Focal adhesion kinase ( FAK 1 ) regulates SHB phosphorylation and its binding with a range of signaling proteins

O. V. Dergai , A. M. Yaruchik, A. V. Rynditch © 2016 O. V. Dergai et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 577.22


Introduction
Focal adhesion kinase 1 (FAK1) is an ubiquitously expressed non-receptor tyrosine kinase which is localized to focal adhesion where a cell is attached to the extracellular matrix.FAK1 operates downstream of the cell surface receptors, such as integrins and receptor tyrosine kinases i.e.EGFR, PDGFR, G-protein coupled receptors (GPCR), EPHA2, netrin receptors and LDL receptors [1,2].Upon incoming activation signal from the above mentioned receptors FAK1 associates with kinase Src, gets phosphorylated by latter and autophosphorylated to gain the maximum of intrinsic kinase activity.Activated FAK1 phosphorylates ACTN1, ARHGEF7, GRB7, RET, WASL and promotes phosphorylation of BCAR1, GIT2 and SHC1, many of these proteins are crucial regulators of the cytoskeleton reorganization, cell spreading, migration, cell cycle progres-sion, and prevention of apoptosis [3][4][5].FAK1 is comprised of a centrally located kinase domain flanked by the large N-and C-terminal non-catalytic domains (Fig. 1).
The N-terminal region of FAK1 contains FERM homology domain, whereas the C-terminal region of FAK1 contains two proline-rich motifs and a focal adhesion targeting (FAT) domain that mediates its discrete localization to focal adhesions upon FAK1 activation [6].In addition to the function as a kinase, FAK1 also serves as a scaffolding protein.FAK1 contains th e binding sites for many signaling proteins (Fig. 1).FAK1 was shown to interact with EGFR, PDGFR, Src, Shc, phosphotidylinositol 3-kinase (PI3K), and phospholipase Cg1 (PLC g1), signaling adaptors GRB2 and GRB7, paxillin, tallin [1,4,7].
FAK1 interacts with and gets activated by signaling scaffold SHB [8].FAK1 and SHB in a concert
Here we show that FAK1 can regulate interaction of SHB scaffold with a range of SH2 domain containing signaling molecules such as CRK, PI3K and с-ABL through the tyrosine phosphorylation of SHB.It has been found that expression of viral protein LMP2A can partially mimic the effect of FAK1 overexpression enhancing binding of SHB to the SH2-domains of CRK, PI3K, GRB2, PLC g1 and с-ABL.An intriguing observation appeared that FAK1 can partially block LMP2A-induced phosphorylation of SHB.

Cell culture and transfection
293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 50 U/ml penicillin and 100 μg/ml streptomycin.The cells were transiently transfected using JetPEI (Polyplus Transfection) according to the manufacturer's instructions and processed 24h after transfection.

Immunoprecipitation
Cell lysis and immunoprecipitation were performed as described previously [15].

Proteins expression, purification and GST-pull down assay
GST-fused SH2-domains of PLCG1, PI3K, ABL, GRB2 and CRK were expressed in E.coli BL21 DE3 and affinity purified using glutathione-sepharose 4B (GE Healthcare) according to the manufacturer's instructions and further used for the GST-pull down assays as described before [13].

