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New partners and phosphorylation sites of focal adhesion kinase identified by mass spectrometry
New partners and phosphorylation sites of Focal Adhesion Kinase identified by Mass Spectrometry Maria del Mar Masdeu, Beatriz G. Armend´ariz, Eduardo Soriano, Jes´us Mariano Ure˜na, Ferran Burgaya PII: DOI: Reference:
S0304-4165(16)30088-5 doi: 10.1016/j.bbagen.2016.02.019 BBAGEN 28436
To appear in:
BBA - General Subjects
Received date: Revised date: Accepted date:
21 July 2015 22 September 2015 23 February 2016
Please cite this article as: Maria del Mar Masdeu, Beatriz G. Armend´ariz, Eduardo Soriano, Jes´ us Mariano Ure˜ na, Ferran Burgaya, New partners and phosphorylation sites of Focal Adhesion Kinase identified by Mass Spectrometry, BBA - General Subjects (2016), doi: 10.1016/j.bbagen.2016.02.019
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ACCEPTED MANUSCRIPT New partners and phosphorylation sites of Focal Adhesion Kinase identified by Mass Spectrometry
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Maria del Mar Masdeu1, 2, Beatriz G Armendáriz1, 2, Eduardo Soriano1, 2, 3, 4, Jesús
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Mariano Ureña1, 2, 5 Ferran Burgaya1, 2, 5, 6.
Developmental Neurobiology and Neural Regeneration Group. Department of Cell
Biology. Faculty of Biology. University of Barcelona. Diagonal 643, 08038 Barcelona,
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Spain.
Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas
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(CIBERNED), ISCIII, 28031 Madrid, Spain
Vall d´Hebron Institute of Research, 08035 Barcelona, Spain.
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Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, Spain
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co-senior authors
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to whom correspondence should be addressed: [email protected]
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KEYWORDS: focal adhesion kinase; mass spectrometry; brain; synapse
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ACCEPTED MANUSCRIPT Abstract: The regulation of focal adhesion kinase (FAK) involves phosphorylation and multiple
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interactions with other signaling proteins. Some of these pathways are relevant for
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nervous system functions such as branching, axonal guidance, and plasticity. In this
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study, we screened mouse brain to identify FAK-interactive proteins and phosphorylatable residues as a first step to address the neuronal functions of this kinase.
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Using mass spectrometry analysis, we identified new phosphorylated sites (Thr 952, Thr 1048, and Ser 1049), which lie in the FAT domain; and putative new partners for FAK,
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which include cytoskeletal proteins such as drebrin and MAP 6, adhesion regulators such as neurabin-2 and plakophilin 1, and synapse-associated proteins such as SynGAP
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FAK in neuronal plasticity.
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and a NMDA receptor subunit. Our findings support the participation of brain-localized
ABBREVIATIONS
FAT
focal adhesion kinase
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FAK
focal adhesion targeting domain
FERM
band four-point-one, ezrin, radixin, and moesin common domain
LC-MS/MS
mass spectrometry coupled to liquid chromatography
MAPK
mitogen-associated protein kinase
P5
postnatal day 5
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ACCEPTED MANUSCRIPT 1. Introduction Focal adhesion kinase (FAK) consists of a canonical 125 KDa polypeptide
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localized mainly on focal adhesions and other types of cellular contacts [1]. FAK is
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ubiquitously expressed in mammalian cells, although it shows particular features in
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brain [2,3], including the presence of alternatively spliced inner fragments [4]. FAK comprises various domains, namely the following [5]: (1) an amino-terminal
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FERM (Band four-point-one, ezrin, radixin, and moesin common) domain, which mediates FAK attachment to the plasma membrane, as well as the interaction with the
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actin cytoskeleton; the FERM domain can also mediate FAK transduction into the nucleus [6]; (2) a central catalytic domain that includes two phosphorylatable residues
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(Tyr 576 and Tyr 577) in the activation loop; and (3) a FAT (focal adhesion targeting) domain, which lies in the carboxyl-terminal end and is responsible for the localization
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of the kinase in focal adhesions. Tyr 925, one of the main phophorylatable residues of FAK, is also found in this region. When phosphorylated, the latter residue acts as a
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binding site for Grb2 and activates the MAPK cascade [7]. Of note, the C-terminal domain of FAK can also be autonomously expressed as an alternatively spliced form of the protein, called FRNK (FAK-related non kinase) [8]. Although the role of FRNK is poorly understood, it seems to compete with FAK for targeting to focal adhesions and to regulate focal adhesion turnover [8] (reviewed in [2]). Tyr 397, the first residue to be phosphorylated upon FAK activation, falls between the FERM and the catalytic domains and is vital for the activity of the protein [9]. Along the polypeptide lie several Pro-rich sequences which are susceptible to binding to SH3 domain-containing proteins, and further phosphorylatable residues such as Tyr 861,
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ACCEPTED MANUSCRIPT which directs the participation of FAK in the formation of lamellipodia [10]. The significance of other phosphorylated residues is less understood.
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FAK often acts as a scaffolding protein, and its kinase activity is just one of the subsets
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of functions that this molecule performs. Therefore, the activities mediated by FAK are
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either kinase-dependent or -independent, both being conditioned by the differential phosphorylation of particular residues, among other events such as its subcellular
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localization or proteolysis [2,3].
FAK depletion generates embryonic lethality [11], thus linking the kinase to vital roles
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during embryogenesis [12]. High amounts of FAK transcript are found in kidney, heart, testicles, ovaries, and brain [4,13]. Furthermore, various alternatively spliced forms of
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FAK have been found in the latter [4,14,15]. These isoforms include the complete
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canonical form of FAK plus one or up to four short insertions found near the major
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autophosphorylated tyrosine and the FAT domain [2], thereby pointing to additional functions of these isoforms. Moreover, among the tasks reported for FAK, several are relevant in the brain, such as its involvement in neuronal guidance, adhesion, migration,
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branching, and plasticity [16,17,18,19,20]. The activation of FAK begins with its autophosphorylation at Tyr 397, which can occur in cis or in trans, depending on the isoform involved [6,21,22]. This phosphorylation allows Src SH2 domain binding [9] and Src-mediated phosphorylation of further residues, including Tyr 576, Tyr 577, Tyr 925 [23] and Tyr 861 [24]. To identify FAK patterns of phosphorylation under diverse conditions, it is of particular interest to unravel its action and regulation. Recently mass spectrometry approaches have reported to be useful for the identification of phosphorylated residues [25,26,27]. These techniques are also suitable for the 4
ACCEPTED MANUSCRIPT detection of peptides from putative partners of the proteins studied [25,26,27]. Here we used mass spectrometry techniques in tandem with immobilized metal affinity
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chromatography of tryptic peptides (known as LC-MS/MS) to characterize the
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phosphorylated residues of FAK in mouse brains of various ages and conditions and
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also to identify new partners of this kinase. We used 5-day postnatal (P5) mice since, at this age, major changes related to neurite guidance and synaptogenesis occur in the actin cytoskeleton of the hippocampus, the cerebral cortex, and the cerebellum. In addition,
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we also worked with either untreated or epileptogenically-stimulated adults in order to
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compare putative changes that may be relevant regarding activity-related
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neosynaptogenesis.
