Protein kinase A-dependent phosphorylation of Rap1 regulates its membrane localization and cell migration

Abstract

The small G protein Rap1 can mediate "inside-out signaling" by recruiting effectors to the plasma membrane that signal to pathways involved in cell adhesion and cell migration. This action relies on the membrane association of Rap1, which is dictated by post-translational prenylation as well as by a stretch of basic residues within its carboxyl terminus. One feature of this stretch of acidic residues is that it lies adjacent to a functional phosphorylation site for the cAMP-dependent protein kinase PKA. This phosphorylation has two effects on Rap1 action. One, it decreases the level of Rap1 activity as measured by GTP loading and the coupling of Rap1 to RapL, a Rap1 effector that couples Rap1 GTP loading to integrin activation. Two, it destabilizes the membrane localization of Rap1, promoting its translocation into the cytoplasm. These two actions, decreased GTP loading and decreased membrane localization, are related, as the translocation of Rap1-GTP into the cytoplasm is associated with its increased GTP hydrolysis and inactivation. The consequences of this phosphorylation in Rap1-dependent cell adhesion and cell migration were also examined. Active Rap1 mutants that lack this phosphorylation site had a minimal effect on cell adhesion but strongly reduced cell migration, when compared with an active Rap1 mutant that retained the phosphorylation site. This suggests that optimal cell migration is associated with cycles of Rap1 activation, membrane egress, and inactivation, and requires the regulated phosphorylation of Rap1 by PKA.

Keywords: Cell Adhesion; Cell Migration; Electrostatic Switch; Geranylgeranylation; Low Molecular Weight G Proteins; Phosphorylation; Protein Kinase A (PKA); Rap1; cAMP.

FIGURE 1.

PKA phosphorylation of the carboxyl-terminal domain of Rap1. A, alignment of the carboxyl-terminal sequences of Rap1a, Rap1b (human, bovine, and mouse), and Rap1 (Drosophila melanogaster). The amino acid residue number is shown below. The conserved cysteines demarcating the sites of geranylgeranyl modification within the CAAX domains are indicated with the arrow. PKA consensus phosphorylation sites are shown in gray. B, phosphorylation of serine 179/180 in Rap1b. HEK293 cells were transfected with wild type FLAG-tagged Rap1b (Flag-Rap1WT) or FLAG-Rap1AA (mutated at the consensus PKA sites Ser-179 and Ser-180), and treated with forskolin (F) or left untreated (U). Forskolin induced the phosphorylation in FLAG-Rap1WT, but not FLAG-Rap1AA, as detected by the PKAS Ab (first panel). Levels of FLAG-Rap1 in the total cell lysate (TCL) are shown (second panel). C, phosphorylation of serine 179/180 in constitutively active Rap1E63 (RapE63). HEK293 cells were transfected with FLAG-tagged RapE63 (FLAG-RapE63) and treated with forskolin (F) or left untreated (U). Forskolin induced the phosphorylation in FLAG-RapE63, as detected by the PKAS Ab, and these actions were prevented by pretreament with H89 (FH) (first panel). Levels of FLAG-RapE63 within the immunoprecipitate (IP) and total cell lysate are shown in the second and third panels, respectively. D, the phosphorylation of endogenous Rap1 can be monitored by PKAS antibody. HEK293 cells were treated with forskolin for the times indicated. Cells were lysed and endogenous Rap1 was immunoprecipitated and assayed for PKA-dependent phosphorylation, detected using PKAS Ab (first panel). Phosphorylation can be seen at 15 min of forskolin and is maximal by 20 min. Rap1 levels within the total cell lysate are shown (second panel). E, the phosphorylation of endogenous Rap1 by forskolin is PKA-dependent. HEK293 cells were untreated (U), treated with forskolin alone (F), or in the presence of pretreatment with H89 (FH). Cells were lysed, endogenous Rap1 was immunoprecipitated and assayed for PKA-dependent phosphorylation, detected using PKAS Ab following Rap1 IP (first panel). Rap1 levels within the immunoprecipitate and total cell lysate are shown in the second and third panels, respectively. F, the phosphorylation of Rap1b by forskolin can be monitored by gel mobility shift. Cells were transfected with FLAG-Rap1b or FLAG-Rap1AA and treated with forskolin for the indicated times. The mobility of FLAG-Rap1b is shown by Western blot and can be seen as a gel shift that is complete by 20 min. This shift requires serines 179/180, as it is absent in FLAG-Rap1AA.

FIGURE 2.

