PAK1 kinase promotes cell motility and invasiveness through CRK-II serine phosphorylation in non-small cell lung cancer cells

Abstract

The role of c-Crk (CRK) in promoting metastasis is well described however the role of CRK phosphorylation and the corresponding signaling events are not well explained. We have observed CRK-II serine 41 phosphorylation is inversely correlated with p120-catenin and E-cadherin expressions in non-small cell lung cancer (NSCLC) cells. Therefore, we investigated the role of CRK-II serine 41 phosphorylation in the down-regulation of p120-catenin, cell motility and cell invasiveness in NSCLC cells. For this purpose, we expressed phosphomimetic and phosphodeficient CRK-II serine 41 mutants in NSCLC cells. NSCLC cells expressing phosphomimetic CRK-II seine 41 mutant showed lower p120-catenin level while CRK-II seine 41 phosphodeficient mutant expression resulted in higher p120-catenin. In addition, A549 cells expressing CRK-II serine 41 phosphomimetic mutant demonstrated more aggressive behavior in wound healing and invasion assays and, on the contrary, expression of phosphodeficient CRK-II serine 41 mutant in A549 cells resulted in reduced cell motility and invasiveness. We also provide evidence that PAK1 mediates CRK-II serine 41 phosphorylation. RNAi mediated silencing of PAK1 increased p120-catenin level in A549 and H157 cells. Furthermore, PAK1 silencing decreased cell motility and invasiveness in A549 cells. These effects were abrogated in A549 cells expressing phosphomimetic CRK-II serine 41. In summary, these data provide evidence for the role of PAK1 in the promotion of cell motility, cell invasiveness and the down regulation of p120-catenin through CRK serine 41 phosphorylation in NSCLC cells.

Figure 1. A- Western blots showing the expression pattern of p120-catenin, E-cadherin, CRK-I, CRK-II, phospho-serine 41 CRK-II and phospho-tyrosine 221 CRK-II in a panel of NSCLC cells and BEAS-2B cells.

B- Quantification of CRK-II, phospho-tyrosine 221 CRK-II and phospho-serine 41 CRK-II signal intensity among NSCLC cell lines and BEAS-2B cells.

Figure 2. A- Relative p120-catenin (CTNND1) promoter activity in A549, Rh2 and H157 cell lines following transient transfection of CRK-II, CRK-II (Ser41Gly) or CRK-II (Ser41Asp) mutants.

(2 tailed student’s t-test: * P<0.05; ** P<0.01; error bars represent ± standard deviation). B- Western blots showing changes in p120-catenin protein level in the above mentioned cell lines following transient transfection of CRK-II and CRK-II mutants.

Figure 3. A-Wound healing assays in A549 cells stably expressing pCMV vector, wild type CRK-II (WT), CRK-II (Ser41Gly) or CRK-II (Ser41Asp) mutants.

Endogenous CRK is silenced in all conditions by siRNA. B- Measurement of wound surface area 24 hours after establishment of the wound among the above mentioned groups. The average is calculated following measurement of three separate experiments. (2 tailed student’s t-test: ** P<0.01; *** P<0.001; error bars represent ± standard deviation).C- Invasion assays following incubation of stably transfected A549 cells expressing either pCMV vector; wild type CRK-II (WT); CRK-II (Ser41Gly) or CRK-II (Ser41Asp) mutants. 1×10⁵ cells were incubated over night as described in the Methods section and stained at 24 hours after incubation. D- Quantitative measurement of the invaded cells. Five separate sections of the invaded cells were counted. (2 tailed student’s t-test: ** P<0.01; *** P<0.001; error bars represent ± standard deviation).

Figure 4. A-Relative p120-catenin (CTNND1) promoter activity in A549 cells following siRNA mediated silencing of PAK1 and PAK2.

B- Quantitative real time PCR measurement of PAK1 and PAK2 mRNA in order to determine the silencing efficiency of siRNA. C-Western blots showing phospho-serine 41 CRK-II and p120-catenin expression following siRNA mediated PAK1 silencing in A549 and H157 cells. D- Pak1 phosphorylation consensus sequence in several Pak1 substrates. Residues that are phosphorylated by Pak1 are highlighted in gray. Circled are the upstream arginine residues that are important for Pak1 target phosphorylation.

Figure 5. Relative p120-catenin (CTNND1) promoter activity in A549 cells stably expressing wild type CRK-II (WT), CRK-II (Ser41Gly) or CRK-II (Ser41Asp) mutants following siRNA mediated silencing of PAK1.

Endogenous CRK is silenced in all conditions by siRNA. (2 tailed student’s t-test: * P<0.05; error bars represent ± standard deviation).

Figure 6. A-Wound healing assays in A549 cells stably expressing wild type CRK-II (WT) or CRK-II phosphomimetic (Ser41Asp) mutants following siRNA mediated PAK1 silencing.

Endogenous CRK is silenced in all conditions by siRNA. B- Measurement of wound surface area 24 hours after the establishment of the wound among the above mentioned groups. The average is calculated following measurement of three separate experiments. (2 tailed student’s t-test: ** P<0.01; *** P<0.001; error bars represent ± standard deviation). C- Invasion assays following incubation of stably transfected A549 cells expressing wild type CRK-II (WT) or CRK-II (Ser41Asp) mutant. Each cell line was treated with PAK1 siRNA or scrambled sequence siRNA. 1×10⁵ cells were incubated over night as described in the Methods section and stained at 36 hours after incubation. D- Quantitative measurement of the invaded cells. Five separate sections of the invaded cells in each Matrigel membrane were counted. (2 tailed student’s t-test: ** P<0.01; *** P<0.001; error bars represent ± standard deviation).

Figure 7. Schematic view of PAK1, CRK-II and SP1 as well as FOXC2 in transcriptional regulation of p120-catenin (CTNND1).