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ACCEPTED MANUSCRIPT 2. Material and Methods
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2.1. Animals: Adult (7-8 weeks of age) or P5 OF1 mice (Charles River, Lyon, France)
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were used. All experimental procedures complied with the guidelines approved by the
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Spanish Ministry of Science and Technology and with the European Community Council Directive 86/609 EEC.
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2.2. Antibodies: The polyclonal antibody against C-FAK was from Santa Cruz Biotechnology (Santa Cruz, CA).
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2.3. PTZ stimulation: Two adult mice were intraperitoneally injected with a first dose of 30 mg/kg of pentylenetetrazol (PTZ), followed by additional 10 mg/kg injections
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every 15 min until a seizure occurred (typically at 60 mg/kg). Control mice were injected intraperitoneally with PBS.
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2.4. Protein extraction: Following dissection, and to ensure amounts of protein high enough for our assays, brains were pooled before homogenization. Ten brains of P5
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mice and two from either untreated or PTZ-treated adult mice were used in each assay. Samples were homogenized in Polytron in 5 times their volume of lysis buffer per weight of tissue. Lysis buffer consisted of 50 mM pH 7.5 HEPES, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton X-100, protease inhibitor cocktail (1x) (Roche, Basel, Switzerland), and the following phosphatase inhibitors: 10 mM Na4P2O7; 200 µM Na3VO4; and 10 mM NaF. The homogenates were centrifuged (20 min at 13,000 rpm at 4ºC), and the supernatants were incubated with anti-C-FAK overnight at 4ºC. G protein-coupled Sepharose beads were then added, and the suspension was then incubated for 2 h at 4ºC, washed 5 times with lysis buffer, and suspended overnight at -20ºC in 1.5 ml of lysis buffer plus 4 6
ACCEPTED MANUSCRIPT volumes of acetone. The next day, the samples were centrifuged, the pellet was incubated under agitation with 2 volumes of 0.1 M glycine pH 2 during 5 min at 4ºC,
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and centrifuged again. The supernatants were neutralized with 0.1 volumes of 1 M pH
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8.5 Tris-HCl and concentrated using 100 KDa Amicon columns (Millipore, Billerica,
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Finally, all the samples were loaded in an 8% polyacrylamide gel and submitted to
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SDS-PAGE electrophoresis. The gel was stained with SyproRuby (Invitrogen, Carlsbad,
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CA) at 4ºC to visualize the bands.
2.5. LC-MS/MS: The bands of the molecular weight of FAK were cut from the gel and digested with trypsin, and this digest was enriched in phosphopeptides using a column
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containing titanium dioxide (TiO2) magnetic beads (GE Healthcare, Barcelona, Spain). The phosphopeptide-enriched fraction was analyzed by LC-MS/MS in a Nanoacquity
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chromatographer (Waters, Cerdanyola del Vallès, Spain) coupled to a LTQOrbitrapVelos mass spectrometer (Thermo Scientific, Walthman, MA). The spectra
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obtained were analyzed by Proteome Discoverer (v1.20) software. Searches were performed by the Sequest search engine using Thermo Proteome Discover (v.1.3.0.339) against the Uniprot-SwissProt database. Percolator [28] was used to discriminate correct from incorrect peptide spectrum matches. Only peptides reported as high confidence (FDR≤1%) were considered for identification. A score value (sum of the score of all the peptides that matched this protein multiplied by the number of different peptides found for this protein) was assigned to the proteins selected. The peptides identified by the software, which may correspond to FAK sequences or to other protein sequences found in the precipitated band –putative partners-, were studied separately.
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ACCEPTED MANUSCRIPT 2.6. Analysis “in silico” of putative kinases: To identify motifs likely to be phosphorylated by specific protein kinases, we used the software ScanSite [29] at
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medium stringency. This program assigns a score value to the motif identified that gets
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closer to 0, the closer he motif is to the known consensus sequence. The software also
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assigns a percentile that indicates the percentage of probability that the candidate motif is phosphorylated by the specific kinase, with respect to all the potential motifs in the protein database. In other words, the database contains thousands of identified
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substrates and consensus sequences and the greater the agreement with only one
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particular consensus, the higher the reliability of being phosphorylated by this kinase and not another, and thus the lower the assigned percentile; therefore, a lower percentile
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indicates major specificity of the candidate motif.
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ACCEPTED MANUSCRIPT 3. Results 3.1. Protein extraction and LC-MS/MS assay. FAK was purified by
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immunoprecipitation from three types of brain samples: untreated or PTZ-treated adult
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brain samples, and untreated P5 brain samples. A band roughly corresponding to the
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size of FAK was cut from the electrophoretically separated proteins. Trypsin digestion of the proteins eluted post-electrophoresis was followed by an enrichment in
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phosphopeptide contents and LC-MS/MS analysis (Fig. 1). In addition to the band corresponding to FAK, a robust band of 40-45KDa was also found. This study
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permitted us to distinguish the peptides of the protein FAK and also discriminate those
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phosphorylated and the residue involved.
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3.2. Phosphorylated residues found in FAK protein. The ionized compounds were
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resolved into a series of mass/charge ratios, from which the composition of the peptide and the phosphorylated residues were deduced by the software. As an example, Suppl. Fig. 1 shows the mass/charge ratios found for the peptide holding Ser 29, and the
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corresponding spectrum.
Three new residues, previously undescribed, were found to be phosphorylated. Specifically, Thr 952, and Ser 1049 were identified in adult brain, both in untreated and PTZ-treated animals, and Thr 1048 only in the former (Fig. 2A). All these residues are placed along the FAT domain (Fig. 2B). Other residues had previously been found to be phosphorylated in tissues or cell types other than nervous cells, whereas others were already characterized in brain (Fig. 2A). Diverse patterns of phosphorylation allowed us to distinguish several categories among the phosphorylated residues. Some appeared: at all ages and treatments, such as Ser 29, Tyr 608, Ser 612, Tyr 614, Tyr 615 and Ser 948; exclusively at P5 (Ser 760 and Ser 9
ACCEPTED MANUSCRIPT 881); only in the adult brain, independently of the state of activation (Ser 606, Ser 715, Ser 716, Ser 878, Thr 952, Tyr 963 and Ser 1049); only in untreated adult brains (Thr
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613 and Thr 1048); or only in PTZ-stimulated adult brains (Ser 618).
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In addition, “in silico” analysis allowed us to determine that Ser 715 matches the
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consensus sequence of CAMK II, Thr 716 and Ser 881 match the kinase PK C ε, and Tyr 963 matches the Lck Kinase (Table 1).
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3.3. Proteins identified in the immunoprecipitated samples of FAK. The LC-MS/MS
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technique identified further proteins –which presumably interact with FAK- in the immunoprecipitated samples from the same assays. The number of putative partners included around 500 potential candidates (the best fits are shown in Suppl. Fig. 2).