The level of activated Rap1 is decreased by PKA phosphorylation. A, inhibition of PKA enhances Rap1 activity. HEK293 cells were transfected with wild type FLAG-Rap1 (Rap1WT) or FLAG-Rap1AA (Rap1AA), along with FLAG-Epac1, and left untreated (U) or treated with forskolin (F), OMe-cAMP (OMe), and H89, as indicated. Activity of FLAG-Rap1 (Flag-Rap1-GTP) was measured by a GST-Ral-GDS pull-down assay (Ral-GDS), followed by a FLAG Western blot (first panel). The expression levels of FLAG-Rap1 (WT or AA) and FLAG-Epac1 within the total cell lysates (TCL) are shown in the second and third panels, respectively. B, the level of Rap1 activity is decreased following by PKA-dependent phosphorylation. HEK293 cells were transfected with wild type FLAG-Rap1 (Rap1WT), FLAG-Rap1AA (Rap1AA), or FLAG-Rap1D (Rap1D), as indicated, along with FLAG-Epac1. Cells were treated with forskolin in the absence of H89 (F), in the presence of H89 pre-treatment (FH), or left untreated (U). Activity of FLAG-Rap1 (Flag-Rap1-GTP) was measured by Ral-GDS assay, followed by a FLAG Western blot (first panel). The expression levels of total FLAG-Rap1 (WT or mutants) and FLAG-Epac1 within the total cell lysates are shown in the second and third panels, respectively. The relative levels of Rap1 activation (Rap-GTP) averaged from four independent experiments are shown in the bar graph, n = 4 ± S.E. *, denotes statistical significance at <0.05. **, denotes statistical significance at <0.01. ns, denotes statistical significance at ≥0.05. C–E, inhibition of PKA enhances endogenous Rap1 activity in multiple cell lines. RCC10 (C), MDM-MB-231 (D), and Ovcar3 (E) cells were treated with H89 alone (H), forskolin in the absence of PKA inhibitors (F), in the presence of either H89 (FH) or myr-PKI (F/PKI), or left untreated (U). Cell lysates were examined for activated Rap1 (Rap-GTP) by a Ral-GDS assay followed by a Rap1 Western blot (first panel) and total Rap protein levels are shown in the second panel.

FIGURE 3.

PKA enhances GAP-dependent inactivation of Rap1-GTP. A, PKA inhibits the duration of Rap1 activation by forskolin. HEK293 cells were transfected with FLAG-Epac1 and wild type FLAG-Rap1 (Rap1WT, left panels) or FLAG-Rap1AA (Rap1AA, right panels), and treated with forskolin for the times indicated. Activity of FLAG-Rap1 (Flag-Rap1-GTP) was measured by a Ral-GDS assay, followed by a FLAG Western blot (first panel). The levels of total FLAG-Rap1 (WT or AA) within the total cell lysates (TCL) are shown in the second panel. The levels of FLAG-Epac1 are also shown (third panel). B, the introduction of basic amino acids restores the activity of the phosphomimetic mutant Rap1D. HEK293 cells were transfected with wild type FLAG-Rap1 (Rap1WT), FLAG-Rap1D (Rap1D), or FLAG-Rap1DK (Rap1DK), as indicated, along with FLAG-Epac1. Cells were treated with forskolin alone (F) or following H89 pre-treatment (FH) or left untreated (U). Activity of FLAG-Rap1 (Flag-Rap1-GTP) was measured by a Ral-GDS assay, followed by a FLAG Western blot (first panel). The expression levels of FLAG-Rap1 (WT or mutants) and FLAG-Epac1 within the total cell lysates are shown in the second and third panels, respectively. C, PKA cannot inhibit the GTP loading of the GAP-insensitive mutant RapE63. HEK293 cells were transfected with FLAG-tagged Rap mutants (E63, E63AA, E63D), treated with forskolin (+) or left untreated (−), and Rap1 activity was measured by Ral-GDS assay followed by a FLAG Western blot (first panel). The levels of FLAG-Rap mutants E63, E63AA, and E63D within the total cell lysates are shown in the second panel.

FIGURE 4.

