Intra- and intermolecular interactions mediated by adaptor protein Ruk/CIN85/SETA

Ruk/CIN85l SETA is a member of a separate and evolutionary conserved family of adapter I scaffold proteins implicated in apoptic and receptor tyrosine kinases signalling, rearrangement of actin cytoskeleton and cell adhesion, podocyte and T cell functions. Self-regulation through intra- and intercellular interactions can be supposed for RuklCIN85l SETA as this protein contains SH3 domains and proline-rich sequence, localized within one polypeptide chain, as well as C-terminal coiled-coil region. The ability of Ruk proline-rich motifs to interact with its own SH3 domains in an intramolecular fashion and coiled-coil region to mediate oligomerization between different isoforms was assessed in GST pull down experiments. It was shown that both Ruk SH3A and to a less extent SH3B domains can interact with its own proline-rich sequences in a cooperative manner, while coiled-coil region provide for isoforms oligomerization. SH3C domain appear exerts conformational constraints, imposed on coiled-coil region, restricting the level of oligomerization. We also demonstrated that the ability of exogenous ligands to interact with Ruk polyprotine motifs is changing during the course of TNFa-induced apoptosis of human myelomonocytic

Up to date, it is well recognized and widely accepted that protein-protein interactions me diated by modular recognition domains represent structural and regulatory framework for the arran gement of functional signalling networks [1]. Srchomology 3 (SH3) domain, is one of the most widespread protein recognition modules in the proteome critically involved in the assembly of many intracellular signalling complexes and pathways [2]. SH3 domains represent independently folding mo dules (of about 60 amino acids residues in length) able to establish intermolecular associations among signalling molecules through binding to prolinerich sequences which usually adopt left-handed polyproline type II (PPII) helices [2 ].
Ruk/CIN85/SETA, a member of a separate and evolutionary conserved family of SH3-containing adaptor/scaffold proteins, was cloned and charac terized in the end of 2000 by three independent scientific groups [3][4][5]. Distinctive features of these proteins are three SH3 domains (A, B and C) located at the N-termini, followed by proline-and serine-rich sequences and C-terminal coiled-coil region [6]. Due to modular organization, Ruk/CIN85/SETA and its splice variants (see Fig. 1) play complex and varied roles in important biological processes such as prolife-  [7] ration and apoptosis [3,8], ligand-induced endocytosis of receptor tyrosine kinases [9][10][11], rearran gement of actin cytoskeleton [12,13], cell cyto kinesis, adhesion and motility of T cells during antigen presentation [13,14], and possibly in nuclear events [15,16]. This means that biological activity of Ruk/CIN85/SETA has to be a subject of tight regulation depending on cellular context. The simul taneous presence of three SH3 domains and four blocks of proline-rich motifs in Ruk/CIN85/SETA structure, suggests the possibility of intra-and hemo philic intermolecular interactions, which in turn may account for autoregulation of the protein. Besides, C-terminal coiled-coil region is suppo-sed to be invol ved in the formation of oligomers between Ruk isoforms, which possess different biological signi ficance. In attempt to further characterize interactions mediated by domains and motifs of Ruk/CIN85/SE-TA we used GST in vitro binding assay and specific polyclonal antibodies. It was shown that both Ruk SH3A and to a less extent SH3B domains can interact with its own proline-rich sequences, while coiled-coil region mediates oligomerization between different isoforms. We also demonstrated that the ability of exogenous ligands to interact with Ruk polyproline motifs changes in the course of TNFa-induced apop tosis of human myelomonocytic U937 cells.
Materials and Methods. pCMV5 expression plasmids encoding various Ruk isoforms and mutants with C-terminal FLAG-tag (DYDDDDK) and pGEX-2T expression plasmids encoding GST-fusion SH3A, SH3B, SH3C domains and Ruk s isoform were des cribed earlier [17]. An expression plasmid encoding GST-3SH3 CIN85 fragment was provided by I. Dikic (Institute of Biochemistry II, Frankfurt, Germany). Anti-Ruk, polyclonal antibodies were raised in rabbit using standard protocol. Specific polyclonal antibodies were purified from serum of an immunized rabbit using GST-Ruk,-Sepharose. An anti-FLAG-tag anti body was from «Sigma» (USA), and secondary horse radish peroxidase-conjugated anti-mouse or anti-rab bit IgG antibodies were purchased from «Promega» (USA). All reagents for cell culture were from Gib-coBRL.
Cell culture and transfection.
