Limits...
The polybasic region of Rac1 modulates bacterial uptake independently of self-association and membrane targeting.

Wong KW, Mohammadi S, Isberg RR - J. Biol. Chem. (2008)

Bottom Line: To determine if there is a role for the PBR in Rac1 effector signaling that was independent of its role in membrane localization or multimerization, we examined the effect of the PBR in the context of a Rac1 derivative that was targeted to the membrane via an NH2-terminal lipid tail.The membrane-targeted Rac1 derivative restored significant invasin-promoted bacterial uptake in a PBR-dependent manner and yet displayed no detectable self-association.This study indicates that, in addition to its role in promoting membrane localization, the PBR exerts a positive effect on Rac1-controlled bacterial uptake that is independent of Rac1 self-association, most likely due to signaling to downstream effectors.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.

ABSTRACT
The COOH-terminal polybasic region (PBR) of Rac1, a Rho family GTPase member, is required for Rac1 self-association, membrane localization, nuclear translocation, and interaction with downstream effectors. We previously demonstrated that phosphatidylinositol-4-phosphate 5-kinase, one of the effectors that requires the polybasic region for interaction, is necessary for efficient invasin-promoted uptake of Yersinia pseudotuberculosis by nonphagocytic cells. Here we further examined the role of this region in invasin-promoted uptake. Using fluorescence resonance energy transfer experiments (FRET), we determined that engagement of integrin receptors by invasin caused elevated levels of Rac1 self-association at the site of bacterial adhesion in a PBR-dependent fashion. Self-association could be disrupted using several strategies: translocation of the Yersinia YopT prenylcysteine protease into host cells, inactivation of the Rac1 isoprenylation signal that is required for membrane localization, and elimination of the PBR. Disruption in each case impaired invasin-promoted uptake. To determine if there is a role for the PBR in Rac1 effector signaling that was independent of its role in membrane localization or multimerization, we examined the effect of the PBR in the context of a Rac1 derivative that was targeted to the membrane via an NH2-terminal lipid tail. The membrane-targeted Rac1 derivative restored significant invasin-promoted bacterial uptake in a PBR-dependent manner and yet displayed no detectable self-association. This study indicates that, in addition to its role in promoting membrane localization, the PBR exerts a positive effect on Rac1-controlled bacterial uptake that is independent of Rac1 self-association, most likely due to signaling to downstream effectors.

Show MeSH

Related in: MedlinePlus

Stable self-association of Rac1 derivatives shows identical profile to FRET assay. A, HA (YFP derivatives) or Myc (CFP derivatives) epitope tags were introduced into each of the constructs used in the FRET assays and used in co-immunoprecipitation experiments to detect stable self-association. Displayed are maps of each of the constructs used. To measure self-association, the noted CFP and YFP derivatives were co-transfected into 293T cells, and lysates were subjected to immunoprecipitation (IP) with anti-HA IgG to precipitate YFP derivatives. Association with the noted CFP derivatives was detected after gel fractionation and Western blotting (IB) with anti-Myc IgG (B), and the total amount precipitated was determined by blotting with HA (D). As controls to determine relative efficiency of immunoprecipitation, the lysates were probed with anti-Myc (C) or anti-HA (E), loading 25% of the total fraction available for immunoprecipitation. IP, immunoprecipitate, noting antibody used. IB, immunoblot, noting antibody used. F, quantitation of the fraction of Rac1 that self-associates. Bands from B and C were subjected to densitometry scanning to determine the fraction of Myc-tagged protein that associated with HA-tagged Rac1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2602895&req=5

fig6: Stable self-association of Rac1 derivatives shows identical profile to FRET assay. A, HA (YFP derivatives) or Myc (CFP derivatives) epitope tags were introduced into each of the constructs used in the FRET assays and used in co-immunoprecipitation experiments to detect stable self-association. Displayed are maps of each of the constructs used. To measure self-association, the noted CFP and YFP derivatives were co-transfected into 293T cells, and lysates were subjected to immunoprecipitation (IP) with anti-HA IgG to precipitate YFP derivatives. Association with the noted CFP derivatives was detected after gel fractionation and Western blotting (IB) with anti-Myc IgG (B), and the total amount precipitated was determined by blotting with HA (D). As controls to determine relative efficiency of immunoprecipitation, the lysates were probed with anti-Myc (C) or anti-HA (E), loading 25% of the total fraction available for immunoprecipitation. IP, immunoprecipitate, noting antibody used. IB, immunoblot, noting antibody used. F, quantitation of the fraction of Rac1 that self-associates. Bands from B and C were subjected to densitometry scanning to determine the fraction of Myc-tagged protein that associated with HA-tagged Rac1.

