New Insights into FAK Phosphorylation Based on a FAT Domain-Defective Mutation

<div><p>Mounting evidence suggests that the FAK N-terminal (FERM) domain controls FAK phosphorylation and function; however, little is known regarding the role of the C terminal (FAT) domain in FAK regulation. We identified a patient-derived FAK mutant, in which a 27-amino acid segment was deleted from the C-terminal FAT domain (named FAK-Del33). When FAK-Del33 was overexpressed in specific tumor cell lines, Y397 phosphorylation increased compared with that observed in cells expressing FAK-WT. Here, we attempt to unveil the mechanism of this increased phosphorylation. Using cell biology experiments, we show that FAK-Del33 is incapable of co-localizing with paxillin, and has constitutively high Y397 phosphorylation. With a kinase-dead mutation, it showed phosphorylation of FAK-Del33 has enhanced through auto-phosphorylation. It was also demonstrated that phosphorylation of FAK-Del33 is not Src dependent or enhanced intermolecular interactions, and that the hyperphosphorylation can be lowered using increasing amounts of transfected FERM domain. This result suggests that Del33 mutation disrupting of FAT's structural integrity and paxillin binding capacity leads to incapable of targeting Focal adhesions, but has gained the capacity for auto-phosphorylation in cis.</p></div

Intermolecular interactions do not play a dominant role in FAK-Del33 auto-phosphorylation.

<p>(A) MDA-MB-468 cells were transfected with 2 µg of plasmids encoding N.C, Y397F, K454R, or Y397F+ K454R with FAK-Del33 background in 6-well plates. The total amount of DNA was kept constant through the addition of empty vector. (B) MDA-MB-468 cells were transfected with FAK (2 µg) and increasing amounts of the FAK Y397F/K454R double mutant (0-4 µg, as indicated) in the FAK-Del33 and FAK-WT backgrounds. The total amount of DNA was kept constant through the addition of empty vector.</p

The FAK-Del33 mutation reduces Src independence.

<p>(<b>A</b>) MDA-MB-468 cells were transfected with FAK-WT, FAK-Del33, FAK-WT/Δ375, and FAK-Del33/Δ375. The cells were serum starved overnight and replated onto FN-coated plates in the presence of the Src inhibitor PP2 or the control compound PP3. (B) Cells transfected with FAK-WT or FAK-Del33 were treated with increasing concentration of Src inhibitor PP2 (0∼40 µm). Whole cell lysates were immunoblotted with antibodies against Y397 (top panel), FAK (middle panel), and GAPDH (bottom panel).</p

Structure and localization of FAK-Del33.

<p>(A) The 3D structure of the FAK FAT domain is shown as a ribbon representation (green) with the deleted portion of Del33 shown in Red. (B) FAT sequence conforms to Fig 1A was shown, with the deleted residues labeled in red. The protein sequence alignment shows that 27 amino acids (969-995Aa) are missing, but an ORF reading frame shift does not occur in FAK-Del33. (C, D) Confocal sections of MDA-MB_468 cells transfected with GFP-tagged FAK or GFP-tagged FAK-Del33. Cells were replated onto glass coverslips, and transfected for 24 h after which time they were fixed and processed for indirect immunofluorescence against either paxillin (C), or p-paxillin (D). Scale bar, 5 µm.</p

The FAK-Del33 mutation inhibits stimulation of adhesion signals and induces constitutive phosphorylation.

<p>(A) FAK-Del33 is insensitive to fibronectin or ColI treatment. MDA-MB-468 cells were transfected with FAK-WT or FAK-Del33 via viral infection and subjected to puromycin selection. The cells were trypsinized and suspended in DMEM with 0.1% BSA for 30 min before plating onto fibronectin (FN)- or ColI-coated dishes for 1 h. Cells plated on poly-L-lysine-coated dishes for 1 h were used as an untreated control (NC). (B) Time-dependent dephosphorylation of FAK-WT and FAK-Del33 in suspended cells. MDA-MB-468 cells stably transfected with FAK-WT or FAK-Del33 were trypsinized and suspended in serum-free medium for various times. Cell lysates were subjected to Western blotting using the indicated antibodies. FAK-Del33 phosphorylation persisted regardless of the suspension time course.</p

Intramolecular interactions contribute to FAK-Del33 auto-phosphorylation.

