Adhesion to the extracellular matrix regulates the coupling of the small GTPase Rac to its effector PAK

Abstract

The small GTPase Rac regulates cytoskeletal organization, cell cycle progression, gene expression and oncogenic transformation, processes that depend upon both soluble growth factors and adhesion to the extracellular matrix (ECM). We now show that growth factors and adhesion to the ECM both contribute independently and approximately equally to Rac activation. However, activated Rac in non-adherent cells failed to stimulate the Rac effector PAK. V12 Rac or Rac activated by serum translocated to the membrane fraction of adherent cells but remained mainly cytoplasmic in suspended cells. An activated Rac mutant lacking a membrane-targeting sequence did not activate PAK in adherent cells, while mutations that forced membrane targeting restored PAK activation in suspended cells. In vitro, V12 Rac showed greater binding to membranes from adherent relative to suspended cells, indicating that cell adhesion regulated membrane binding sites for Rac. These results show that ECM regulates the ability of Rac to couple with PAK via an effect on membrane binding sites that facilitate their interaction.

Fig. 1. Rac activation by cell adhesion and serum. (A) Serum-starved NIH 3T3 cells were detached and held in suspension for 3 h, then plated on fibronectin-coated dishes. Cell lysates were incubated with GST–PBD beads or GST-only beads, and bound GTP-Rac was detected by Western blotting. Right panel: cells transiently transfected with V12 Rac were used as a positive control to demonstrate specific binding to GST–PBD but not to GST. Lower panel: total cell lysates probed for Rac demonstrate equal amounts of total Rac. Data are representative of four independent experiments. (B) Attached (Att) or suspended (Sus) serum-starved cells were stimulated with 10% calf serum for 10 min, and Rac activity was assayed as described. Data are representative of four experiments. (C) Densitometric quantification of (B). Relative Rac activity was calculated from the amount of PBD-bound Rac normalized to the amount of Rac in whole-cell lysates, and then expressed as a percentage of the maximum in each experiment. Bar graphs represent means ± SEM of four experiments.

Fig. 2. PAK activation requires integrin-mediated cell adhesion. (A) Stably adherent serum-starved cells or cells in suspension for 3 h were stimulated with 10% serum for 10 min. PAK protein was immunoprecipitated and its kinase activity was assayed. Data are representative of six independent experiments. (B) Cells were maintained in suspension for 3 h and then replated on plastic coated with anti-mouse CD44 IgG or anti-mouse β1 integrin IgG. After 2 h, cells were stimulated with 10% serum for 10 min and PAK kinase activity assayed. Data are representative of two experiments. (C) PAK immunoprecipitates from serum-starved attached (Att) or suspended (Sus) cells were incubated without (CON) or with recombinant GST–Cdc42 loaded with GDP or GTPγS as indicated. PAK in vitro kinase activity was then assayed using MBP as substrate. Kinase activity from adherent cells immunoprecipitated with pre-immune serum (PRE) and incubated with GTPγS-loaded GST–Cdc42 is shown as a control. Data are representative of three experiments.

Fig. 3. V12 Rac is adhesion dependent. Rat-1 cells stably expressing Myc-tagged V12 Rac were cultured for 48 h in the presence or absence of tetracycline (tet) and extracts prepared. (A) Lysates were analyzed by Western blotting with anti-Rac and anti-myc mAbs to assess cellular levels of the V12 and endogenous Rac. (B) Control Rat-1 cells or V12 Rac cells were cultured with or without tet in low serum, then placed in suspension for 2 h or kept stably adherent. PAK was then immunoprecipitated and its kinase activity determined using the in-gel kinase assay. Data are representative of three independent experiments. (C) Densitometric quantitation of PAK kinase activity normalized for PAK protein levels; values are means ± SD from three experiments.

Fig. 4. Rac translocation to the particulate fraction. (A) Serum-starved attached (Att) or suspended (Sus) cells were stimulated with 10% serum and subjected to hypotonic lysis. The particulate (P) and soluble (S) fractions were isolated and samples with 10 µg of protein were analyzed. These amounts typically represent 1–2% of the cytoplasmic fraction and 10–20% of the membrane fraction. Samples were subjected to SDS–PAGE and Western blotting with anti-Rac, anti-PAK or anti-Cdc42. The anti-integrin β1 subunit and anti-RhoGDI were used as markers for the membrane and cytosol, respectively. Results are representative of three independent experiments. The shift in mobility of the β1 subunit is consistent with previously described changes in integrin processing in suspended cells (Dalton et al., 1995). (B) Densitometric quantification of Rac translocation. The amount of Rac was corrected for total protein and the percentage in the membrane fraction calculated. Values are means ± SEM from three separate experiments. (C) Soluble and particulate fractions from attached or suspended Rat-1-V12 Rac cells in the presence of tetracycline (10 µg each) were analyzed by Western blotting. The upper band co-migrates with the Myc-positive V12 Rac while the lower band co-migrates with endogenous Rac in untransfected cells. As in (A), 10 µg of the particulate and soluble fractions represents 10–20% and 1–2% of the total, respectively.

Fig. 5. Membrane targeting is required for PAK activation. Normal V12 Rac or V12 Rac with a mutated CAAX sequence in the vector pBK-CMV were transiently transfected into NIH 3T3 cells. Transfection efficiency was ∼30%. (A) Western blots of lysate from transfected cells demonstrate equivalent expression of the Rac constructs. (B) At 24 h, cells were transferred to medium with 0.2% serum. At 48 h, the adherent cells were harvested and total endogenous PAK was immunoprecipitated. Both PAK protein and kinase activity were assayed and specific activity (kinase activity normalized for PAK protein) calculated. Values are means ± SD (n = 3).

Fig. 6. Restoring PAK activity in suspended cells. (A) Diagram of membrane-targeted Rac mutants. HA-tagged wild-type Rac was fused to the transmembrane domain of the Tac subunit of the IL-2 receptor or the myristylation sequence from c-src. (B) Upper panel: a Western blot with anti-HA antibody of total cell lysate from transfected cells. The myristylated and IL2R–Rac chimeras were expressed at substantially lower levels than wild-type HA-Rac. Inclusion of cDNA for green fluorescent protein showed that transfection efficiency was ∼30%. Lower panel: the transfected Rac proteins in the soluble and particulate fractions of non-adherent cells subjected to homogenization and fractionation. Wild-type Rac was primarily cytosolic whereas both mutants show enhanced localization to the particulate fraction. (C) Serum-starved cells were placed in suspension for 3 h, stimulated with serum for 10 min and total endogenous PAK immunoprecipitated. PAK protein and kinase activity were determined and the specific activity calculated. Values are means ± SD (n = 4).

Fig. 7. In vitro membrane binding of V12 Rac. Cytosol from attached or suspended V12 Rac cells was incubated with equal amounts of membranes from attached or suspended NIH 3T3 cells. Membranes were collected by centrifugation and V12 Rac detected by Western blotting with anti-Myc. Western blotting for the integrin β1 subunit demonstrates equivalent levels of membrane protein in each sample. Data are representative of three independent experiments.