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Some of the highest scoring proteins, listed in Table 2, are linked to cytoskeletal organization and dynamics, in agreement with the well-established functions of FAK
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[2,3,30]. This is the case of drebrin [31], which is involved in plasticity events [32], and also of an isoform of the heavy chains of kinesin 5C ([33]) and kinesin 1 [34], the
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microtubule-associated protein MAP 6 [35], and myosin-1 subtypes [36]. Other putative partners are involved in adhesion, such as neurabin-2 [37] and plakophilin 1 [38], while others link FAK to synaptic transmission, such as SynGAP [39] and the θ-1 subunit of the NMDA receptor [40]. Of note, whereas various proteins were found at all ages, such as drebrin, MAP 6, and neurabin-2, some motor proteins were detected only in the adult (kinesin 1 heavy chain was found in untreated and also in PTZ-treated adults, kinesin 5A only in untreated adults, and myosin-1d only in PTZ-treated adults). Particular myosins of type 1 were found at P5 only, and while the association with the NMDA receptor was detected only
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ACCEPTED MANUSCRIPT in untreated adults, SynGAP was observed in both untreated and PTZ-treated adults
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(Table 2).
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ACCEPTED MANUSCRIPT 4. Discussion 4.1. Phosphopeptide mapping of FAK. The regulation, functions and activation
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mechanisms of FAK show bulky density and have just been partly described. However,
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it is evident that a part of the functions and regulation of FAK depend on its degree of
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phosphorylation and, most specifically, on the combination of residues phosphorylated. FAK can be phosphorylated by several kinases such as Src, or in response to growth
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factors such as BDNF [17,41]. This kinase shows many other phosphorylated residues, and a plethora of conditions can modify FAK phosphorylation and/or activity [2]. In
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this study, we characterize the phosphorylation of three residues that had not been described previously in vivo: namely Thr 952 and Thr 1049, identified in untreated and
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PTZ-activated adult mouse brain, and Thr 1048, found in untreated adult mouse brain
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only. All these residues lie along the FAT domain, which fulfils FAK recruitment to
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focal adhesions through paxillin association [42,43]. Interestingly, even while the crucial residues and path of association between FAK and paxillin are well described, FAK association with talin is less understood, although it is
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also related to the FAT domain [30,44]. The FAK-talin association could be influenced by the FAT phosphorylation pattern. In addition, the phosphorylation of Tyr 925 disrupts focal adhesion targeting of FAK [45]. It has recently been shown that the first helix of the four-helix bundle of FAT associates with Tyr 925 and must be dissociated prior to Tyr 925 phosphorylation and MAPK pathway activation [44]. It is likely that the phosphorylation of one or several of the three residues cited above affects FAT folding, talin association and Tyr 925 uncovering, thus influencing signal transduction pathways. Of note, the lack of focal adhesions in neurons and the multiplicity of pathways exerted by them in the brain during development or neuronal activation [2] are probably related to the new phosphorylation sites described herein. Other studies 12
ACCEPTED MANUSCRIPT report the decrease in phosphorylated Tyr-925 found in oligodendrocytes prior to myelination [2,46], an observation that is consistent with the lack of phosphorylation on
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that residue at P5 (Fig. 2), which would also impede the presence of paxillin and Src in
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the extracts at that age [45]. The significance of these changes in terms of FAK
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competition with FRNK—which contains the complete FAT domain—also deserves additional attention.
In addition, Ser 29, Ser 606, Tyr 608, Ser 612, Tyr 615, Ser 715, Thr 716, Ser 760, Ser
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878 and Ser 881 have not been previously described in mouse brain in vivo, although
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reports in other tissues or organisms are available [17, 47,48,49,50,51,52,53] (see Fig. 2). The function of some of these residues has been partially characterized; however, for others there is scarce information about the role that their phosphorylation plays
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[46,53,54,55]. The adscription to the catalytic domain relates most of them to the fine regulation of kinase-dependent functions of brain FAK. Ser 29 lies N-terminal to the
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FERM domain, and the significance of its phosphorylation is unknown [47]. Here we also provide new information on the differential phosphorylation of FAK
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under various paradigms, in agreement with the patterns of phosphorylation found depending on brain activity. This is the case of Ser 618, located in the catalytic domain, and found phosphorylated in PTZ-stimulated brains only, whereas the neighboring Thr 613 and the FAT domain-located Thr 1048 were found phosphorylated only in untreated samples. Further studies are needed to determine whether these changes are related to the neosynaptogenesis that occurs during epileptogenic brain stimulation [56,57]. Finally, the simultaneous involvement of FAK in various signaling pathways is also feasible during development. Ser 760 and Ser 881 were phosphorylated in P5 samples only, whereas Ser 606, Ser 618, Ser 715, Thr 716, Ser 878, Thr 952, Tyr 963, Thr 1048, 13
ACCEPTED MANUSCRIPT and Ser 1049, were phosphorylated only in adult samples. Noteworthy, when phosphorylated, Tyr 963—the equivalent of the well described Tyr 925 of canonical
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FAK—mediates FAK detachment from focal adhesions [45], Grb2 association, and
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MAPK pathway activation [58,59], and these results are consistent with the needs of
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MAPK activation at adult age. The significance of other phosphorylated requires further attention.
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The resolution of the technique used is constrained by the weight and negative charge of the peptides obtained during trypsinization [60,61]. Therefore, there may be further
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phosphorylated sites. Tyr 397 may be such a site since it is the main phosphorylated residue of active FAK [9,21]. Nor did we detect phosphorylated Tyr 861, which is
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phosphorylated mostly during Rac activation and lamellipodial formation. Its absence
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can also be due to this constraint of resolution. In contrast, we found phosphorylated Tyr 576 and Tyr 577, whose phosphorylation is dependent on previous Tyr397
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phosphorylation, in our assays, and also phosphorylated Tyr 925.
4.2. Putative new partners of FAK identified by MS. Several putative new partners found here point to the participation of FAK in novel functions and additional pathways. The observation that many of these partners are involved in cytoskeletal functions is in agreement with the description of FAK as a cytoskeleton modulator [30,62]. Since this kinase modulates cell adhesion, these results are consistent with the association of FAK to other proteins involved in junctional adhesions. It is worth noting that the polyclonal antibody used for this study is directed against the C-terminal domain of FAK. It can compete and eventually disturb some associations between FAK and C-terminal domain-binding partners. Therefore, 14
ACCEPTED MANUSCRIPT additional information could be obtained from a similar study performed with N terminus-directed antibodies.
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Because of the ages and state of activation chosen, it could be envisaged that most of
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the FAK partners that we found are neuronal. However, it must be pointed out that a
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significant amount of brain FAK is of glial origin. In fact, the presence of at least three distinct gap junction-associated proteins in the extracts (Suppl. Fig. 2) points to the
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participation of FAK in the connexon-mediated communication between neurons and astrocytes.