<p>(A) MDA-MB-468 cells were transfected with FAK or FAK/Δ375 plasmids. Then, 30 µg of whole cell lysates were immunoblotted with Y397 and total FAK antibodies. (B)The FERM domain inhibited Y397 phosphorylation in cis in FAK-Del33. MDA-MB-468 cells were co-transfected with HA-tagged FAK (2 µg) and increasing amounts of Flag-tagged FERM domain fragments (consisting of residues 1–396, 1-4 µg, as indicated). The total amount of DNA was kept constant by the addition of empty vector. In total, 30 µg of whole cell lysates was immunoblotted with the indicated antibodies.</p

FAK-Del33 is hyper-phosphorylated in a cell-line dependent fashion.

<p>(A) Lysates containing 1 mg of total cellular protein were prepared from HA-tagged FAK-WT (lanes 1-2) and FAK-Del33 stable transfected cells (lanes 3-4). The cells overexpressing FAK were grown in the absence (induction of expression, even-numbered lanes) or presence (suppression of expression, odd-numbered lanes) of 5 ng/ml doxycycline (Dox). The lysates were immunoblotted with either anti-HA polyclonal (panels A, C, E) or anti-Tyr397 monoclonal (panels B, D, F) antibodies. (B) Different cells lines were transfected with each of the pCDNA3.1⁺-FAK-WT/FAK-Del33 plasmids separately. Equal amounts of the cellular lysates were subjected to Western blotting using the indicated antibodies. (C) The cells were transfected with pCDNA3.1⁺-GFP-FAK-WT/FAK-Del33 plasmids. A GFP tag was added to the N-terminus of FAK, and the resulting fusion protein has a larger mass than endogenous FAK (Arrow a shows GFP-FAK, arrow b shows endogenous FAK). (D) The cells were transfected with increasing amount of pCDNA3.1⁺-GFP-FAK-Del33 plasmids (0, 0.5, 1.0, 2.0 µg in 6-well plates.). The phosphorylation of endogenous FAK does not change with increasing expression and phosphorylation of exogenous FAK-Del33 in cells.</p

The chromatin remodeling factor Brg-1 is essential for serum starvation-induced DRAM expression.

<p>(A) Brg-1 is bound to the proximal region of the DRAM promoter. ChIP assays were performed in Hep3B and HepG2 cells. (B) Serum deprivation enhances Brg-1 and Pol II binding to the DRAM promoter locus. (C) Depletion of Brg-1 using specific targeting shRNA abolishes serum deprivation induced DRAM expression.</p

Serum deprivation induces changes in histone modification at the DRAM promoter locus in Hep3B cells.

<p>Hep3B cells were prepared for chromatin IP (ChIP) assays. The chromatin DNAs were immunoprecipitated with antibodies specific to H3K4me3 (A), anti-di-acetyl-H3 (B), anti-H3K9me2 (C) and anti-tetra-acetyl-H4 (D), and the enriched DNA fragments flanking the DRAM promoter were analyzed by quantitative PCR. Data were presented as the amount of DNA recovered by specific antibodies relative to DNA enriched by the appropriate IgG controls. The results were expressed as the means±standard deviations of three independent experiments. *: P<0.05.</p

The putative cis regulatory elements responsive to serum deprivation is resided in the DRAM promoter region flanking −19/+28 nucleotides.

<p>(A) Hep3B and HepG2 cells were transiently transfected with reporter plasmids containing truncated versions of the promoter region of the <i>DRAM</i> gene as indicated. Luc-DCP is defined as the reporter containing the shortest promoter region (−19∼+28). (B) The core promoter region contains a putative NF-κB binding site. Transcription factor binding sites presented in the core promoter region (-19∼+28) were predicated by web software TFSEARCH and MatInspector and the mutation or deletion plasmids were generated using the site-directed mutagenesis method. (C) Mutation or deletion of the NF-κB binding site does not affect the core promoter activity.</p