Interaction between adaptor proteins Ruk / CIN 85 and Tks 4 in normal and tumor cells of different tissue origins

Aim. To study the interaction of adaptor protein Ruk/CIN85 SH3 domains with endogenous adaptor protein Tks4 in normal and tumor cells of different tissue origins. Methods. GST in vitro pull-down assay was performed using total cell lysates of cell lines of different origins. Results. Using GST in vitro pull-down assay, we have determined that SH3A domain of adaptor protein Ruk/CIN85 precipitated full-length form of adaptor protein Tks4 (M r 120 kDa) from lysates of human breast (MCF-7, MDA-MB-231), melanoma (MM-4), colon (HT-29, DLD-1) tumor cells as well as from lysates of mouse Lewis lung carcinoma cells (LLC) and fibroblasts (NIH 3T3). It has been also revealed that all Ruk/CIN85 SH3 domains (A, B and C) with high efficiency precipitate the additional forms of Tks4 with M r 75, 90 and 160 kDa from lysates of human colon carcinoma cells and mouse fibroblasts. The molecular nature of new multiple forms of Tks4 has not been determined to date. Conclusions. New multiple molecular forms of adaptor protein Tks4 with M r 75, 90 and 160 kDa, able to interact with high affinity with SH3 domains of Ruk/CIN85, were identified using GST in vitro pull-down assay. The data obtained suggest that interaction between Ruk/CIN85 SH3 domains with Tks4 endogenous forms is determined by cellular context while a level of this interaction can be regulated in the course of physiological cellular responses.