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In addition, our results suggest that, in the adult, FAK associates with synapse-related partners, such as SynGAP [39], or with a subunit of the NMDA receptor [40], the latter
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present only in untreated adult brains. In addition to the high scores reported for some
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proteins such as drebrin, kinesin heavy chain isoforms 5A and 5C, and neurabin 2,
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others show lower scores. LC-MS/MS is not a quantitative method and variations must be interpreted with care. They imply that distinct peptides were found in distinct assays. This does not mean that a higher or lower amount of protein was bound nor must this
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observation be interpreted as a lower reliability of these results. Conversely, the multiplicity of forms and functions of FAK are consistent with a functional connection between FAK and numerous regulatory proteins such as Rho GEF, synGAP and the plasticity-related NMDA receptor, which are found only in particular circumstances. The presence or absence of these partners points to changes in the regulation of pathways related to actin dynamics and synaptic efficiency. Taken together, these data support the notion that FAK participates in plasticity [63].
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ACCEPTED MANUSCRIPT ACKNOWLEDGEMENTS The proteomics work was done at the Proteomics Platform of the Barcelona Science
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Park, a member of the ProteoRed-ISCIII network. We thank Dr Eliandre Oliveira and
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Maria Antònia Òdena for technical assistance and helpful comments and discussions.
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Thanks go also to the SCT-UB for technical assistance. We thank Tanya Yates for editorial help. This work was granted by projects PI12/02108 to FB, PI10/01750 to
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JMU, and SAF2013-42445R to ES.
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outgrowth and attraction. Nat Neurosci. 2004; 7:1222-1232. 50.
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Jiang X, Sinnett-Smith J, Rozengurt E. Differential FAK phosphorylation at Ser-910, Ser-843 and Tyr-397 induced by angiotensin II, LPA and EGF in intestinal epithelial cells. Cell Signal. 2007; 19:1000-1010.
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transduction linked to Ras pathway by GRB2 binding to FAK. Nature. 1994; 372:786–
Schlaepfer D D, Hunter T. Evidence for in vivo phosphorylation of the Grb2 SH2domain binding site on FAK by Src-family protein-tyrosine kinases. Mol Cell Biol. 1996; 16:5623–5633.
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Steen H, Jebanathirajah JA, Rush J et al. Phosphorylation analysis by mass spectrometry: myths, facts, and the consequences for qualitative and quantitative measurements. Mol Cell Proteomics. 2006; 5:172-181.
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Zhang B, Liu JY. Mass spectrometric identification of in vivo phosphorylation sites of differentially expressed proteins in elongating cotton fiber cells. PLoS One. 2013; 8:
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63.
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during axonal remodeling. Mol Cell Neurosci. 2010; 44:30-42.
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ACCEPTED MANUSCRIPT Figure Legends
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Figure 1. Protein extraction and LC-MS/MS analysis: Brains were dissected,
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homogenized, and immunoprecipitated with polyclonal antibodies against FAK. The
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products were subjected to SDS-PAGE, the gel was stained with SyproRuby, and the bands corresponding to the molecular weight of FAK (signaled by an arrow) were cut. The lanes containing BSA correspond to negative controls and were performed with
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albumin instead of antibody. The molecular weight is indicated on the left side of the
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gel (only the most relevant sizes are shown). The italic letters in blue, on the right of the gel, show various relevant bands. “a” shows albumin, “b” and “d” the heavy and light
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chains of immunoglobulins, and “c” most probably corresponds to the C-terminal
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domain of FAK, which can also be autonomously expressed, and called FRNK (see also text). The eluted samples were trypsinized, and the digest was enriched in
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phosphopeptides in a column containing TiO2 magnetic beads. Finally, the
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phosphopeptides obtained were analyzed by LC-MS/MS.
Figure 2. Phosphorylated residues identified in the protein FAK: (A) Summary of the phosphorylated residues found. The shades in gray highlight the novel sites. Note that the numbering of residues in the first column corresponds to the brain form of mouse FAK (1090 amino acids). The second column indicates the equivalent numbering for the canonical forms of FAK in either human, mouse, or rat, which consist of 1052 amino acids (sequences available at http://www.uniprot.org/uniprot/Q05397). The ages and conditions in which a residue was been found phosphorylated are also shown. FDR (“false discovery rate”) index indicates the degree of confidence of the result, that is to say, the probability that the
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ACCEPTED MANUSCRIPT spectrum obtained corresponded to a different peptide from the database; it is a value directly estimated by the software. The three columns on the right indicate the function
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described for each phosphorylation, the lab model used, and the corresponding
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reference. The residues found phosphorylated for the first time in this study are
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highlighted in bold. (B) Diagrammatic structure of FAK. The main domains characterized in the polypeptide are shown, and the phosphorylated residues identified.
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Those found phosphorylated for the first time are highlighted in bold.
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Table 1. Analysis “in silico” of the putative kinases that may phosphorylate the corresponding residues of FAK. Summary of the highest scoring phosphopeptides
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found, when they appropriately matched the assigned kinases. The table also shows the
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score and percentile values.
Table 2. Proteins identified by LC-MS/MS in the FAK-immunoprecipitated pellets
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under the experimental conditions tested. Summary of the proteins identified in the immunoprecipitates of FAK that are relevant in the central nervous system and present the highest scores. The proteins are grouped on the basis of whether they were identified in all the experimental conditions or not.
