A serine kinase associates with the RAL GTPase and phosphorylates RAL-interacting protein 1

NRC publication: Ye

Characterization and Functional Analysis of the Calmodulin-Binding Domain of Rac1 GTPase

<div><p>Rac1, a member of the Rho family of small GTPases, has been shown to promote formation of lamellipodia at the leading edge of motile cells and affect cell migration. We previously demonstrated that calmodulin can bind to a region in the C-terminal of Rac1 and that this interaction is important in the activation of platelet Rac1. Now, we have analyzed amino acid residue(s) in the Rac1-calmodulin binding domain that are essential for the interaction and assessed their functional contribution in Rac1 activation. The results demonstrated that region 151–164 in Rac1 is essential for calmodulin binding. Within the 151–164 region, positively-charged amino acids K153 and R163 were mutated to alanine to study impact on calmodulin binding. Mutant form of Rac1 (K153A) demonstrated significantly reduced binding to calmodulin while the double mutant K153A/R163A demonstrated complete lack of binding to calmodulin. Thrombin or EGF resulted in activation of Rac1 in CHRF-288-11 or HeLa cells respectively and W7 inhibited this activation. Immunoprecipitation studies demonstrated that higher amount of CaM was associated with Rac1 during EGF dependent activation. In cells expressing mutant forms of Rac1 (K153A or K153A/R163A), activation induced by EGF was significantly decreased in comparison to wild type or the R163A forms of Rac1. The lack of Rac1 activation in mutant forms was not due to an inability of GDP-GTP exchange or a change in subcelllular distribution. Moreover, Rac1 activation was decreased in cells where endogenous level of calmodulin was reduced using shRNA knockdown and increased in cells where calmodulin was overexpressed. Docking analysis and modeling demonstrated that K153 in Rac1 interacts with Q41 in calmodulin. These results suggest an important role for calmodulin in the activation of Rac1 and thus, in cytoskeleton reorganization and cell migration.</p> </div

CaM is required for thrombin-induced activation of Rac1 in CHRF-288-11 cells and EGF-induced activation of Rac1 in HeLa cells.

<p>(A) CHRF-288-11 cells or (B) HeLa cells were serum starved for 12 h and incubated with W7 (150 μM) for 10 min followed by addition of thrombin to CHRF-288-11 cells and EGF to HeLa cells for 1 min or 3 min. At the end of the incubation cells were lysed using RIPA buffer. After centrifugation, 60 µl of the supernatant was suspended in 20 µl 4X Laemmli's sample buffer to determine level of endogenous Rac1 in various samples by western blotting. The rest of the supernatant was incubated with GST-PAK1 for 2 h at 4°C. After incubation, the beads were washed three times with Rac1 washing buffer. The final bead pellet was suspended in 30 µl of Laemmli's sample buffer and heated at 100°C for 5 min. Western blotting was performed using mouse anti-Rac1 antibody. Quantification (adjusted for endogenous level of Rac1) was carried out using Bio-Rad “quantity one” program and *p<0.05 were considered significantly different. #p<0.05 was considered significantly different compared with corresponding thrombin or EGF treatment. The experiments were repeated a minimum of three times. In part (C), an equal amount of lysate (500 µg) from HeLa cells transiently expressing wild type HA-Rac1 and stimulated for various times with EGF was incubated for 2 hrs at 4°C with anti-HA antibody coupled to agarose beads. At the end of incubation beads were washed and bound proteins analyzed using SDS-PAGE and western blotting using anti-CaM antibody. Quantification was carried out using Bio-Rad “quantity one” program and *p<0.05 were considered significantly different when compared to EGF at 0 min.</p

Predicted model for the interaction between various forms of Rac1 and CaM.

<p>The Apo-CaM (PDB id: 1CFD) was docked with Rac1 151–164 amino acids of Rac1 WT (PDB id: 1FOE), Rac1 K153A, Rac1 R163A, and K153A/R163A using ZDock server (<a href="http://zdock.bu.edu/" target="_blank">http://zdock.bu.edu/</a>). The docking calculations were carried out using Fast fourier transform based protein docking method using ZDock. All possible binding modes in the translational and rotational space between two proteins were searched and each was evaluated by an energy scoring function. The poses with the best energy scores were chosen for further analysis. The models were visualized using PyMol (<a href="http://www.pymol.org/" target="_blank">http://www.pymol.org/</a>). CaM is shown in green and Rac1 is in red. The figure above represents interaction between: (A) WT Rac1 and CaM; (B) Rac1 K153A and CaM; (C) Rac1 R163A and CaM and (D) Rac1 K153A/R163A and CaM.</p

Binding of pure CaM to Rac1(K153A), Rac1(R163A), and Rac1(K153A/R163A).

<p>Equal amount (20 µg) of wild type (WT) GST-Rac1 and different GST-Rac1 mutants were incubated with purified CaM (20 µg) in MOPS buffer and allowed to shake for 2 h at 4°C. GST beads were used as negative control. The incubation conditions were beads containing WT GST-Rac1 or different GST-Rac1 mutants with buffer alone, buffer plus 5 mM Ca²⁺ or buffer plus 10 mM EGTA. At the end of the incubation, beads were washed three times and bound proteins were eluted using Laemmli's sample buffer. Western blot analysis was carried out using anti-CaM antibodies. Quantification was carried out using Bio-Rad Quantity one program and *p<0.05 values were considered significantly different (n = 3). In the figure above double refers to Rac1(K153A/R163A).</p

Effect of deletion of Rac1 putative CaM binding domain on interaction with purified bovine brain CaM.

