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Differential Rac1 signalling by guanine nucleotide exchange factors implicates FLII in regulating Rac1-driven cell migration.

Marei H, Carpy A, Woroniuk A, Vennin C, White G, Timpson P, Macek B, Malliri A - Nat Commun (2016)

Bottom Line: This calls for the identification of factors that influence Rac1-driven cell motility.Here we show that Tiam1 and P-Rex1, two Rac GEFs, promote Rac1 anti- and pro-migratory signalling cascades, respectively, through regulating the Rac1 interactome.In particular, we demonstrate that P-Rex1 stimulates migration through enhancing the interaction between Rac1 and the actin-remodelling protein flightless-1 homologue, to modulate cell contraction in a RhoA-ROCK-independent manner.

View Article: PubMed Central - PubMed

Affiliation: Cell Signalling Group, Cancer Research UK Manchester Institute, The University of Manchester, Manchester M204BX, UK.

ABSTRACT
The small GTPase Rac1 has been implicated in the formation and dissemination of tumours. Upon activation by guanine nucleotide exchange factors (GEFs), Rac1 associates with a variety of proteins in the cell thereby regulating various functions, including cell migration. However, activation of Rac1 can lead to opposing migratory phenotypes raising the possibility of exacerbating tumour progression when targeting Rac1 in a clinical setting. This calls for the identification of factors that influence Rac1-driven cell motility. Here we show that Tiam1 and P-Rex1, two Rac GEFs, promote Rac1 anti- and pro-migratory signalling cascades, respectively, through regulating the Rac1 interactome. In particular, we demonstrate that P-Rex1 stimulates migration through enhancing the interaction between Rac1 and the actin-remodelling protein flightless-1 homologue, to modulate cell contraction in a RhoA-ROCK-independent manner.

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P-Rex1 and Rac1 bind preferentially to the GEL and LRR domain of FLII, respectively.(a) Schematic representation of FLII domain structure showing 16 leucine-rich repeats (LRR) comprising the N-terminal LRR domain and six gelsolin-like repeats (S1-S6) representing the C-terminal GEL domain together with the amino acid number (a.a. #) range for each domain. FLII FL, full-length FLII; FLII GEL, gelsolin domain only; FLII LRR, LRR domain only. (b) FLAG immunoprecipitation (IP) from HEK293T cells expressing the different FLAG-tagged FLII domain mutants outlined in a alone or together with P-Rex1 WT. Co-precipitated exogenous P-Rex1 was detected by western blot analysis. α-Tubulin was used as a loading control. Representative western blot from three independent experiments. (c) GST pulldown using purified GST (EV) or GST-tagged Rac1 WT (Rac1 WT) loaded with GTPγS and incubated with HEK293T lysates expressing the different FLAG-tagged FLII domain mutants outlined in a. Co-precipitated FLAG-tagged FLII domain mutants were detected by western blot analysis. Ponceau staining was used as a loading control. Representative western blot from three independent experiments.
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f5: P-Rex1 and Rac1 bind preferentially to the GEL and LRR domain of FLII, respectively.(a) Schematic representation of FLII domain structure showing 16 leucine-rich repeats (LRR) comprising the N-terminal LRR domain and six gelsolin-like repeats (S1-S6) representing the C-terminal GEL domain together with the amino acid number (a.a. #) range for each domain. FLII FL, full-length FLII; FLII GEL, gelsolin domain only; FLII LRR, LRR domain only. (b) FLAG immunoprecipitation (IP) from HEK293T cells expressing the different FLAG-tagged FLII domain mutants outlined in a alone or together with P-Rex1 WT. Co-precipitated exogenous P-Rex1 was detected by western blot analysis. α-Tubulin was used as a loading control. Representative western blot from three independent experiments. (c) GST pulldown using purified GST (EV) or GST-tagged Rac1 WT (Rac1 WT) loaded with GTPγS and incubated with HEK293T lysates expressing the different FLAG-tagged FLII domain mutants outlined in a. Co-precipitated FLAG-tagged FLII domain mutants were detected by western blot analysis. Ponceau staining was used as a loading control. Representative western blot from three independent experiments.