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ACCEPTED MANUSCRIPT SITE
KINASE
S715 CAM KII T716 PK C ε
CONSENSUS SEQUENCE FOUND IN FAK 707 TELKAQLSTILEEEK 723
SCORE
PERCENTILE
0.4441
0.197 0.654
ELKAQLSTILEEEKV 724
0.4371
S881 PK C ε
873
DVRLSRGSIDREDGS 889
0.3979
0.222
Y963 Lck
955
DRSNDKVYENVTGLV 971
0.5012
0.886
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708
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Table 1
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370.85 168.34 83.45 42.98 63.68 79.70 41.16
46.43
78.14
76.97 54.66 -----
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77.59 53.12 41.24 9.09 68.05 66.34 51.53
Function
Cytoskeleton Cytoskeleton
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15.31 189.88 128.16 78.91 47.09 19.69 19.46
Cytoskeleton Cell Adhesion Cytoskeleton Cytoskeleton Cytoskeleton Cell Adhesion Cytoskeleton Cytoskeleton
----213.60 145.29
55.37 20.90 23.47 143.83
Cytoskeleton Cell Adhesion Cytoskeleton Cytoskeleton
105.08
54.33
---
195.40
---
Synaptic transmission Cytoskeleton
---
40.93
---
---
34.96
---
55.20
---
---
RNA regulation Synaptic transmission Cytoskeleton
41.82 36.60 -------
-----------
----34.89 31.11 29.06
Cytoskeleton Cytoskeleton Cytoskeleton Cytoskeleton Endocytosis
---
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---
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Kinesin heavy chain isoform 5A Rho guanine nucleotide exchange factor Glutamate [NMDA] receptor subunit θ-1 Myosin phosphatase Rho-interacting protein Myosin-1b Myosin-1c Myosin-1d Synaptopodin AP-1 complex subunit β-1
Ad mouse Ad mouse (untreated) (PTZ-treated) score score 556.16 170.26 421.68 52.09
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Drebrin Kinesin heavy chain isoform 5C MAP 6 Neurabin-2 β-adducin α-catenin 2 α-actinin 1 Plakophilin 1 Neurofilament medium polypeptide WD repeat-containing protein 47 α-actinin 4 Cytospin-B Kinesin 1 heavy chain MAP 7 domaincontaining protein 1 SynGAP
P5 mouse score 805.65 41.13
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PROTEIN IDENTIFIED
Table 2
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S0304-4165(16)30088-5 doi: 10.1016/j.bbagen.2016.02.019 BBAGEN 28436
To appear in:
BBA - General Subjects
Received date: Revised date: Accepted date:
21 July 2015 22 September 2015 23 February 2016
Please cite this article as: Maria del Mar Masdeu, Beatriz G. Armend´ariz, Eduardo Soriano, Jes´ us Mariano Ure˜ na, Ferran Burgaya, New partners and phosphorylation sites of Focal Adhesion Kinase identified by Mass Spectrometry, BBA - General Subjects (2016), doi: 10.1016/j.bbagen.2016.02.019
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ACCEPTED MANUSCRIPT New partners and phosphorylation sites of Focal Adhesion Kinase identified by Mass Spectrometry
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Maria del Mar Masdeu1, 2, Beatriz G Armendáriz1, 2, Eduardo Soriano1, 2, 3, 4, Jesús
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1
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Mariano Ureña1, 2, 5 Ferran Burgaya1, 2, 5, 6.
Developmental Neurobiology and Neural Regeneration Group. Department of Cell
Biology. Faculty of Biology. University of Barcelona. Diagonal 643, 08038 Barcelona,
2
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Spain.
Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas
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(CIBERNED), ISCIII, 28031 Madrid, Spain
Vall d´Hebron Institute of Research, 08035 Barcelona, Spain.
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Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, Spain
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co-senior authors
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to whom correspondence should be addressed: [email protected]
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KEYWORDS: focal adhesion kinase; mass spectrometry; brain; synapse
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ACCEPTED MANUSCRIPT Abstract: The regulation of focal adhesion kinase (FAK) involves phosphorylation and multiple
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interactions with other signaling proteins. Some of these pathways are relevant for
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nervous system functions such as branching, axonal guidance, and plasticity. In this
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study, we screened mouse brain to identify FAK-interactive proteins and phosphorylatable residues as a first step to address the neuronal functions of this kinase.
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Using mass spectrometry analysis, we identified new phosphorylated sites (Thr 952, Thr 1048, and Ser 1049), which lie in the FAT domain; and putative new partners for FAK,
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which include cytoskeletal proteins such as drebrin and MAP 6, adhesion regulators such as neurabin-2 and plakophilin 1, and synapse-associated proteins such as SynGAP
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FAK in neuronal plasticity.
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and a NMDA receptor subunit. Our findings support the participation of brain-localized
ABBREVIATIONS
FAT
focal adhesion kinase
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FAK
focal adhesion targeting domain
FERM
band four-point-one, ezrin, radixin, and moesin common domain
LC-MS/MS
mass spectrometry coupled to liquid chromatography
MAPK
mitogen-associated protein kinase
P5
postnatal day 5
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ACCEPTED MANUSCRIPT 1. Introduction Focal adhesion kinase (FAK) consists of a canonical 125 KDa polypeptide
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localized mainly on focal adhesions and other types of cellular contacts [1]. FAK is
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ubiquitously expressed in mammalian cells, although it shows particular features in
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brain [2,3], including the presence of alternatively spliced inner fragments [4]. FAK comprises various domains, namely the following [5]: (1) an amino-terminal
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FERM (Band four-point-one, ezrin, radixin, and moesin common) domain, which mediates FAK attachment to the plasma membrane, as well as the interaction with the
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actin cytoskeleton; the FERM domain can also mediate FAK transduction into the nucleus [6]; (2) a central catalytic domain that includes two phosphorylatable residues
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(Tyr 576 and Tyr 577) in the activation loop; and (3) a FAT (focal adhesion targeting) domain, which lies in the carboxyl-terminal end and is responsible for the localization
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of the kinase in focal adhesions. Tyr 925, one of the main phophorylatable residues of FAK, is also found in this region. When phosphorylated, the latter residue acts as a
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binding site for Grb2 and activates the MAPK cascade [7]. Of note, the C-terminal domain of FAK can also be autonomously expressed as an alternatively spliced form of the protein, called FRNK (FAK-related non kinase) [8]. Although the role of FRNK is poorly understood, it seems to compete with FAK for targeting to focal adhesions and to regulate focal adhesion turnover [8] (reviewed in [2]). Tyr 397, the first residue to be phosphorylated upon FAK activation, falls between the FERM and the catalytic domains and is vital for the activity of the protein [9]. Along the polypeptide lie several Pro-rich sequences which are susceptible to binding to SH3 domain-containing proteins, and further phosphorylatable residues such as Tyr 861,
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ACCEPTED MANUSCRIPT which directs the participation of FAK in the formation of lamellipodia [10]. The significance of other phosphorylated residues is less understood.
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FAK often acts as a scaffolding protein, and its kinase activity is just one of the subsets
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of functions that this molecule performs. Therefore, the activities mediated by FAK are
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either kinase-dependent or -independent, both being conditioned by the differential phosphorylation of particular residues, among other events such as its subcellular
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localization or proteolysis [2,3].
FAK depletion generates embryonic lethality [11], thus linking the kinase to vital roles
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during embryogenesis [12]. High amounts of FAK transcript are found in kidney, heart, testicles, ovaries, and brain [4,13]. Furthermore, various alternatively spliced forms of
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FAK have been found in the latter [4,14,15]. These isoforms include the complete
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canonical form of FAK plus one or up to four short insertions found near the major
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autophosphorylated tyrosine and the FAT domain [2], thereby pointing to additional functions of these isoforms. Moreover, among the tasks reported for FAK, several are relevant in the brain, such as its involvement in neuronal guidance, adhesion, migration,
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branching, and plasticity [16,17,18,19,20]. The activation of FAK begins with its autophosphorylation at Tyr 397, which can occur in cis or in trans, depending on the isoform involved [6,21,22]. This phosphorylation allows Src SH2 domain binding [9] and Src-mediated phosphorylation of further residues, including Tyr 576, Tyr 577, Tyr 925 [23] and Tyr 861 [24]. To identify FAK patterns of phosphorylation under diverse conditions, it is of particular interest to unravel its action and regulation. Recently mass spectrometry approaches have reported to be useful for the identification of phosphorylated residues [25,26,27]. These techniques are also suitable for the 4
ACCEPTED MANUSCRIPT detection of peptides from putative partners of the proteins studied [25,26,27]. Here we used mass spectrometry techniques in tandem with immobilized metal affinity
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chromatography of tryptic peptides (known as LC-MS/MS) to characterize the
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phosphorylated residues of FAK in mouse brains of various ages and conditions and
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also to identify new partners of this kinase. We used 5-day postnatal (P5) mice since, at this age, major changes related to neurite guidance and synaptogenesis occur in the actin cytoskeleton of the hippocampus, the cerebral cortex, and the cerebellum. In addition,
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we also worked with either untreated or epileptogenically-stimulated adults in order to
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compare putative changes that may be relevant regarding activity-related
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neosynaptogenesis.