Mentions: Formation of a Stable Rac1-Rac1 Complex Requires the Presence of the PBR and Prenylation Signal—To support the data from the FRET assay, an independent test of Rac1 self-association was performed using a coimmunoprecipitation procedure (Fig. 6A). The power of the FRET readout is that it provides spatial information and may allow the detection of transient interactions. If the observed self-association is transient, then it may not survive other detection strategies, such as immunoaffinity techniques, that require slow dissociation rates. To determine if interaction could be observed using more restrictive conditions, several of the constructs used in the microscopic assay were tagged with either Myc or HA epitopes and expressed in mammalian cells (Fig. 6A). For each condition, two constructs were co-transfected into 293T cells, each having different tags to allow detection of self-association. Extracts from the transfectants were then subjected to immunoprecipitation with anti-HA, followed by immunoblotting with anti-Myc, to determine if a fraction of the Myc-tagged partner was found in the precipitate (see “Materials and Methods”). When HA-tagged CFP-Rac1(WT) was immunoprecipitated, it was able to pellet either Myc-tagged YFP-Rac1(WT) or Myc-tagged YFP-Rac1(R66A) based on immunoblotting, consistent with the FRET observations (Fig. 6, B-F). On the other hand, any disruption of the prenylation site (Rac1(C189S)) or the PBR (Rac1(6Q)) was sufficient to interfere with co-immunoprecipitation (Fig. 6, B-F). This loss of association was true even if only one of the partners was lacking the critical carboxyl-terminal sequences (Fig. 6, B and F; wt + 6Q and wt + C189S) or if membrane localization was forced by adding a Lyn myristoylation site (Fig. 6, B and F; Lyn constructs). Therefore, there is concordance between the two assays for detecting interaction, since Rac1 self-association is sufficiently long lived to survive detergent extraction and immunoprecipitation. Furthermore, these results provide direct evidence that Rac1 self-association requires the PBR and COOH-terminal prenylation signal.


The polybasic region of Rac1 modulates bacterial uptake independently of self-association and membrane targeting.

Wong KW, Mohammadi S, Isberg RR - J. Biol. Chem. (2008)

Stable self-association of Rac1 derivatives shows identical profile to FRET assay. A, HA (YFP derivatives) or Myc (CFP derivatives) epitope tags were introduced into each of the constructs used in the FRET assays and used in co-immunoprecipitation experiments to detect stable self-association. Displayed are maps of each of the constructs used. To measure self-association, the noted CFP and YFP derivatives were co-transfected into 293T cells, and lysates were subjected to immunoprecipitation (IP) with anti-HA IgG to precipitate YFP derivatives. Association with the noted CFP derivatives was detected after gel fractionation and Western blotting (IB) with anti-Myc IgG (B), and the total amount precipitated was determined by blotting with HA (D). As controls to determine relative efficiency of immunoprecipitation, the lysates were probed with anti-Myc (C) or anti-HA (E), loading 25% of the total fraction available for immunoprecipitation. IP, immunoprecipitate, noting antibody used. IB, immunoblot, noting antibody used. F, quantitation of the fraction of Rac1 that self-associates. Bands from B and C were subjected to densitometry scanning to determine the fraction of Myc-tagged protein that associated with HA-tagged Rac1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2602895&req=5