Results and Discussion
Taking into consideration the fact that SHB regulates the FAK1 [8] activity through its tyrosine phosphorylation we asked whether there is a reciprocal loop of SHB regulation by FAK1.At first we performed in silico prediction of the FAK1 capability to phosphorylate SHB undertaken by means of GPS3.0 software (Group-based Prediction System) [16].
According to this analysis 8 tyrosine residues were predicted to be phosphorylated by FAK1 at high stringency, namely Y96, Y162, Y178 Y246, Y272, Y297, Y336, Y444 (Fig. 2A).So, we took advantage of the experimental data from PhosphoSitePlus database (http://www.phosphosite.org/)and found that 5 tyrosine residues (namely Y96, Y246, Y272, Y297, Y336) out of 8 predicted ones were found to be phosphorylated in human and mouse samples.Moreover, the indicated five tyrosine residues are conserved in Vertebrates in contrast to tyrosines at the positions Y162, Y178, Y444 (Fig. 2A) To test experimentally the hypothesis that FAK1 may phosphorylate SHB we co-expressed the full-length HAtagged FAK1 or its truncated omni-tagged FAK1-Cterm lacking kinase domain (see Fig. 1) together with omni-tagged SHB in 293 cells.The basal level of SHB phosphorylation in 293 cells was below of the detection limit.Despite the equal amount of precipitated SHB (Fig. 2B, second panel), a level of its phosphorylation is greatly increased in the cells transfected with full-length FAK1 but not in the cells with expression of FAK1-Cterm lacking a kinase domain (Fig. 2B, first panel).Next, we asked whether the FAK1-induced phosphorylation of SHB has a functional consequence.It's widely accepted that phosphorylation in general serves to switch proteins conformations or to create interaction interfaces which can be recognized by the specialized phospho-binding protein domains.One of the well-known class of phosphotyrosine recognition modules is SH2-domain (Src homology2), which specifically binds the phosphotyrosine residues [17].Some of the established SHB binding substrates such as GRB2, ABL, PLCg1 contain SH2domain [18].We studied whether the FAK1-induced phosphorylation of SHB can modulate its interaction with SH2 domains of the binding partners.To do so, we used a set of GST-fused SH2-domains as a bait in pull-down assay to precipitate SHB from the cell extracts transfected with HA-FAK1.As Fig. 3A shows the overexpression of FAK1 enhanced the interaction of SHB with SH2 domains of CRK, PI3K and ABL while no interaction with SH2 domain of GRB2 was detected.This fact may evidence that the FAK1dependent SHB phosphorylation specifically triggers the association of different SH2 domains containing signaling proteins with SHB.Additionally, the interaction of SHB with SH2 domain of PLCg1 was significantly increased upon the FAK1 overexpression but the binding of PLCg1 SH3 domain to SHB was decreased suggesting that the tyrosine phosphorylation of SHB can regulate binding properties of its proline-rich sequences (Fig. 3A, lanes 9, 10).
The interaction of SHB with PI3K that is mediated by SH2 domains of latter is of great interest as SHB is required for the activation of the PI3K/AKT pathway [11] and the regulation of cell survival [18].ABL is a proto-oncogene that encodes a protein tyrosine kinase involved in a variety of cellular processes, including cell division, adhesion, differentiation, and response to stress.In the signaling pathways ABL is located upstream of PI3K/AKT [19].A functional significance of phosphorylation-dependent interaction with ABL kinase is enigmatic but recently SHB has been shown to enhance FAK1 activity in the BCR-ABL aberrant cells that caused accelerated disease progression emphasizing the importance of ABL/SHB interplay [9].It was demonstrated that the interactions between SHB and ABL regulates the ABL kinase activity.The data suggest that ABL binds to tyrosine phosphorylated SHB via a concerted effort involving both ABL SH3-and SH2-domains and these interactions modulate cell death in response to the genotoxic agents [20].In general the place of SHB in signaling network is depicted at the Fig. 3D.The activated receptor tyrosine kinases transduce a signal to ABL, SRC, phospholipase C and PI3K that results in the cell proliferation or survival depending on the signaling context.SHB is required for the signaling downstream of RTKs and its role depends on the interaction with the protein-partners such as ABL, SRC, GRB2, PI3K and PLCg1.FAK1 is activated by integrins (Fig. 3D) and indirectly by RTKs signaling that leads to the cytoskeleton changes and cell migration [18].We suggest that the FAK1-induced SHB phosphorylation may be a part of signaling cross-talk between RTKs-and integrin-controlled pathways.Additionally, the augmentation of FAK activity in human tumors, in which it is often overexpressed, occurs through its elevated expression [1], so described here the FAK1-induced effects may be a part of pathological phenotype, e.g. the resistance to apoptosis and invasiveness.
Previously we have shown that the expression of viral protein LMP2A induces the tyrosine phosphorylation of SHB [12].To address whether the LMP2Ainduced phosphorylation of SHB may have an impact on the SHB binding to the SH2-domain containing proteins we applied a before mentioned approach.As Fig. 3B shows the expression LMP2A strongly enhanced the binding of SHB to a range of the SH2 domains of such proteins as CRK, GRB2, PI3K, PLCg1 and ABL.One can suggest that FAK1 and LMP2A caused the phosphorylation of nonoverlapping sets of tyrosine residues as far as the LMP2A expression enhanced the SHB association with GRB2 but for FAK1 (Fig3 A and B).The described effects of LMP2A and FAK1 expression on the SHB in vitro interaction with before mentioned SH2 and SH3 domains could be explained in two ways: 1) both FAK1 and LMP2A cause the SHB phosphorylation at certain sites that are recognized by respective SH2 domains or 2) FAK1 and LMP2A serve as a scaffold to bring together SHB and before mentioned domains.The later statement is supported by our unpublished observation that LMP2A and FAK1 can interact with SH2-domains of PI3K, ABL and GRB2.From the other hand, the fact that FAK1 negatively regulated SHB binding to the SH3domain of PLCg1 challenges the idea that FAK1 serves simply as an adaptor.Unfortunately, so far it is difficult to uncouple the phosphorylation-induced and potential scaffolding effects of FAK1 on the SHB interaction with its partners.
As far as the expression of both LMP2A and FAK1 strongly upregulated the SHB tyrosine phosphorylation we expected to detect a synergetic effect of co-expression of both proteins with SHB.In addition while the expression of individual proteins elevated SHB phosphorylation level, the co-expression of LMP2A and FAK1 had remarkable negative cooperation effect (Fig. 4).The reason for this kind of conclusion is the fact that the most robust effect on the SHB phosphorylation was caused by LMP2A while the co-expression of FAK1 decreased this effect to the FAK1-induced level.One could suggest that FAK1 partially inhibits the activity of Syk and Lyn tyrosine kinases which carry out the LMP2Ainduced SHB phosphorylation [12].
SHB is required for the tonic and serum-independent AKT activation caused by Epstein-Barr virus protein LMP2A [10] and in line with this observation it has been shown that LMP2A induces the SHB tyrosine phosphorylation [12].We suggest that described here LMP2A-induced interaction of SHB with SH2 domains of PI3K, ABL, PLCg1 may be responsible for the downstream signaling towards AKT and RAS activation.Moreover, it has been shown that LMP2A induces the epithelial cell migration [21] in FAK1-dependent manner [22].We assume that signaling downstream of FAK1 and LMP2A may converge on SHB.The fact that LMP2A can induce the SHB phosphorylation that triggers SHB binding to GRB2, PLCG1, PI3K and other proteins in this way mimicking the FAK1 activity may represent an important aspect of the LMP2A-induced pathogenicity providing a link between the LMP2Adriven SHB activation and cell migration.К л юч е в ы е с л ов а: FAK1, SHB, LMP2A, фосфорилирование.