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ACCEPTED MANUSCRIPT 2. Material and Methods
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2.1. Animals: Adult (7-8 weeks of age) or P5 OF1 mice (Charles River, Lyon, France)
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were used. All experimental procedures complied with the guidelines approved by the
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Spanish Ministry of Science and Technology and with the European Community Council Directive 86/609 EEC.
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2.2. Antibodies: The polyclonal antibody against C-FAK was from Santa Cruz Biotechnology (Santa Cruz, CA).
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2.3. PTZ stimulation: Two adult mice were intraperitoneally injected with a first dose of 30 mg/kg of pentylenetetrazol (PTZ), followed by additional 10 mg/kg injections
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every 15 min until a seizure occurred (typically at 60 mg/kg). Control mice were injected intraperitoneally with PBS.
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2.4. Protein extraction: Following dissection, and to ensure amounts of protein high enough for our assays, brains were pooled before homogenization. Ten brains of P5
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mice and two from either untreated or PTZ-treated adult mice were used in each assay. Samples were homogenized in Polytron in 5 times their volume of lysis buffer per weight of tissue. Lysis buffer consisted of 50 mM pH 7.5 HEPES, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton X-100, protease inhibitor cocktail (1x) (Roche, Basel, Switzerland), and the following phosphatase inhibitors: 10 mM Na4P2O7; 200 µM Na3VO4; and 10 mM NaF. The homogenates were centrifuged (20 min at 13,000 rpm at 4ºC), and the supernatants were incubated with anti-C-FAK overnight at 4ºC. G protein-coupled Sepharose beads were then added, and the suspension was then incubated for 2 h at 4ºC, washed 5 times with lysis buffer, and suspended overnight at -20ºC in 1.5 ml of lysis buffer plus 4 6
ACCEPTED MANUSCRIPT volumes of acetone. The next day, the samples were centrifuged, the pellet was incubated under agitation with 2 volumes of 0.1 M glycine pH 2 during 5 min at 4ºC,
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and centrifuged again. The supernatants were neutralized with 0.1 volumes of 1 M pH
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8.5 Tris-HCl and concentrated using 100 KDa Amicon columns (Millipore, Billerica,
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MA). Negative controls consisted of treatment of the same samples with BSA instead of anti-C-FAK antibody.
Finally, all the samples were loaded in an 8% polyacrylamide gel and submitted to
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SDS-PAGE electrophoresis. The gel was stained with SyproRuby (Invitrogen, Carlsbad,
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CA) at 4ºC to visualize the bands.
2.5. LC-MS/MS: The bands of the molecular weight of FAK were cut from the gel and digested with trypsin, and this digest was enriched in phosphopeptides using a column
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containing titanium dioxide (TiO2) magnetic beads (GE Healthcare, Barcelona, Spain). The phosphopeptide-enriched fraction was analyzed by LC-MS/MS in a Nanoacquity
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chromatographer (Waters, Cerdanyola del Vallès, Spain) coupled to a LTQOrbitrapVelos mass spectrometer (Thermo Scientific, Walthman, MA). The spectra
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obtained were analyzed by Proteome Discoverer (v1.20) software. Searches were performed by the Sequest search engine using Thermo Proteome Discover (v.1.3.0.339) against the Uniprot-SwissProt database. Percolator [28] was used to discriminate correct from incorrect peptide spectrum matches. Only peptides reported as high confidence (FDR≤1%) were considered for identification. A score value (sum of the score of all the peptides that matched this protein multiplied by the number of different peptides found for this protein) was assigned to the proteins selected. The peptides identified by the software, which may correspond to FAK sequences or to other protein sequences found in the precipitated band –putative partners-, were studied separately.
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ACCEPTED MANUSCRIPT 2.6. Analysis “in silico” of putative kinases: To identify motifs likely to be phosphorylated by specific protein kinases, we used the software ScanSite [29] at
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medium stringency. This program assigns a score value to the motif identified that gets
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closer to 0, the closer he motif is to the known consensus sequence. The software also
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assigns a percentile that indicates the percentage of probability that the candidate motif is phosphorylated by the specific kinase, with respect to all the potential motifs in the protein database. In other words, the database contains thousands of identified
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substrates and consensus sequences and the greater the agreement with only one
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particular consensus, the higher the reliability of being phosphorylated by this kinase and not another, and thus the lower the assigned percentile; therefore, a lower percentile
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indicates major specificity of the candidate motif.
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ACCEPTED MANUSCRIPT 3. Results 3.1. Protein extraction and LC-MS/MS assay. FAK was purified by
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immunoprecipitation from three types of brain samples: untreated or PTZ-treated adult
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brain samples, and untreated P5 brain samples. A band roughly corresponding to the
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size of FAK was cut from the electrophoretically separated proteins. Trypsin digestion of the proteins eluted post-electrophoresis was followed by an enrichment in
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phosphopeptide contents and LC-MS/MS analysis (Fig. 1). In addition to the band corresponding to FAK, a robust band of 40-45KDa was also found. This study
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permitted us to distinguish the peptides of the protein FAK and also discriminate those
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phosphorylated and the residue involved.
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3.2. Phosphorylated residues found in FAK protein. The ionized compounds were
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resolved into a series of mass/charge ratios, from which the composition of the peptide and the phosphorylated residues were deduced by the software. As an example, Suppl. Fig. 1 shows the mass/charge ratios found for the peptide holding Ser 29, and the
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corresponding spectrum.
Three new residues, previously undescribed, were found to be phosphorylated. Specifically, Thr 952, and Ser 1049 were identified in adult brain, both in untreated and PTZ-treated animals, and Thr 1048 only in the former (Fig. 2A). All these residues are placed along the FAT domain (Fig. 2B). Other residues had previously been found to be phosphorylated in tissues or cell types other than nervous cells, whereas others were already characterized in brain (Fig. 2A). Diverse patterns of phosphorylation allowed us to distinguish several categories among the phosphorylated residues. Some appeared: at all ages and treatments, such as Ser 29, Tyr 608, Ser 612, Tyr 614, Tyr 615 and Ser 948; exclusively at P5 (Ser 760 and Ser 9
ACCEPTED MANUSCRIPT 881); only in the adult brain, independently of the state of activation (Ser 606, Ser 715, Ser 716, Ser 878, Thr 952, Tyr 963 and Ser 1049); only in untreated adult brains (Thr
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613 and Thr 1048); or only in PTZ-stimulated adult brains (Ser 618).
