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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

Localization and translocation dynamics of GxcC ARM coincides with Rap activation. (A) Confocal images of starved cells expressing GFP-RalGDS, GFP-ARM or GFP-GxcC in buffer, 4 sec after cAMP stimulation or in a gradient of cAMP (B) Time-course of RalGDS, GFP-ARM or GFP-FL GxcC translocation from cytoplasm to the membrane upon stimulation with 1μM cAMP. Shown are the mean and SEM of at least 9 cells.
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Figure 2: Localization and translocation dynamics of GxcC ARM coincides with Rap activation. (A) Confocal images of starved cells expressing GFP-RalGDS, GFP-ARM or GFP-GxcC in buffer, 4 sec after cAMP stimulation or in a gradient of cAMP (B) Time-course of RalGDS, GFP-ARM or GFP-FL GxcC translocation from cytoplasm to the membrane upon stimulation with 1μM cAMP. Shown are the mean and SEM of at least 9 cells.

Mentions: Rap activation in vivo can be monitored with the previously published GFP-RalGDS reporter, which specifically binds to Rap-GTP [22]. Upon uniform stimulation with cAMP, GFP-RalGDS transiently translocates uniformly to the cell boundary, maximum peaking after 3–6 sec after stimulation, followed by a return to the cytoplasm around 20 sec (Figure 2A + B) [13]. N-terminal GxcC shows a highly similar response; it is recruited from the cytoplasm to the cell boundary upon cAMP stimulation, with a maximum drop in the cytoplasm after 5 sec after stimulation and return to basal after 20 sec (Figure 2A + B). Upon application of a chemoatractant gradient to wild-type cells, both GFP-RalGDS and N-terminal GxcC localize at the side of the cell facing the gradient (Figure 2A). On the contrary, full length GxcC is uniformly distributed in the cytosol and doesn’t alter localization upon stimulation with cAMP (Figure 2A + B), suggesting GxcC is in an auto-inhibited state and may need additional specific inputs for translocation to the membrane and/or RapA is locally activating GxcC rather than regulating its localization. These data show that the localization and kinetics of RapA activation coincides with the localization of N-terminal GxcC, indicating an interaction in vivo.


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)

Localization and translocation dynamics of GxcC ARM coincides with Rap activation. (A) Confocal images of starved cells expressing GFP-RalGDS, GFP-ARM or GFP-GxcC in buffer, 4 sec after cAMP stimulation or in a gradient of cAMP (B) Time-course of RalGDS, GFP-ARM or GFP-FL GxcC translocation from cytoplasm to the membrane upon stimulation with 1μM cAMP. Shown are the mean and SEM of at least 9 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Localization and translocation dynamics of GxcC ARM coincides with Rap activation. (A) Confocal images of starved cells expressing GFP-RalGDS, GFP-ARM or GFP-GxcC in buffer, 4 sec after cAMP stimulation or in a gradient of cAMP (B) Time-course of RalGDS, GFP-ARM or GFP-FL GxcC translocation from cytoplasm to the membrane upon stimulation with 1μM cAMP. Shown are the mean and SEM of at least 9 cells.
Mentions: Rap activation in vivo can be monitored with the previously published GFP-RalGDS reporter, which specifically binds to Rap-GTP [22]. Upon uniform stimulation with cAMP, GFP-RalGDS transiently translocates uniformly to the cell boundary, maximum peaking after 3–6 sec after stimulation, followed by a return to the cytoplasm around 20 sec (Figure 2A + B) [13]. N-terminal GxcC shows a highly similar response; it is recruited from the cytoplasm to the cell boundary upon cAMP stimulation, with a maximum drop in the cytoplasm after 5 sec after stimulation and return to basal after 20 sec (Figure 2A + B). Upon application of a chemoatractant gradient to wild-type cells, both GFP-RalGDS and N-terminal GxcC localize at the side of the cell facing the gradient (Figure 2A). On the contrary, full length GxcC is uniformly distributed in the cytosol and doesn’t alter localization upon stimulation with cAMP (Figure 2A + B), suggesting GxcC is in an auto-inhibited state and may need additional specific inputs for translocation to the membrane and/or RapA is locally activating GxcC rather than regulating its localization. These data show that the localization and kinetics of RapA activation coincides with the localization of N-terminal GxcC, indicating an interaction in vivo.

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