFP or GST, respectively (B) The amount of detected proteins was quantified using ImageJ and normalized to the amount of GxcC protein used in the assay. Shown is the percentage of bound GxcC as mean and SEM of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3675359&req=5

Figure 3: GxcC binds to a subset of Dictyostelium Rac proteins. (A) GST and various purified GST-Rac proteins were incubated with GFP-GxcC cell lysate. Beads were precipitated and the amount of GFP-GxcC and bait was detected by western blotting using antibody specific for GFP or GST, respectively (B) The amount of detected proteins was quantified using ImageJ and normalized to the amount of GxcC protein used in the assay. Shown is the percentage of bound GxcC as mean and SEM of three independent experiments.

Mentions: The Dictyostelium genome encodes for 18 Rac proteins, whereas Rho and CDC42 are absent [20]. To determine the downstream targets of GxcC, binding to Dictyostelium Rac proteins was determined. The Rac proteins were expressed and purified from E. coli, bound to GSH beads and subsequently incubated with lystate of vegetative GFP-GxcC Dictyostelium cells in the presence of EDTA. Imunnoblotting was used to detect bait (GST-Rac) and prey (GFP-GxcC) protein. GxcC showed the highest affinity to RacG, RacH, RacE, RacI and RacL whereas hardly binding to Rac1A, RacF2 and RacC could be detected (Figure 3), indicating specificity of GxcC binding in the pull-down. To get complete insight in the downstream targets of GxcC we tried to perform in vitro nucleotide exchange assays, but unfortunately we were not able to isolate a stable recombinant GxcC DH-PH fragment. We were able to isolate small quantities of full length GxcC from Dictyostelium cells, but this protein didn’t show exchange activity on any of the tested Rac proteins. This lack of activity could be due to the quality of the isolated protein, or consistent with the translocation data may suggest that full length GxcC is in an auto-inhibited state (unpublished results).


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 binds to a subset of Dictyostelium Rac proteins. (A) GST and various purified GST-Rac proteins were incubated with GFP-GxcC cell lysate. Beads were precipitated and the amount of GFP-GxcC and bait was detected by western blotting using antibody specific for GFP or GST, respectively (B) The amount of detected proteins was quantified using ImageJ and normalized to the amount of GxcC protein used in the assay. Shown is the percentage of bound GxcC as mean and SEM of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: GxcC binds to a subset of Dictyostelium Rac proteins. (A) GST and various purified GST-Rac proteins were incubated with GFP-GxcC cell lysate. Beads were precipitated and the amount of GFP-GxcC and bait was detected by western blotting using antibody specific for GFP or GST, respectively (B) The amount of detected proteins was quantified using ImageJ and normalized to the amount of GxcC protein used in the assay. Shown is the percentage of bound GxcC as mean and SEM of three independent experiments.
Mentions: The Dictyostelium genome encodes for 18 Rac proteins, whereas Rho and CDC42 are absent [20]. To determine the downstream targets of GxcC, binding to Dictyostelium Rac proteins was determined. The Rac proteins were expressed and purified from E. coli, bound to GSH beads and subsequently incubated with lystate of vegetative GFP-GxcC Dictyostelium cells in the presence of EDTA. Imunnoblotting was used to detect bait (GST-Rac) and prey (GFP-GxcC) protein. GxcC showed the highest affinity to RacG, RacH, RacE, RacI and RacL whereas hardly binding to Rac1A, RacF2 and RacC could be detected (Figure 3), indicating specificity of GxcC binding in the pull-down. To get complete insight in the downstream targets of GxcC we tried to perform in vitro nucleotide exchange assays, but unfortunately we were not able to isolate a stable recombinant GxcC DH-PH fragment. We were able to isolate small quantities of full length GxcC from Dictyostelium cells, but this protein didn’t show exchange activity on any of the tested Rac proteins. This lack of activity could be due to the quality of the isolated protein, or consistent with the translocation data may suggest that full length GxcC is in an auto-inhibited state (unpublished results).

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