Mentions: As a member of the gelsolin protein superfamily, FLII possesses the characteristic gelsolin-like (GEL) domain. However, unlike other superfamily members, FLII also contains an N-terminal leucine-rich repeats (LRR) domain (Fig. 5a) with the two domains governing distinct functions. The GEL domain is mainly responsible for FLII-mediated actin remodelling48. On the other hand, LRR domains serve as protein–protein interaction motifs via forming a doughnut- or horseshoe-like conformation that serves as a hydrophobic pocket for protein binding495051. To gain functional insight into the Rac1-FLII and P-Rex1-FLII interactions, previously described FLAG-tagged full-length FLII (FLII FL), a GEL only mutant (FLII GEL), and an LRR only mutant (FLII LRR) (ref. 52) (Fig. 5a) were used to determine the FLII domain responsible for mediating its interaction with Rac1 and P-Rex1. Intriguingly, FLAG co-immunoprecipitation of the different FLII domain mutants revealed that P-Rex1 binds to the GEL domain (Fig. 5b), whereas Rac1 binds preferentially to the LRR domain of FLII (Supplementary Fig. 5a). Using purified proteins we showed that P-Rex1 binds directly to the GEL domain of FLII (Supplementary Fig. 5b). Rac1 binding to the LRR domain of FLII was also further confirmed by GST pulldown of purified GTPγS loaded GST-Rac1 incubated with lysates from HEK293T cells expressing the different FLAG-tagged FLII domain mutants (Fig. 5c). Together, the above data imply that P-Rex1, Rac1 and FLII might form a ternary complex in cells that is functionally important for eliciting P-Rex1-Rac1-driven cellular phenotypes.


Differential Rac1 signalling by guanine nucleotide exchange factors implicates FLII in regulating Rac1-driven cell migration.

Marei H, Carpy A, Woroniuk A, Vennin C, White G, Timpson P, Macek B, Malliri A - Nat Commun (2016)