PKA effects binding of Rap1 to the Rap1 effector RapL. A, the binding of Rap1 to its effector RapL is decreased by PKA-dependent phosphorylation. HEK293 cells were transfected with FLAG-RapL and wild type GFP-Rap1 (Rap1WT), GFP-Rap1AA, or GFP-Rap1D, as indicated, along with FLAG-Epac1. Cells were treated with forskolin in the absence of H89 (F), in the presence of H89 pre-treatment (FH), or left untreated (U). RapL binding to GFP-Rap1 and mutants was shown by a FLAG Western blot following GFP immunoprecipitation (IP) (first panel). Levels of GFP-Rap WT and mutants in the immunoprecipitation are shown in the second panel. The levels of expression of FLAG-RapL (third panel), GFP-Rap1WT and mutants (fourth panel), and FLAG-Epac1 (fifth panel) in the total cell lysates (TCL) are shown. The last two lanes include cells transfected with FLAG-Epac1 and GFP-Rap1 (C1) or FLAG-Epac1 and FLAG-RapL (C2). B, the binding of constitutively activated Rap1 to its effector RapL is not affected by PKA-dependent phosphorylation. HEK293 cells were transfected with FLAG-RapL and either wild type GFP-Rap1 (WT) or mutants E63, E63AA, or E63D, as indicated. Cells were treated with forskolin (F) or left untreated (U). RapL binding to GFP-Rap1 mutants was determined by a FLAG Western blot following GFP IP (first panel). Levels of Rap1 mutants within the IP are shown in the second panel. The expression levels of FLAG-RapL (third panel) and RapE63 mutants (fourth panel) within the total cell lysates are shown. The last lane includes cells transfected with Rap1WT (WT) and left untreated (U), showing the GTP dependence of the interaction with RapL.

FIGURE 5.

Rap1 localization is regulated by PKA phosphorylation. A, Rap1 translocates from the membrane to the cytosol upon forskolin treatment, whereas Rap1-GAP is always cytosolic. HEK293 cells were transfected with FLAG-Rap1-GAP and FLAG-Rap1WT and were left untreated (−) or treated with forskolin (+), then fractionated into soluble (S, left) and particulate (P, right) fractions. FLAG-Rap1-GAP (first panel) and FLAG-Rap1 (second panel) were detected by a FLAG Western blot. B and C, forskolin induces the translocation of endogenous Rap1 from the plasma membrane and into the cytosol in HEK293 (B) and H1299 cells (C). Cells were treated with forskolin for 20 min, and lysates were separated into particulate (P) and soluble (S) fractions. The first and second panels show the levels of the cytoplasmic protein B-Raf and the membrane protein cadherin as markers for S and P fractions, respectively. The third panel shows the localization of endogenous Rap1 protein within the S and P fractions, as detected by a Rap1 Western blot. The bottom inset shows the levels of ERK2 (upper panel) and Rap1 (lower panel) within the total cell lysates (TCL) before and after forskolin treatment. D, forskolin-induced translocation of Rap1 from the plasma membrane to the cytosol requires Rap1 phosphorylation. Forskolin induces a relocalization of FLAG-RapE63 (E63), but not FLAG-RapE63AA (E63AA) to the soluble fraction. HEK293 cells were transfected with the indicated FLAG-tagged proteins, treated with forskolin for 20 min, and lysates were separated into soluble and particulate fractions. The first and second panels show the levels of the cytoplasmic protein B-Raf and the membrane protein Cadherin as markers for S and P fractions, respectively. The third panel shows the localization of FLAG-Rap mutants within S and P fractions, as detected by a FLAG Western blot. The bottom inset shows the levels of FLAG-Rap mutant proteins (upper panel) and ERK2 (lower panel) within the total cell lysates before and after forskolin treatment. E, immunofluorescence of SK-MEL-24 cells. Left, a representative untreated cell showing endogenous Rap1 at the plasma membrane. Right, a representative cell treated with forskolin (20 min). The upper and lower panels represent two optical sections through the same cells. The optical planes in the upper panels are 0.32 μm above those in the lower panels. The scale bar is 5 μm in length. F, time course of Rap1 relocalization by forskolin. HEK293 cells were transfected with GFP-Rap1WT (first panel) or GFP-RapAA (second panel). Cells were left untreated (Un) or treated with forskolin for 10 and 30 min, as indicated. The scale bar is 5 μm in length. G, quantification of Rap1 at the plasma membrane in real time. The fluorescence intensities of GFP-Rap1WT or GFP-RapAA at the plasma membrane and the cytosol at 0, 10, and 30 min after forskolin treatment were quantified as described under “Experimental Procedures.” The data are plotted as the ratio of cytoplasmic to plasma membrane intensities, normalized to that ratio calculated for 0 min, n = 3, ± S.E. ***, denotes statistical significance at <0.001.

FIGURE 6.

Localization of Rap1 mutants in cells. A, the introduction of basic amino acids restores plasma membrane relocalization of the Rap1D mutant. Micrographs of HEK293 cells transfected with GFP-tagged Rap1 constructs, GFP-Rap1WT, GFP-Rap1D, and GFP-Rap1DK, are shown. Images were taken before and 20 min after forskolin treatment. For each transfection, a representative cell is shown. The scale bar is 5 μm in length. B, localization of RapE63 is regulated by PKA phosphorylation. Shown here are micrographs of HEK293 cells transfected with the GFP-tagged RapE63 constructs, GFP-RapE63 WT, GFP-RapE63AA, and GFP-RapE63D. For each transfection, a representative cell is shown before and after forskolin treatment. The scale bar is 5 μm in length.

