Minimal features of paxillin that are required for the tyrosine phosphorylation of focal adhesion kinase

Abstract

Tyrosine phosphorylation of FAK (focal adhesion kinase) regulates signalling that results from the interaction of integrins with extracellular matrix and growth factor receptors. A critical step in this process is the phosphorylation of Tyr397 of FAK, which creates a binding site for Src family kinases, PI3K (phosphoinositide 3-kinase) and Shc (Src homology and collagen homology). An intact Tyr397 site is required for FAK-mediated regulation of cell migration, survival signals and full responsiveness to soluble growth factors. We showed previously that the adaptor protein paxillin is required for the overall tyrosine phosphorylation of FAK in embryonic stem cells [Wade, Bohl and Vande Pol (2002) Oncogene 21, 96-107]. In the present paper, we identify the minimal structural features of paxillin that are required to support overall FAK tyrosine phosphorylation and Tyr397 phosphorylation. Paxillin contains N-terminal leucine-rich LD motifs that bind directly to FAK and four LIM (Lin-11, Isl-1 and Mec-3) domains in the C-terminus. We show that paxillin LIM domains 1, 2 and 3 are each required for FAK tyrosine phosphorylation, while LIM4 is dispensable. In addition to paxillin LIM domains 1, 2 and 3, a single LD motif on paxillin is required to support FAK tyrosine phosphorylation in embryonic stem cells. Both sequence and spatial requirements exist for LD motifs to support FAK tyrosine phosphorylation. Interestingly, synthetic LD motifs that fail to bind FAK in vitro are able to fully support FAK tyrosine phosphorylation, indicating that minimal interactions of LD motifs with FAK suffice. Our results demonstrate at least four distinct structural domains of paxillin support at least three distinct functions that are each required for FAK tyrosine phosphorylation.

Figure 1. Paxillin^−/− clone 17 cells are deficient in FAK phosphorylation at Tyr³⁹⁷ and Tyr⁸⁶¹

(A) FAK was immunoprecipitated (Ip) from clone 43 (^+/−) or 17 (^−/−) cells and Western-blotted (Wb) with antibodies against phosphotyrosine (pY20 and 4G10) and FAK as indicated. (B) Whole-cell lysates from clone 43 or 17 cells were probed with the indicated antibodies. FAK is minimally phosphorylated at Tyr³⁹⁷ and Tyr⁸⁶¹ in clone 17 (^−/−) cells.

Figure 2. Diagram and localization of paxillin mutants

(A) Paxillin mutants used in the present study showing the relative location of LD motifs, tyrosine phosphorylation sites (Tyr³¹ and Tyr¹¹⁸) and the LIM domains. (B) Localization of wild-type and mutant paxillin molecules expressed transiently in paxillin^−/− T17 cells. Localization of the expressed paxillin mutants was visualized with the rabbit anti-paxillin CU4 antibody (Pax.). Endogenous vinculin (Vin.) localization is also shown corresponding to the same cell as for paxillin.

Figure 3. Paxillin LIM domains 1–3 are required for FAK tyrosine phosphorylation

In the top row, FAK immunoprecipitates (IP) from clone 17 cell lines stably expressing the indicated paxillin molecules were analysed by Western blotting (WB) with the anti-phosphotyrosine antibody 4G10. The remaining strips show the same whole-cell lysates analysed by separate Western blots with the indicated antibodies. ΔLIM4 is able to support total FAK phosphorylation as well as phosphorylation at Tyr³⁹⁷ and Tyr⁸⁶¹, while loss of LIM1, LIM2 or LIM3 abrogates FAK tyrosine phosphorylation. All paxillin constructs were FLAG-tagged at the N-terminus. Wt, wild-type.

Figure 4. FAK tyrosine phosphorylation does not require LD motifs 2 and 4

The truncation mutants of paxillin shown in Figure 2(A) were used to define the minimal domain for FAK tyrosine phosphorylation. Mutants listed as ΔLD5 have amino acids 302–309 deleted. Clone 17 cells stably expressing the indicated paxillin mutant were grown and lysed, and FAK was recovered by immunoprecipitation (IP). The immunoprecipitated FAK was split, and SDS/10% (w/v) polyacrylamide gels were Western-blotted (WB) and probed with the 4G10, p397 and p861 antibodies. The p397 blot was then stripped and reprobed with the FAK antibody. The bottom panel shows the expression of the paxillin mutants by Western blotting whole-cell lysates with anti-FLAG antibody. The paxillin N-terminus is almost completely dispensable for FAK tyrosine phosphorylation.

Figure 5. FAK tyrosine phosphorylation requires one LD motif and LIM domains 1, 2 and 3

The experiments were carried out as in Figure 4. The LD1 and LD4 swap mutants contain amino acids 4–10 and 267–273 respectively fused in-frame to LIM domains 1–3 (310–502).

Figure 6. LD motifs that support FAK tyrosine phosphorylation contain the hydrophobic LD core leucines

(A, B) Experiments were carried out as in Figure 4. All mutants listed are point mutations in the 302–502 construct. Mutations of the leucines of the LD motif abrogated FAK tyrosine phosphorylation.

Figure 7. Stable association of FAK with paxillin is not required for FAK tyrosine phosphorylation

(A) Paxillin mutants were tested for their ability to associate with FAK in vitro as described in the Materials and methods section. Although wild-type (Wt) paxillin associates with GST–FAK, none of the 302–502 mutants of paxillin associate with FAK, irrespective of their ability to promote FAK tyrosine phosphorylation. ‘PD’ refers to pull-down with GST–FAK. (B) HEK-293 (human embryonic kidney) cells were transfected with the indicated paxillin construct and were lysed and subjected to immunoprecipitation (IP) with the anti-FLAG antibody, and Western blots (WB) were probed with an antibody against FAK. Paxillin LD2 and LD4 are required for direct association of FAK and paxillin in vivo. M.W., molecular mass (sizes are given in kDa).

Figure 8. Paxillin mutants that support FAK tyrosine phosphorylation support cell spreading on fibronectin

(A) ES clone 17 (^−/−) cells spreading on fibronectin. Clone 17 cells were transiently co-transfected with GFP (green fluorescent protein) and the indicated plasmid for 24 h and then plated on to a fibronectin-coated coverslip for 7 h. Clone 17 cells transfected with vector had not appreciably spread at 7 h when compared with cells expressing wild-type paxillin. (B) Cell spreading assays of clone 17 (^−/−) cells transfected with paxillin or paxillin mutants were performed as described (see the Materials and methods section). GFP-positive cells were counted, and spreading was determined. A cell was counted as spread if the diameter of the cell was at least twice the diameter of the DAPI (4,6-diamidino-2-phenylindole)-stained nucleus. Results are the means±S.D. for three independent experiments. FAK tyrosine phosphorylation and cell spreading are closely related. (C) Expression of the paxillin mutants used in (B) was determined by probing Western blots (WB) of cell lysates with anti-FLAG antibody.