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In addition, “in silico” analysis allowed us to determine that Ser 715 matches the
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consensus sequence of CAMK II, Thr 716 and Ser 881 match the kinase PK C ε, and Tyr 963 matches the Lck Kinase (Table 1).
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3.3. Proteins identified in the immunoprecipitated samples of FAK. The LC-MS/MS
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technique identified further proteins –which presumably interact with FAK- in the immunoprecipitated samples from the same assays. The number of putative partners included around 500 potential candidates (the best fits are shown in Suppl. Fig. 2).
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Some of the highest scoring proteins, listed in Table 2, are linked to cytoskeletal organization and dynamics, in agreement with the well-established functions of FAK
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[2,3,30]. This is the case of drebrin [31], which is involved in plasticity events [32], and also of an isoform of the heavy chains of kinesin 5C ([33]) and kinesin 1 [34], the
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microtubule-associated protein MAP 6 [35], and myosin-1 subtypes [36]. Other putative partners are involved in adhesion, such as neurabin-2 [37] and plakophilin 1 [38], while others link FAK to synaptic transmission, such as SynGAP [39] and the θ-1 subunit of the NMDA receptor [40]. Of note, whereas various proteins were found at all ages, such as drebrin, MAP 6, and neurabin-2, some motor proteins were detected only in the adult (kinesin 1 heavy chain was found in untreated and also in PTZ-treated adults, kinesin 5A only in untreated adults, and myosin-1d only in PTZ-treated adults). Particular myosins of type 1 were found at P5 only, and while the association with the NMDA receptor was detected only
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ACCEPTED MANUSCRIPT in untreated adults, SynGAP was observed in both untreated and PTZ-treated adults
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(Table 2).
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ACCEPTED MANUSCRIPT 4. Discussion 4.1. Phosphopeptide mapping of FAK. The regulation, functions and activation
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mechanisms of FAK show bulky density and have just been partly described. However,
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it is evident that a part of the functions and regulation of FAK depend on its degree of
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phosphorylation and, most specifically, on the combination of residues phosphorylated. FAK can be phosphorylated by several kinases such as Src, or in response to growth
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factors such as BDNF [17,41]. This kinase shows many other phosphorylated residues, and a plethora of conditions can modify FAK phosphorylation and/or activity [2]. In
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this study, we characterize the phosphorylation of three residues that had not been described previously in vivo: namely Thr 952 and Thr 1049, identified in untreated and
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PTZ-activated adult mouse brain, and Thr 1048, found in untreated adult mouse brain
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only. All these residues lie along the FAT domain, which fulfils FAK recruitment to
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focal adhesions through paxillin association [42,43]. Interestingly, even while the crucial residues and path of association between FAK and paxillin are well described, FAK association with talin is less understood, although it is
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also related to the FAT domain [30,44]. The FAK-talin association could be influenced by the FAT phosphorylation pattern. In addition, the phosphorylation of Tyr 925 disrupts focal adhesion targeting of FAK [45]. It has recently been shown that the first helix of the four-helix bundle of FAT associates with Tyr 925 and must be dissociated prior to Tyr 925 phosphorylation and MAPK pathway activation [44]. It is likely that the phosphorylation of one or several of the three residues cited above affects FAT folding, talin association and Tyr 925 uncovering, thus influencing signal transduction pathways. Of note, the lack of focal adhesions in neurons and the multiplicity of pathways exerted by them in the brain during development or neuronal activation [2] are probably related to the new phosphorylation sites described herein. Other studies 12
ACCEPTED MANUSCRIPT report the decrease in phosphorylated Tyr-925 found in oligodendrocytes prior to myelination [2,46], an observation that is consistent with the lack of phosphorylation on
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that residue at P5 (Fig. 2), which would also impede the presence of paxillin and Src in
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the extracts at that age [45]. The significance of these changes in terms of FAK
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competition with FRNK—which contains the complete FAT domain—also deserves additional attention.
In addition, Ser 29, Ser 606, Tyr 608, Ser 612, Tyr 615, Ser 715, Thr 716, Ser 760, Ser
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878 and Ser 881 have not been previously described in mouse brain in vivo, although
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reports in other tissues or organisms are available [17, 47,48,49,50,51,52,53] (see Fig. 2). The function of some of these residues has been partially characterized; however, for others there is scarce information about the role that their phosphorylation plays
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[46,53,54,55]. The adscription to the catalytic domain relates most of them to the fine regulation of kinase-dependent functions of brain FAK. Ser 29 lies N-terminal to the
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FERM domain, and the significance of its phosphorylation is unknown [47]. Here we also provide new information on the differential phosphorylation of FAK
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under various paradigms, in agreement with the patterns of phosphorylation found depending on brain activity. This is the case of Ser 618, located in the catalytic domain, and found phosphorylated in PTZ-stimulated brains only, whereas the neighboring Thr 613 and the FAT domain-located Thr 1048 were found phosphorylated only in untreated samples. Further studies are needed to determine whether these changes are related to the neosynaptogenesis that occurs during epileptogenic brain stimulation [56,57]. Finally, the simultaneous involvement of FAK in various signaling pathways is also feasible during development. Ser 760 and Ser 881 were phosphorylated in P5 samples only, whereas Ser 606, Ser 618, Ser 715, Thr 716, Ser 878, Thr 952, Tyr 963, Thr 1048, 13
ACCEPTED MANUSCRIPT and Ser 1049, were phosphorylated only in adult samples. Noteworthy, when phosphorylated, Tyr 963—the equivalent of the well described Tyr 925 of canonical
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FAK—mediates FAK detachment from focal adhesions [45], Grb2 association, and
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MAPK pathway activation [58,59], and these results are consistent with the needs of
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MAPK activation at adult age. The significance of other phosphorylated requires further attention.
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The resolution of the technique used is constrained by the weight and negative charge of the peptides obtained during trypsinization [60,61]. Therefore, there may be further
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phosphorylated sites. Tyr 397 may be such a site since it is the main phosphorylated residue of active FAK [9,21]. Nor did we detect phosphorylated Tyr 861, which is
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phosphorylated mostly during Rac activation and lamellipodial formation. Its absence
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can also be due to this constraint of resolution. In contrast, we found phosphorylated Tyr 576 and Tyr 577, whose phosphorylation is dependent on previous Tyr397
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phosphorylation, in our assays, and also phosphorylated Tyr 925.
4.2. Putative new partners of FAK identified by MS. Several putative new partners found here point to the participation of FAK in novel functions and additional pathways. The observation that many of these partners are involved in cytoskeletal functions is in agreement with the description of FAK as a cytoskeleton modulator [30,62]. Since this kinase modulates cell adhesion, these results are consistent with the association of FAK to other proteins involved in junctional adhesions. It is worth noting that the polyclonal antibody used for this study is directed against the C-terminal domain of FAK. It can compete and eventually disturb some associations between FAK and C-terminal domain-binding partners. Therefore, 14
ACCEPTED MANUSCRIPT additional information could be obtained from a similar study performed with N terminus-directed antibodies.
