Mapping Interactions between p27 and RhoA that Stimulate Cell Migration

Abstract

p27 mediates cell cycle arrest by binding to and inhibiting cyclin-dependent kinase/cyclin complexes, but p27 can also contribute to pro-oncogenic signaling upon mislocalization to the cytoplasm. Cytoplasmic p27 stimulates cell migration by associating with RhoA and interfering with the exchange of GDP from RhoA stimulated by guanine nucleotide exchange factors. We used biophysical methods to show that the N-terminus of p27 directly interacts with RhoA in vitro. The affinity of p27 for RhoA is low, with an equilibrium dissociation constant of hundreds of micromolar; however, at high concentrations, p27 interfered with guanine nucleotide exchange factor-mediated nucleotide exchange from RhoA. We also show that promotion of cell migration in scratch wound cell healing assays requires full-length p27 despite the C-terminus being dispensable for the direct interaction between p27 and RhoA in vitro. These results suggest that there may be an unidentified factor(s) that associates with the C-terminus of p27 to enhance its interactions with RhoA and promote cell migration.

Keywords: IDPs; Rho; cell migration; p27(Kip1); protein interactions.

Figure 1. 2D NMR shows that p27 binds weakly to the Switch I & II regions of RhoA

(a) Domain architecture showing functional regions of p27 and important phosphorylation sites. The kinase interacting domain (KID) that binds to and inhibits Cdk/cyclin complexes is shown in red. The nuclear export signal (NES) and nuclear localization signal (NLS) are shown in salmon and grey, respectively. Phosphorylation by non-receptor tyrosine kinases (NRTKs) at Y74 and Y88 partially reactivate bound Cdk/cyclin complexes leading to intracomplex phosphorylation of p27 at T187, creating a phosphodegron that recruits the Skp2 ubiquitin ligase. (b) 2D ¹H-¹⁵N HSQC spectra of 200 μM ¹⁵N-RhoA upon titration of unlabeled, full-length p27 show chemical shift perturbations and resonance broadening consistent with weak binding. The spectra of 200 μM ¹⁵N-RhoA in the presence of 0, 200, 400, 600, 800 and 1000 μM p27 are shown in blue, cyan, green, yellow, orange, and red, respectively. (c) Representative binding isotherms of several individual resonances. The resonances with chemical shift perturbations greater than one standard deviation above the average for a majority of titration points were fit individually to a 1:1 binding model. Individual fits for A61, E64, and D65 are illustrated. The average K_D is 280 ± 170 μM where the error represents the standard deviation of the mean for individually fit K_D values. Ten resonances (V35, V38, A44, A61, E64, D65, S73, W99, H126, and R150) could be fit confidently to a 1:1 binding model. (d) The residues that fit the above criteria are depicted in red on the GDP bound structure of RhoA (PDB code: 1FTN). The surface representation does not include Mg²⁺-GDP, which is shown explicitly. Several of the most perturbed resonances are located in the Switch I and Switch II regions, which are known to be important for GDP exchange. Residues A61, E64, and D65 are located in the Switch II region. (e) Chemical shift perturbations of 200 μM RhoA upon addition of 1000 μM p27 plotted versus the residue position. Residues shifted by more than 1 standard deviation above the average (denoted by the dashed line) are shown in red.

Figure 2. NMR titration of ¹³C/¹⁵N p27 with RhoA localizes the binding region of p27 to approximately residues 55–95

(a) Overlay of ¹³C-detected 2D CoN correlation spectra of ¹³C/¹⁵N-labeled p27 with increasing concentrations of RhoA. The spectra of 500 μM¹³C/¹⁵N-labeled p27 in the presence of 0, 200, 400, 600, 800 and 1000 μM RhoA are shown in blue, cyan, green, yellow, orange, and red, respectively. Select resonance assignments are shown. Due to slight pH changes during the titration, the resonance of a serine residue in the non-native N-terminal segment of the RhoA construct (N-GSHM; S_tag) exhibited chemical shift changes. (b) The extent of resonance broadening with respect to the apo spectrum is plotted as stacked columns along the primary sequence with residues that are broadened above one standard deviation in a majority of titration points and the position of histidine residues denoted in the box above as black and magenta bars, respectively. The color scheme is the same as in (a).

Figure 3. p27 interferes with GDP exchange from RhoA induced by p115

Nucleotide exchange from 1 μM MantGDP-loaded RhoA (RhoA*) in the presence of 100 μM unlabeled GDP and increasing concentrations of p27 was initiated by rapid addition of p115 to a final concentration of 1 μM. The exchange of MantGDP for unlabeled GDP was monitored by the change in fluorescence intensity over time. Panels a, b and c show titrations with p27, p27-C and p27^50–105, respectively. The maximum p27 concentration tested in (a) and (b) was 0.5 mM. The potency of p27^50–105 is slightly lower than that of full-length p27. The maximum concentration in (c) is 1.5 mM. The lower concentrations of p27 are 2-fold dilutions from these maxima. GDP exchange assays in the presence of p27 are depicted as circles with colors changing from red to blue to represent the serial 2-fold dilutions from the maximum concentration shown in red. The squares and triangles depicted in black are exchange assays conducted in the absence of p27 with and without p115, respectively. Error bars represent standard deviations from triplicate measurements.

Figure 4. Rescue of the cell migration phenotype of p27^−/− MEFs requires intact p27

Representative images and quantification of scratch wound cell migration assays performed with immortalized p27^−/− MEFs transfected with empty vector, p27, p27-N, or p27-C. The darker grey areas show initial wound masks and dotted lines outline the migration fronts. The scale bar is drawn to 300 μm. The gray and black columns in the graph below depict quantification of the relative wound density of the 12 and 24 hour time points, respectively. Error bars represent the standard error of the mean for independent experiments (n=5 empty vector, n=4 for p27, n=7 for p27-N and p27-C). p < 0.005 for all comparisons between p27^−/− MEFs transfected with empty vector, p27-N, or p27-C with respect to full-length p27 transfections.