<p>Equal amount (20 µg) of wild type (WT) GST-Rac1 and GST-Rac1 mutant (amino acids 151 to 164 deleted) were incubated with purified CaM (20 µg) in MOPS buffer and allowed to shake for 2 h at 4°C. GST beads were used as negative control. The incubation conditions were WT GST-Rac1 or GST-Rac1 mutant beads with buffer alone, buffer plus 5 mM Ca²⁺ or buffer plus 10 mM EGTA. At the end of the incubation, beads were washed three times and bound proteins were eluted using Laemmli's sample buffer. Western blot analysis was carried out using anti-CaM antibodies. A representative autoradiograph and quantitation is shown above. The experiment was repeated a minimum of three times.</p

Effect of CaM over-expression or down-regulation on activation of Rac1.

<p>(A) Control HeLa cells and HeLa cells transiently over-expressing HA-CaM were serum starved for 12 h and stimulated with EGF for 3 min and analyzed for level of Rac1-GTP using GST-PAK1 pull-down assay. The total lysates were subjected to SDS-PAGE and immunoblotting performed using anti-Rac1 antibody. Anti-HA antibody was used to detect HA-CaM expression. β-actin antibody was used to establish equal loading of protein in different samples. Data presented is a representative immunoblot of at least three independent experiments. Quantification was carried out using Bio-Rad “quantity one” program and *p<0.05 were considered significantly different. #p<0.05 was considered significantly different compared with corresponding non-treatment samples. (B) HeLa cells were stably co-transfected with shRNAs targeting human CaM2 and human CaM3 or non-target shRNA as a negative control. HeLa cells were serum starved for 12 h and stimulated with EGF for 3 min. The level of Rac1-GTP was assessed using GST-PAK1 pull down assay. The total lysates were subjected to SDS-PAGE and immunoblotting using anti-Rac1 antibody or anti-CaM antibody or β-actin antibody. Data presented is one representative immunoblot of at least three independent experiments. Quantification was carried out using Bio-Rad “quantity one” program and *p<0.05 were considered significantly different. #p<0.05 was considered significantly different compared with corresponding non-treatment sample.</p

Activation of HA-Rac1 mutants is induced by EGF in HeLa cells.

<p>(A) Various HA-Rac1 mutants were transiently expressed in HeLa cells. 48 h post transfection cells were serum starved for 12 h and stimulated for 3 min with EGF and lysed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042975#s2" target="_blank">Materials & Methods</a>. After centrifugation, the supernatant was incubated with GST-PAK1 for 2 h at 4°C and the beads were washed three times with washing buffer. The final bead pellet was suspended in 30 µl of Laemmli's sample buffer and heated at 100°C for 5 min. Western blotting was performed using mouse anti-HA antibody. Data presented is a representative immunoblot of at least three independent experiments. Quantification was carried out using Bio-Rad “quantity one” program and key * p<0.05 were considered significantly different. (B) GTP loading of WT Rac1 and mutants of Rac1 was tested by the GST-PAK1 pull-down assay. HeLa cells expressing various forms of HA-Rac1 were lysed in buffer as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042975#s2" target="_blank">Materials & Methods</a>. After centrifugation, guanine nucleotides (100 µM GTPγS or 100 µM GDPβS) plus 10 mM EGTA (final concn.) were added to the supernatant and the mixture was incubated at 30°C for 15 min. At the end of the incubation, magnesium chloride (MgCl₂) was added to a final concentration of 60 mM to lock in nucleotides. The mixture was incubated with 100 µl GST-PAK1 beads for 2 h at 4°C. Unbound proteins were removed by washing three times with binding buffer. 30 µl of Laemmli's sample buffer was added to beads and heated at 100°C for 5 min. Western blotting was performed using mouse anti-HA antibody. In the figure above double refers to the Rac1 (K153A/R163A) mutant.</p

Amino acid derivatives as bitter taste receptor (T2R) blockers

In humans, the 25 bitter taste receptors (T2Rs) are activated by hundreds of structurally diverse bitter compounds. However, only five antagonists or bitter blockers are known. In this study, using molecular modeling guided site-directed mutagenesis, we elucidated the ligand-binding pocket of T2R4. We found seven amino acids located in the extracellular side of transmembrane 3 (TM3), TM4, extracellular loop 2 (ECL2), and ECL3 to be involved in T2R4 binding to its agonist quinine. ECL2 residues Asn-173 and Thr-174 are essential for quinine binding. Guided by a molecular model of T2R4, a number of amino acid derivatives were screened for their ability to bind to T2R4. These predictions were tested by calcium imaging assays that led to identification of \u3b3-aminobutryic acid (GABA) and N\u3b1,N\u3b1-bis(carboxymethyl)-L-lysine (BCML) as competitive inhibitors of quinine-activated T2R4 with an IC50of 3.2 \ub1 0.3 \u3bcM and 59 \ub1 18 nM, respectively. Interestingly, pharmacological characterization using a constitutively active mutant of T2R4 reveals that GABA acts as an antagonist, whereas BCML acts as an inverse agonist on T2R4. Site-directed mutagenesis confirms that the two novel bitter blockers share the same orthosteric site as the agonist quinine. The signature residues Ala-90 and Lys-270 play important roles in interacting with BCML and GABA, respectively. This is the first report to characterize a T2R endogenous antagonist and an inverse agonist. The novel bitter blockers will facilitate physiological studies focused on understanding the roles of T2Rs in extraoral tissues.Peer reviewed: YesNRC publication: Ye