Introduction.
Adaptor proteins consist of domains and motifs, which mediate intermolecular interactions and are able to form intracellular protein complexes in a regular and selective manner.It determines their important role in the maintenance of specificity and efficiency of all known signal transduction pathways [1].In particular, adaptor proteins Ruk/CIN85 (Regulator for ubiquitous kinase/Cbl-interacting protein of 85 kDa) and Tks4 (Tyrosine kinase substrate with 4 SH3 domains) contain multiple SH3 domains and Pro-rich regions in their structure [2,3].Of note, these adaptor proteins are involved in the control of fundamental cellular and sig-naling events as well as in carcinogenesis [4,5].More than 100 protein partners of Ruk/CIN85 SH3 domains were identified in HeLa cells in vitro using the highthroughput technique combining affinity purification of interacting partners from cell lysates with liquid chromatography-coupled tandem mass spectrometry (LS-MS/MS).Adaptor protein Tks4 was revealed as one of the main protein partners according to the number of unique peptides matching to protein sequence and percentage of protein sequence covered by matching peptides.In general, 21 peptides, which overlap 27 % of 911 amino acids of Tks4, were identified [6].However, no data regarding the peculiarities of interaction of the individual Ruk/CIN85 SH3 domains with the endogenous Tks4 protein in cell context dependent manner have been reported so far.
Preparation of total cell lysates.For obtaining total cell lysates, monolayer cultures of cells were washed in ice-cold PBS and scraped into lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 50 mM NaF, 1 % Triton X-100, 5 mM EDTA supplemented with complete protease inhibitor cocktail tablet («Roche», France), 1 mM sodium orthovanadate, and 1 mM phenylmethylsulfonylfuoride (PMSF) («Fluka», Switzerland).The lysates were kept at 4 °C for 20 min and then were clarified by centrifugation at 12000 rpm at 4 °C.Protein content in supernatants was determined using bicinchoninic acid protein assay kit (Pierce ® BCA Protein Assay Kit, «Thermo Scientific», USA) according to the manufacturer's instructions.
Expression of recombinant proteins was induced by 1 mM IPTG (isopropyl-b-D-thiogalactoside) with further incubation at 25-27 °C for 4 h.Cells were pelleted by centrifugation at 3000 rpm, washed with icecold PBS, lysed in appropriate amount of lysis buffer (see above) at the ratio 1 : 10 for 20 min on ice.Cell lysates were sonicated 3 times for 20 s and centrifuged at 12000 rpm at 4 °C for 30 min.
Supernatants were added to Glutathione-Sepharose beads 4B («Amersham Biosciences», USA) in lysis buffer.Samples were incubated for 2 h at 4 °C.Glutathione-Sepharose beads were washed by PBS supplemented with 0.05 % Tween-20 and pelleted by centrifugation at 2000 rpm for 1-1.5 min.Amount of recombinant proteins bound to Glutathione-Sepharose beads was verified using SDS-PAGE.For the pull-down experiments, equal amounts of fusion proteins (about 15 µg at density 0.5 µg per 1 µl of Glutathione-Sepharose beads 4B) were used.
Cell lysates (about 1.5 mg of total protein) were incubated with 60 µl of 50 % Glutathione-Sepharose beads conjugated with corresponding recombinant proteins.Protein complexes were pelleted by centrifugation at 3000 rpm.Unbound proteins were removed by washing the beads 5 times with lysis buffer, whereas retained proteins were resolved by SDS-PAGE [9] and analyzed by Western blotting [10].For detection of Tks4 on blots, the affinity purified polyclonal rabbit antibodies against SH3AB fragment of Tks4 were used.These antibodies were obtained and characterized in our laboratory.Anti-rabbit secondary antibodies (peroxidaseconjugated IgG from «Sigma», USA) were used.The enhanced chemiluminescence kit («Amersham Farmacia Biotech», USA) was used for detection of immunoreactive protein bands.
Results and discussion.Cell lines of different tissue origins, in particular, MCF-7, MDA-MB 231, HT-29, DLD-1, MM-4, NIH 3T3 and LLC were investigated.According to Fig. 1, the obtained antibodies to SH3AB fragment of Tks4 (dilution 1:5000) effectively recognize the endogenous full-length form of Tks4 (M r 120 kDa) in all analyzed cell lysates.The highest levels of Tks4 were identified in MDA-MB-231, LLC, MM-4 and DLD-1 cells, while lower levels -in MCF-7 and HT-29 cells.
Based on these results we can conclude that the level of Tks4 expression is specific for each cell line and does not depend on tissue origin.As can be seen in Fig. 1, the additional form of Tks4 (M r 160 kDa) is detected in MDA-MB-231, LLC, MM-4, DLD-1 and NIH 3T3 cell lysates.
Progress in the field of functional proteomics during the last decades is determined by the development and further improvement of methodological approa-ches for decoding intermolecular interactions networks.It is known that these methods are divided into two groups, synthetic and analytical.GST in vitro pulldown assay is one of the analytical methods, which allow identification of the complexes usually purified by affinity chromatography.The components of purified supramolecular complexes can be recognized by specific antibodies or mass-spectrometry.The aim of analytical methods is the identification of protein partners in cellulo under physiological conditions.As a result, the data on protein composition of purified complexes provide an immediate success on the way to the determination of their functional significance.
GST-fused SH3A, SH3B, SH3C, SH3AB, SH3BC, SH3ABC domains, Pro-rich, Ser-rich, coiled-coil regions, which overlapped all amino acid sequence of Ruk l , were used for GST in vitro pull-down assay.According to the results obtained (Fig. 2), only SH3A domain precipitates Tks4 from lysates of breast adenocarcinoma (MCF-7 and MDA-MB-231) and melanoma (MM-4) cells.In mouse Lewis lung carcinoma cells (LLC) Tks4 is preferentially precipitated by SH3A domain, whereas weak immunoreactive bands are detected in SH3C-and SH3BC-precipitates.Tks4 is not detected in SH3B-and SH3AB-precipitates.However, a significant reduction of Tks4 binding in SH3ABCprecipitate in comparison with SH3A domain is observed.The performed analysis allows suggesting that at the level of three-dimensional structure of Ruk/ CIN85 SH3ABC fragment it is SH3B domain that can screen the binding surface of SH3A domain for Prorich sequences of Tks4 in LLC cells.We also suggest that phosphorylation of specific Ser/Thr residues localized in SH3AB domains as well as C-terminally from Pro-rich region in endogenous Ruk/CIN85 mediated by input signal may lead to destabilization of its autoinhibitory conformation and provides the capacity to form complexes with specific binding partners.In mouse fibroblasts, the full-length form of Tks4 is mainly precipitated by SH3A and weakly by SH3BC and SH3ABC domains.
Interesting results were obtained using pull down assay for colonic cell lines such as DLD-1 and HT-29.As can be seen in Fig. 2, the main Tks4 form (M r 120 kDa) is detected only in SH3A-precipitate.Simultaneously, all used GST-SH3 fragments of Ruk/CIN85 (A, B, C, AB, BC and ABC) with high efficiency precipitate the additional form of 90 kDa, the content of which in cells is extremely low.This form is revealed in Westernblotting only in the case of increased protein addition into the pockets of PAA gel.It is possible that the form with M r 160 kDa, which is detected in precipitates, could be a result of posttranslational modification of Tks4 by ubiquitination or related protein modifications.New additional form in NIH 3T3 cells is characterized by higher mobility (M r about 75 kDa).It is precipitated by all GST-SH3 fragments of Ruk/CIN85 (A, B, C, AB, BC and ABC).It should be noted that the high molecular weight form of Tks4 is detected in NIH 3T3 cell lysates but it is not precipitated by SH3 domains of Ruk/CIN85.For more careful examination of the specificity of the obtained anti-Tks4 polyclonal antibodies, we performed an antigen competition experiment.For this purpose, anti-Tks4 antibodies were preincubated with purified GST-Tks4/SH3AB fragment, and then this mixture was used for Western blotting of Tks4 endogenous forms.We found that detection of all immunoreactive bands was abrogated by antigen competition (data not shown).
The molecular nature of new multiple Tks4 forms with M r 75, 90 and 160 kDa identified in our study is unknown.At present there are no data about posttranslational modifications of Tks4 and additional mRNAs, which could be formed as a result of alternative splicing or alternative promoter usage in TKS4 gene.The database analysis of expressed sequence tags (ESTs) allows identifying some ESTs, which could encode the

Fig. 1 .
Fig. 1.Expression of adaptor protein Tks4 in cell lines of different tissue origins.Immunoblot analysis of cell lysates was performed using the affinity purified rabbit polyclonal anti-Tks4 antibodies produced against Tks4 SH3AB fragment