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Inhibition of PYK2-induced actin cytoskeleton reorganization, PYK2 autophosphorylation and focal adhesion targeting by FAK

Quan-Sheng Du1, Xiu-Rong Ren1, Yi Xie1, Qiang Wang2, Lin Mei2 and Wen-Cheng Xiong1,*

1 Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
2 Departments of Neurobiology, Pathology and Physical Medicine and Rehabilitation, University of Alabama at Birmingham, Birmingham, AL 35294, USA
* Author for correspondence (e-mail: wxiong{at}path.uab.edu )



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  		Fig. 1. Expression and focal adhesion localization of PYK2 in fibroblasts. (A) Western blot analysis of HEK 293 cell lysates expressing Myc-tagged PYK2 and FAK using antibodies against Myc, FAK (monoclonal, 2A7) and PYK2 (polyclonal, from UBI). (B) Western blot analysis of PYK2 expression in different cell types. Solid and open arrows indicate PYK2 and FAK, respectively. In (A) and (B), cell lysates were resolved on SDS-PAGE, transferred to nitrocellulose and probed with antibodies as indicated. (C) Co-immunostaining of PYK2 with FAK or paxillin in fibroblasts. 10T1/2 (a-f) and fak-/- fibroblasts (g-l) were immunostained with the polyclonal antibody against PYK2 (UBI) (a,d,g,j) and the monoclonal antibody against paxillin (b,h) or FAK (e,k); c, f, i and l are overlay pictures. Bar, 50 µm. (D) Time course of fibronectin treatment on PYK2 focal adhesion localization. 10T1/2 fibroblasts were trypsinized and re-plated on fibronectin-coated coverslips for indicated time. These cells were then immunostained with the polyclonal antibody against PYK2 (UBI) (a,d,g) and monoclonal antibody against paxillin (b,e,h); c, f and i are overlay pictures. Scale for D same as C.

 


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  		Fig. 2. Alterations of cell morphology and focal adhesions in Swiss 3T3 cells expressing PYK2. Swiss 3T3 fibroblasts were microinjected with plasmids encoding Myc-tagged PYK2 (A-C,G-I) or FAK (D-F,J-L). Cells were fixed for 2-4 hours after injection and immunostained with anti-PYK2 (polyclonal, against PYK2 amino acids 587-988) (A,G) and anti-vinculin (monoclonal) (B,E) or anti-FAK (monoclonal) (D,J) antibodies, or with phalloidin (H,K); C, F, I and L are overlay pictures. Bar, 50 µm.

 


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  		Fig. 3. Alterations of focal adhesions in 3Y1, 10T1/2 and MDCK cells expressing PYK2. 3Y1 (A,B), 10T1/2 (C,D) and MDCK (E,F) cells were transiently transfected with plasmid encoding Myc-tagged PYK2. Cells were fixed 24 hours after transfection and immunostained with the polyclonal antibodies against PYK2 (amino acids 587-988) (A,C,E) or monoclonal antibodies against vinculin (B,D,F).

 


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  		Fig. 4. The N terminus and FAT domain of PYK2 are required for PYK2-mediated actin cytoskeleton reorganization. (a) Mapping domains of PYK2 required for actin cytoskeleton reorganization and focal adhesion targeting. Plasmids encoding wild-type PYK2 (A,A') and its deletion mutants including PYK2{Delta}1-88 (B,B'), PYK2{Delta}1-416 (C,C'), PYK2{Delta}1-868 (D,D'), PYK2{Delta}1-902 (E,E') and PYK2{Delta}936-1009 (F,F') were microinjected into Swiss 3T3 cells. The microinjected cells were fixed 2-4 hours after injection and immunostained with antibodies against PYK2 (polyclonal, against PYK2 amino acids 587-988) (A-F) and paxillin (A'-F'). (b) PYK2 and PYK2 mutants and a summary of the phenotypes of Swiss 3T3 cells expressing these mutants. The numbers on the diagrams represent the amino acid residues in PYK2. The proline-rich sequences, Band 4.1, kinase and FAT domains are indicated. The percentage of cytoskeleton reorganizing activity was determined by counting the number of PYK2-expressing cells with podosome-like focal adhesions and dividing by the total number of PYK2-expressing cells. More than 200 injected cells of each construct were counted. The percentage of total PYK2-expressing cells with normal or podosome-like focal adhesion localization of PYK2 is also listed.