Fig. 1 .
Fig. 1.A schematic representation of FAK1 and SHB proteins.The most important phosphotyrosine sites of FAK1 are shown above with indication of proteins which recognize them.FAK1-C term lacking N-terminal portion with kinase domain is demonstrated below the full-length one.SHB comprises the proline-rich domain (PRD), phosphotyrosine-binding domain (PTB) and SH2 domain, the two latter domains of SHB are hosphotyrosine-recognition modules.The tyrosine residues predicted by GPS3.0 to be phosphorylated by FAK1 are show above SHB scheme.

Fig. 2 .
Fig.2.Overexpression of FAK1 kinase induces tyrosine phosphorylation of SHB protein.A. Table representing a list of phosphotyrosine sites of SHB predicted with highest stringency by GPS3.0 to be phosphorylated by FAK1 kinase.Category "mammals" contains Homo sapiens, Mus musculus, Rattus norvegicus, Canis lupus.B. 293 cells were co-transfected with omni-SHB-HA, HA-FAK1 or omni-FAK1-Cterm.24h after transfection cells were harvested, lysed and omni-SHB-HA immunoprecipitated with monoclonal a-omni antibodies from the respective cell extracts.Precipitated material was analyzed by western blot: tyrosine phosphorylated species were detected by monoclonal a-phosphotyrosine antibody, total omni-SHB-HA was visualized by aomni antibody.Two lower panel show immunodetection of omni-SHB-HA, full-length and omni-FAK1-Cterm in cell extracts.

Fig. 3 .
Fig.3.FAK1 and LMP2A regulate association of SHB with SH2-domains.A. Bacterially expressed GST-fused SH2 (A, B and C) or SH3-PLCg1 (A, lanes 9 and 10) of indicated proteins were immobilized on glutathione-beads and incubated with 293 lysates cotransfected with omni-SHB-HA and HA-FAK1 (A and B) or Flag-LMP2A (C) or vector.D. Schematic representation of SHB role in RTKs-and integrin-driven signaling.SHB may directly interact with depicted proteins, with red dashed arrow is shown newly discovered phosphorylation event, FAK1-induced interactions are shown with bold back arrows.Indirect effects are shown with dashed arrows.WCE stands for the whole cell extract.

Fig. 4 .
Fig.4.Expression of FAK1 and LMP2A displays negative cooperation effect on the tyrosine phosphorylation levels of SHB.293 cells were co-transfected with omni-SHB-HA, HA-FAK1+control vector or HA-FAK1 and Flag-LMP2A.Levels of SHB tyrosine phosphorylation were estimated as described at figure 2B.