P-Rex1 and Rac1 bind preferentially to the GEL and LRR domain of FLII, respectively.(a) Schematic representation of FLII domain structure showing 16 leucine-rich repeats (LRR) comprising the N-terminal LRR domain and six gelsolin-like repeats (S1-S6) representing the C-terminal GEL domain together with the amino acid number (a.a. #) range for each domain. FLII FL, full-length FLII; FLII GEL, gelsolin domain only; FLII LRR, LRR domain only. (b) FLAG immunoprecipitation (IP) from HEK293T cells expressing the different FLAG-tagged FLII domain mutants outlined in a alone or together with P-Rex1 WT. Co-precipitated exogenous P-Rex1 was detected by western blot analysis. α-Tubulin was used as a loading control. Representative western blot from three independent experiments. (c) GST pulldown using purified GST (EV) or GST-tagged Rac1 WT (Rac1 WT) loaded with GTPγS and incubated with HEK293T lysates expressing the different FLAG-tagged FLII domain mutants outlined in a. Co-precipitated FLAG-tagged FLII domain mutants were detected by western blot analysis. Ponceau staining was used as a loading control. Representative western blot from three independent experiments.
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f5: P-Rex1 and Rac1 bind preferentially to the GEL and LRR domain of FLII, respectively.(a) Schematic representation of FLII domain structure showing 16 leucine-rich repeats (LRR) comprising the N-terminal LRR domain and six gelsolin-like repeats (S1-S6) representing the C-terminal GEL domain together with the amino acid number (a.a. #) range for each domain. FLII FL, full-length FLII; FLII GEL, gelsolin domain only; FLII LRR, LRR domain only. (b) FLAG immunoprecipitation (IP) from HEK293T cells expressing the different FLAG-tagged FLII domain mutants outlined in a alone or together with P-Rex1 WT. Co-precipitated exogenous P-Rex1 was detected by western blot analysis. α-Tubulin was used as a loading control. Representative western blot from three independent experiments. (c) GST pulldown using purified GST (EV) or GST-tagged Rac1 WT (Rac1 WT) loaded with GTPγS and incubated with HEK293T lysates expressing the different FLAG-tagged FLII domain mutants outlined in a. Co-precipitated FLAG-tagged FLII domain mutants were detected by western blot analysis. Ponceau staining was used as a loading control. Representative western blot from three independent experiments.
Mentions: As a member of the gelsolin protein superfamily, FLII possesses the characteristic gelsolin-like (GEL) domain. However, unlike other superfamily members, FLII also contains an N-terminal leucine-rich repeats (LRR) domain (Fig. 5a) with the two domains governing distinct functions. The GEL domain is mainly responsible for FLII-mediated actin remodelling48. On the other hand, LRR domains serve as protein–protein interaction motifs via forming a doughnut- or horseshoe-like conformation that serves as a hydrophobic pocket for protein binding495051. To gain functional insight into the Rac1-FLII and P-Rex1-FLII interactions, previously described FLAG-tagged full-length FLII (FLII FL), a GEL only mutant (FLII GEL), and an LRR only mutant (FLII LRR) (ref. 52) (Fig. 5a) were used to determine the FLII domain responsible for mediating its interaction with Rac1 and P-Rex1. Intriguingly, FLAG co-immunoprecipitation of the different FLII domain mutants revealed that P-Rex1 binds to the GEL domain (Fig. 5b), whereas Rac1 binds preferentially to the LRR domain of FLII (Supplementary Fig. 5a). Using purified proteins we showed that P-Rex1 binds directly to the GEL domain of FLII (Supplementary Fig. 5b). Rac1 binding to the LRR domain of FLII was also further confirmed by GST pulldown of purified GTPγS loaded GST-Rac1 incubated with lysates from HEK293T cells expressing the different FLAG-tagged FLII domain mutants (Fig. 5c). Together, the above data imply that P-Rex1, Rac1 and FLII might form a ternary complex in cells that is functionally important for eliciting P-Rex1-Rac1-driven cellular phenotypes.

Bottom Line: This calls for the identification of factors that influence Rac1-driven cell motility.Here we show that Tiam1 and P-Rex1, two Rac GEFs, promote Rac1 anti- and pro-migratory signalling cascades, respectively, through regulating the Rac1 interactome.In particular, we demonstrate that P-Rex1 stimulates migration through enhancing the interaction between Rac1 and the actin-remodelling protein flightless-1 homologue, to modulate cell contraction in a RhoA-ROCK-independent manner.

View Article: PubMed Central - PubMed

Affiliation: Cell Signalling Group, Cancer Research UK Manchester Institute, The University of Manchester, Manchester M204BX, UK.

ABSTRACT
The small GTPase Rac1 has been implicated in the formation and dissemination of tumours. Upon activation by guanine nucleotide exchange factors (GEFs), Rac1 associates with a variety of proteins in the cell thereby regulating various functions, including cell migration. However, activation of Rac1 can lead to opposing migratory phenotypes raising the possibility of exacerbating tumour progression when targeting Rac1 in a clinical setting. This calls for the identification of factors that influence Rac1-driven cell motility. Here we show that Tiam1 and P-Rex1, two Rac GEFs, promote Rac1 anti- and pro-migratory signalling cascades, respectively, through regulating the Rac1 interactome. In particular, we demonstrate that P-Rex1 stimulates migration through enhancing the interaction between Rac1 and the actin-remodelling protein flightless-1 homologue, to modulate cell contraction in a RhoA-ROCK-independent manner.

Show MeSH
Related in: MedlinePlus