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Because of the ages and state of activation chosen, it could be envisaged that most of
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the FAK partners that we found are neuronal. However, it must be pointed out that a
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significant amount of brain FAK is of glial origin. In fact, the presence of at least three distinct gap junction-associated proteins in the extracts (Suppl. Fig. 2) points to the
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participation of FAK in the connexon-mediated communication between neurons and astrocytes.
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In addition, our results suggest that, in the adult, FAK associates with synapse-related partners, such as SynGAP [39], or with a subunit of the NMDA receptor [40], the latter
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present only in untreated adult brains. In addition to the high scores reported for some
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proteins such as drebrin, kinesin heavy chain isoforms 5A and 5C, and neurabin 2,
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others show lower scores. LC-MS/MS is not a quantitative method and variations must be interpreted with care. They imply that distinct peptides were found in distinct assays. This does not mean that a higher or lower amount of protein was bound nor must this
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observation be interpreted as a lower reliability of these results. Conversely, the multiplicity of forms and functions of FAK are consistent with a functional connection between FAK and numerous regulatory proteins such as Rho GEF, synGAP and the plasticity-related NMDA receptor, which are found only in particular circumstances. The presence or absence of these partners points to changes in the regulation of pathways related to actin dynamics and synaptic efficiency. Taken together, these data support the notion that FAK participates in plasticity [63].
15
ACCEPTED MANUSCRIPT ACKNOWLEDGEMENTS The proteomics work was done at the Proteomics Platform of the Barcelona Science
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Park, a member of the ProteoRed-ISCIII network. We thank Dr Eliandre Oliveira and
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Maria Antònia Òdena for technical assistance and helpful comments and discussions.
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Thanks go also to the SCT-UB for technical assistance. We thank Tanya Yates for editorial help. This work was granted by projects PI12/02108 to FB, PI10/01750 to
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JMU, and SAF2013-42445R to ES.
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ACCEPTED MANUSCRIPT Figure Legends
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Figure 1. Protein extraction and LC-MS/MS analysis: Brains were dissected,
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homogenized, and immunoprecipitated with polyclonal antibodies against FAK. The
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products were subjected to SDS-PAGE, the gel was stained with SyproRuby, and the bands corresponding to the molecular weight of FAK (signaled by an arrow) were cut. The lanes containing BSA correspond to negative controls and were performed with
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albumin instead of antibody. The molecular weight is indicated on the left side of the
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gel (only the most relevant sizes are shown). The italic letters in blue, on the right of the gel, show various relevant bands. “a” shows albumin, “b” and “d” the heavy and light
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chains of immunoglobulins, and “c” most probably corresponds to the C-terminal
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domain of FAK, which can also be autonomously expressed, and called FRNK (see also text). The eluted samples were trypsinized, and the digest was enriched in
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phosphopeptides in a column containing TiO2 magnetic beads. Finally, the
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phosphopeptides obtained were analyzed by LC-MS/MS.
Figure 2. Phosphorylated residues identified in the protein FAK: (A) Summary of the phosphorylated residues found. The shades in gray highlight the novel sites. Note that the numbering of residues in the first column corresponds to the brain form of mouse FAK (1090 amino acids). The second column indicates the equivalent numbering for the canonical forms of FAK in either human, mouse, or rat, which consist of 1052 amino acids (sequences available at http://www.uniprot.org/uniprot/Q05397). The ages and conditions in which a residue was been found phosphorylated are also shown. FDR (“false discovery rate”) index indicates the degree of confidence of the result, that is to say, the probability that the
25
ACCEPTED MANUSCRIPT spectrum obtained corresponded to a different peptide from the database; it is a value directly estimated by the software. The three columns on the right indicate the function
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described for each phosphorylation, the lab model used, and the corresponding
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reference. The residues found phosphorylated for the first time in this study are
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highlighted in bold. (B) Diagrammatic structure of FAK. The main domains characterized in the polypeptide are shown, and the phosphorylated residues identified.
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Those found phosphorylated for the first time are highlighted in bold.
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Table 1. Analysis “in silico” of the putative kinases that may phosphorylate the corresponding residues of FAK. Summary of the highest scoring phosphopeptides
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found, when they appropriately matched the assigned kinases. The table also shows the
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score and percentile values.
Table 2. Proteins identified by LC-MS/MS in the FAK-immunoprecipitated pellets
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under the experimental conditions tested. Summary of the proteins identified in the immunoprecipitates of FAK that are relevant in the central nervous system and present the highest scores. The proteins are grouped on the basis of whether they were identified in all the experimental conditions or not.
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ACCEPTED MANUSCRIPT SITE
KINASE
S715 CAM KII T716 PK C ε
CONSENSUS SEQUENCE FOUND IN FAK 707 TELKAQLSTILEEEK 723
SCORE
PERCENTILE
0.4441
0.197 0.654
ELKAQLSTILEEEKV 724
0.4371
S881 PK C ε
873
DVRLSRGSIDREDGS 889
0.3979
0.222
Y963 Lck
955
DRSNDKVYENVTGLV 971
0.5012
0.886
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Table 1
ACCEPTED MANUSCRIPT
370.85 168.34 83.45 42.98 63.68 79.70 41.16
46.43
78.14
76.97 54.66 -----
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77.59 53.12 41.24 9.09 68.05 66.34 51.53
Function
Cytoskeleton Cytoskeleton
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15.31 189.88 128.16 78.91 47.09 19.69 19.46
Cytoskeleton Cell Adhesion Cytoskeleton Cytoskeleton Cytoskeleton Cell Adhesion Cytoskeleton Cytoskeleton
----213.60 145.29
55.37 20.90 23.47 143.83
Cytoskeleton Cell Adhesion Cytoskeleton Cytoskeleton
105.08
54.33
---
195.40
---
Synaptic transmission Cytoskeleton
---
40.93
---
---
34.96
---
55.20
---
---
RNA regulation Synaptic transmission Cytoskeleton
41.82 36.60 -------
-----------
----34.89 31.11 29.06
Cytoskeleton Cytoskeleton Cytoskeleton Cytoskeleton Endocytosis
---
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---
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Kinesin heavy chain isoform 5A Rho guanine nucleotide exchange factor Glutamate [NMDA] receptor subunit θ-1 Myosin phosphatase Rho-interacting protein Myosin-1b Myosin-1c Myosin-1d Synaptopodin AP-1 complex subunit β-1
Ad mouse Ad mouse (untreated) (PTZ-treated) score score 556.16 170.26 421.68 52.09
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Drebrin Kinesin heavy chain isoform 5C MAP 6 Neurabin-2 β-adducin α-catenin 2 α-actinin 1 Plakophilin 1 Neurofilament medium polypeptide WD repeat-containing protein 47 α-actinin 4 Cytospin-B Kinesin 1 heavy chain MAP 7 domaincontaining protein 1 SynGAP
P5 mouse score 805.65 41.13
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PROTEIN IDENTIFIED
Table 2
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