fig6: Stable self-association of Rac1 derivatives shows identical profile to FRET assay. A, HA (YFP derivatives) or Myc (CFP derivatives) epitope tags were introduced into each of the constructs used in the FRET assays and used in co-immunoprecipitation experiments to detect stable self-association. Displayed are maps of each of the constructs used. To measure self-association, the noted CFP and YFP derivatives were co-transfected into 293T cells, and lysates were subjected to immunoprecipitation (IP) with anti-HA IgG to precipitate YFP derivatives. Association with the noted CFP derivatives was detected after gel fractionation and Western blotting (IB) with anti-Myc IgG (B), and the total amount precipitated was determined by blotting with HA (D). As controls to determine relative efficiency of immunoprecipitation, the lysates were probed with anti-Myc (C) or anti-HA (E), loading 25% of the total fraction available for immunoprecipitation. IP, immunoprecipitate, noting antibody used. IB, immunoblot, noting antibody used. F, quantitation of the fraction of Rac1 that self-associates. Bands from B and C were subjected to densitometry scanning to determine the fraction of Myc-tagged protein that associated with HA-tagged Rac1.
Mentions: Formation of a Stable Rac1-Rac1 Complex Requires the Presence of the PBR and Prenylation Signal—To support the data from the FRET assay, an independent test of Rac1 self-association was performed using a coimmunoprecipitation procedure (Fig. 6A). The power of the FRET readout is that it provides spatial information and may allow the detection of transient interactions. If the observed self-association is transient, then it may not survive other detection strategies, such as immunoaffinity techniques, that require slow dissociation rates. To determine if interaction could be observed using more restrictive conditions, several of the constructs used in the microscopic assay were tagged with either Myc or HA epitopes and expressed in mammalian cells (Fig. 6A). For each condition, two constructs were co-transfected into 293T cells, each having different tags to allow detection of self-association. Extracts from the transfectants were then subjected to immunoprecipitation with anti-HA, followed by immunoblotting with anti-Myc, to determine if a fraction of the Myc-tagged partner was found in the precipitate (see “Materials and Methods”). When HA-tagged CFP-Rac1(WT) was immunoprecipitated, it was able to pellet either Myc-tagged YFP-Rac1(WT) or Myc-tagged YFP-Rac1(R66A) based on immunoblotting, consistent with the FRET observations (Fig. 6, B-F). On the other hand, any disruption of the prenylation site (Rac1(C189S)) or the PBR (Rac1(6Q)) was sufficient to interfere with co-immunoprecipitation (Fig. 6, B-F). This loss of association was true even if only one of the partners was lacking the critical carboxyl-terminal sequences (Fig. 6, B and F; wt + 6Q and wt + C189S) or if membrane localization was forced by adding a Lyn myristoylation site (Fig. 6, B and F; Lyn constructs). Therefore, there is concordance between the two assays for detecting interaction, since Rac1 self-association is sufficiently long lived to survive detergent extraction and immunoprecipitation. Furthermore, these results provide direct evidence that Rac1 self-association requires the PBR and COOH-terminal prenylation signal.

Bottom Line: To determine if there is a role for the PBR in Rac1 effector signaling that was independent of its role in membrane localization or multimerization, we examined the effect of the PBR in the context of a Rac1 derivative that was targeted to the membrane via an NH2-terminal lipid tail.The membrane-targeted Rac1 derivative restored significant invasin-promoted bacterial uptake in a PBR-dependent manner and yet displayed no detectable self-association.This study indicates that, in addition to its role in promoting membrane localization, the PBR exerts a positive effect on Rac1-controlled bacterial uptake that is independent of Rac1 self-association, most likely due to signaling to downstream effectors.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.

ABSTRACT
The COOH-terminal polybasic region (PBR) of Rac1, a Rho family GTPase member, is required for Rac1 self-association, membrane localization, nuclear translocation, and interaction with downstream effectors. We previously demonstrated that phosphatidylinositol-4-phosphate 5-kinase, one of the effectors that requires the polybasic region for interaction, is necessary for efficient invasin-promoted uptake of Yersinia pseudotuberculosis by nonphagocytic cells. Here we further examined the role of this region in invasin-promoted uptake. Using fluorescence resonance energy transfer experiments (FRET), we determined that engagement of integrin receptors by invasin caused elevated levels of Rac1 self-association at the site of bacterial adhesion in a PBR-dependent fashion. Self-association could be disrupted using several strategies: translocation of the Yersinia YopT prenylcysteine protease into host cells, inactivation of the Rac1 isoprenylation signal that is required for membrane localization, and elimination of the PBR. Disruption in each case impaired invasin-promoted uptake. To determine if there is a role for the PBR in Rac1 effector signaling that was independent of its role in membrane localization or multimerization, we examined the effect of the PBR in the context of a Rac1 derivative that was targeted to the membrane via an NH2-terminal lipid tail. The membrane-targeted Rac1 derivative restored significant invasin-promoted bacterial uptake in a PBR-dependent manner and yet displayed no detectable self-association. This study indicates that, in addition to its role in promoting membrane localization, the PBR exerts a positive effect on Rac1-controlled bacterial uptake that is independent of Rac1 self-association, most likely due to signaling to downstream effectors.

Show MeSH
Related in: MedlinePlus