 


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  		Fig. 5. Effect of the catalytic activity and autophosphorylation of PYK2 on cytoskeletal reorganization. (A) Swiss 3T3 cells were microinjected with plasmids encoding Myc-tagged PYK2 (PYK2-WT), catalytically inactive PYK2 (PYK2-KD) or the autophosphorylation mutant (PYK2-Y402F) and stained with antibodies against PYK2 (polyclonal, UBI) and vinculin (monoclonal, Sigma). White arrows indicate the microinjected cells. Bar, 50 µm. (B) PYK2-WT, PYK2-KD and PYK2-Y402F protein were immunoprecipitated from transfected HEK293 cells and subjected to SDS-PAGE or to an in vitro kinase assay using GST-paxillin N-terminus as a substrate. (C) Increased tyrosine phosphorylation of several proteins including p130Cas was observed in PYK2-expressing cells. HEK 293 cells lysates expressing Myc-tagged PYK2-WT and PYK2-KD were either directly immunoblotted with antibodies against Myc and phosphotyrosine (RC20, Transductions Labs) or immunoprecipitated with antibodies against p130Cas (Transduction Labs) and immunoblotted with antibodies against p130Cas and phosphotyrosine. Arrows indicate proteins with increased tyrosine phosphorylation in PYK2-WT expressing cells. (D) Histograms summarizing results in (A). These show the percentages of means ± s.d. of three or more different samples (total of 200 microinjected cells). The asterisk indicates a P value of <0.05 compared with expression of (PYK2-WT) (Mann-Whitney u test).

 


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  		Fig. 6. Suppression of PYK2-induced cytoskeletal reorganization by FAK. Plasmids encoding GFP (50 ng), FAK (25 ng, 50 ng and 100 ng), FAK-Y397F (50 ng) or FRNK (50 ng) were co-injected with Myc-tagged PYK2 (50 ng) into Swiss 3T3 cells. (A) Immunostaining of injected cells with antibodies against PYK2 (monoclonal, Transduction Labs), FAK (BC3) or FRNK (BC3). (B) Histograms summarizing results from the co-injection experiments. Microinjected cells with normal focal adhesion or cell shape are shown as percentages of means ± s.d. of three or more different samples (total of 200 microinjected cells). PYK2+FAK (1:0.5), (1:1) and (1:2) represent ratios of plasmid DNA between PYK2 and FAK, which are 50:25 ng, 50:50 ng and 50:100 ng, respectively. Asterisk represents a P value of <0.05 and double asterisks a P value of <0.01 compared with the expression of PYK2 alone (Mann-Whitney u test).

 


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  		Fig. 7. Inhibition of PYK2 autophosphorylation by FAK. (A) FAK N-terminal domain is required for inhibition of PYK2 autophosphorylation. (B) FAK tyrosine phosphorylation was not required for inhibition of PYK2 autophosphorylation. HEK 293 cells were transfected with constructs encoding Myc-tagged PYK2, PYK2 plus FAK or PYK2 plus FAK mutants as indicated. Cell lysates were immunoblotted with anti-Myc antibodies to detect the production of PYK2, FAK or FAK mutants and anti-phosphotyrosine 402 (PY402, Biosource). Solid arrows indicate PYK2 and open arrows indicate FAK or FAK mutants.

 


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  		Fig. 8. Reduction of PYK2 focal adhesion targeting by FAK. (a) First, fak-/- fibroblasts were transiently transfected with the empty vector (A-C) or vectors encoding FAK (D-F) or FRNK (G-I). They were then stained with polyclonal antibodies against PYK2 (UBI) (A,D,G) or monoclonal antibodies against paxillin (B) or FAK (E,H); C, F and I are overlay pictures. Arrows indicate the FAK- or FRNK-expressing cells. Bar, 50 µm. (b) Summary of results from (a). More than 100 transfected cells were examined for endogenous PYK2 subcellular localization. Although most fak-/- cells (>90%) exhibited PYK2 focal adhesion targeting (+++), most fak-/- cells expressing FAK or FRNK (>80%) showed reduced staining intensity (+) compared with fak-/- cells without FAK or FRNK expression in the same image field.

 

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