FIGURE 7.

RapE63AA and RapE63 stimulate adhesion in H1299 cells. A, adhesion assay. Stably transfected H1299 cells expressing pcDNA3 vector (Vector) or FLAG-RapE63 (RapE63) or FLAG-RapE63AA (RapE63AA) were resuspended and plated in quadruplicate onto fibronectin (1 μg/ml)-coated wells and adherent cells were quantitated as described under “Experimental Procedures.” The values shown are the average of 4 independent experiments, ± S.E. **, denotes statistical significance at <0.01. ***, denotes statistical significance at <0.001. B, adhesion assay. Stably transfected H1299 cells expressing pcDNA3 vector (V) or FLAG-RapE63 (E63) were incubated with H89 40 min before being plated onto fibronectin (1.5 μg/ml)-coated wells and cells were allowed to adhere for 60 min. The percentage of adherent cells were quantitated as described under “Experimental Procedures.” The values shown are the average of 5 independent experiments, ± S.E. *, denotes statistical significance at <0.05. **, denotes statistical significance at <0.01. ns, denotes statistical significance at ≥0.05. C, stable cells lines. H1299 cells expressing pcDNA3 vector (V), FLAG-RapE63 (E63), or FLAG-RapE63AA (E63AA) were stably selected as described. The levels of transfected Rap proteins are shown in the first panel. The levels of ERK2 are shown in the second panel as a loading control.

FIGURE 8.

Cell migration assays show Rap1-dependent migration. A, wound healing assay. Stably transfected H1299 cells expressing pcDNA3 vector (V), FLAG-RapE63 (E63), or FLAG-RapE63AA (E63AA) were examined by a wound healing assay as described under “Experimental Procedures.” The bar graph shows the percentage of wound closure across the scratch at 8 h. The values shown are the average of 5 independent experiments, ± S.E. **, denotes statistical significance at <0.01. B, wound healing assay. Stably transfected H1299 cells expressing pcDNA vector (V) or FLAG-RapE63 (E63) were prepared for a wound healing assay as described under “Experimental Procedures.” Then cells were incubated in 1% serum with (+) or without (−) H89. Cell migration across the gap was monitored and analyzed after 8 h. The bar graph shows the percentage of wound closure. n = 3, ± S.E. *, denotes statistical significance at <0.05. ***, denotes statistical significance at <0.001. C, modified Boyden chamber assay. Stably transfected H1299 cells expressing pcDNA vector (Vector), FLAG-RapE63 (E63), or FLAG-RapE63AA (E63AA) were loaded into duplicate wells of a 24-well BD FluoroBlock^TM plates (modified Boyden chamber) and migrating cells were quantitated as described under “Experimental Procedures.” n = 4, ± S.E. *, denotes statistical significance at <0.05. **, denotes statistical significance at <0.01. D, modified Boyden chamber assay. Stably transfected H1299 cells expressing pcDNA vector (V), FLAG-Rap1WT (WT), or FLAG-RapAA (AA) were serum staved overnight and loaded into duplicate wells, then allowed to migrate into 1% serum contain medium with (+) or without (−) forskolin (10 μm)/3-isobutyl-1-methylxanthine (100 μm) of a 24-well BD FluoroBlock^TM plates (modified Boyden chamber). Migrating cells were quantitated as described under “Experimental Procedures.” n = 3, ± S.E. **, denotes statistical significance at <0.01. ***, denotes statistical significance at <0.001. E, stable cells lines used in S180D. H1299 cells expressing pcDNA3 vector (V), FLAG-Rap1 (WT), or FLAG-RapAA (AA) were stably selected as described. The levels of transfected Rap proteins are shown in the first panel. The levels of ERK2 are shown in the second panel as a loading control. F, wound healing assay. H1299 cells expressing either pcDNA3 vector (V), scrambled (scr), or Rap1a shRNA (sh-Rap1a) were examined by a wound healing assay as described under “Experimental Procedures.” The bar graph shows the percentage of wound confluence at 6 h. n = 4, ± S.E. *, denotes statistical significance at <0.05. G, stable cells lines were as described in F. The efficiencies of knockdown of endogenous Rap1a by pcDNA3 vector (V), scrambled (scr), or Rap1a shRNA (sh-Rap1a) are shown in the first panel and total endogenous ERK is shown as a loading control in the second panel.