Human embryonic kidney (HEK) 293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supple mented with 10 % fetal calf serum (FCS), 2 mM L-glutamine, 50 IU/ml penicillin, and 50 fig/ml streptomycin in humidified atmosphere containing 5 % C0 2 at 37 °C. Human myelomonocytic leukemia U937 cells were grown in RPMI-1640 medium con taining 10 % FCS. HEK293 cells were plated on 10 cm diameter dishes and transfected with 10 pg of plasmid DNA by a modified calcium phosphate me thod [18]. 24 hours after transfection, cells were washed twice with cold phosphate buffered saline (PBS) and lysed in the lysis buffer (LB) containing 10 mM Tris, pH 7.5, 150 mM NaCl, 1 % Triton X-100, 5 mM EDTA, 20 mM NaF, 1 mM Naorthovanadate, 1 mM PMSF, and a cocktail of pro tease inhibitors from Roche at 4 °C for 20 min. Lysates were cleared by centrifugation at 17000 g for 20 min at 4 °C and supernatants were used for further experiments. Before induction, U937 cells were cul tured in RPMI 1640 medium supplemented with 2 % FCS for 24 hours and then plated at 1 • 10 s cells/ml in RPMI 1640 with 10 % FCS. Cells were induced with TNFa at the final concentration of 10 ng/ml. Cell lysates were prepared at the 2 nd , 4 th and 6 ,h hour after induction. GST-fusion constructs were expressed in IPTG-induced Escherichia coli BL21 at 30 °C for 4 hrs. Cells were pelleted at 3000 g for 15 min at 4 °C. Pellets were washed in PBS and resuspended in the buffer containing 50 mM Tris, pH 7,5, 150 mM NaCl, 1 % Triton X-100, 1 mg/ml lysozyme, and protease inhibitors. After one freeze-thaw cycle, the bacteria were sonicated on ice and cleared from cell debris by centrifugation at 13000 g for 30 min at 4 "C. The efficiency of the bacterial lysis was checked by SDS-PAGE. The GST-fusion proteins were isolated from the supernatants using glutathione-Sepharose beads, as recommended by the manufacturer («Amersham Pharmacia Biotech*, UK). For pull-down experiments, GST or GST-fusion proteins (7 pg of protein) bound to glutathione-Sepharose (approximately 20 fi\ of packed beads equilibrated in 50 mM Tris, pH 7.5, 150 mM NaCl, 1 % Triton X-100) were incubated for four hours with normalized cleared lysates (1.5 mg of total protein) of HEK293 cells transiently transfected with Ruk plasmids. The cleared lysates were prepared as described above. The beads were washed three times with PBS/1 % Triton X-100, 3 times with PBS and boiled in 50 ml of SDS-loading buffer. Eluted proteins were separated by SDS-PAGE [19] and transferred onto PVDF membrane for Western blot analysis with anti-FLAG or anti-Ruk, antibodies. All Western blots were performed according to a standard protocol [20 ]. The final detection was done with ECL system («Amersham Pharmacia Biotech*, UK).
Results and Discussion. Protein-protein in teractions are ubiquitous regulatory events in cell biology. Therefore, it is not surprising that nature has evolved and coordinated multiple mechanisms to exquisitely control the biological activity of adap tor/scaffold proteins which function as the main organizing centers of signalling networks. These me chanisms usually include different types of posttranslational modification and intermolecular or in tramolecular associations with inhibitory molecules or domains [1 ]. These inhibitory interactions may be disrupted in response to appropriate stimuli allowing the adaptor/scaffold protein to adopt a biologically active conformation.
Since adaptor protein Ruk/CIN85/SETA con tains four blocks of proline-rich motifs, we were interested whether its SH3 domains can bind to these motifs in an autoregulatory fashion.
To evaluate the possibility of intramolecular in teractions between different Ruk SH3 domains and its own C-terminally located proline-rich stretches, GST in vitro binding assay was used. For this purpose, the GST-fused A, B, and C SH3 domains, 3SH3 (ABC) fragment, and a shortest Ruk, isoform which includes only the C-terminal coiled-coil region were incubated with lysates of HEK293 cells transiently transfected with constructs encoding different Ruk isoforms and mutants. Proteins bound to glutathione-Sepharose beads were analysed by Western blotting using anti-  Fig. 2 show that Ruk SH3A and, to a lesser extent, SH3B domains were able to bind to proline-rich motif-bearing Ruk, (full length) and Ruk m (without two of three N-terminal SH3 domains) isoforms and Ruk^^ mutant (Fig. 2, a, c, e) but not to Ruk APro or Ruk,, (heart specific Ruk isoform) (Fig. 2, b, d) which don't contain SH3 recognition sequences.
The increase of binding was detected in pre cipitates using GST-3SH3 fragment in comparison with using GST-SH3A and B domains as baits, separately. It was also shown that Ruk SH3C domain does not bind to Ruk, and Rul^ isoforms (Fig. 2, a,  c) but pulls down Ruk^Hj mutant quite effectively (Fig. 2, e). In similar experiments using human orthologues of Ruk, CIN85 [21 ] and CD2BP3 [14], binding of GST-SH3B to their own proline-rich stret ches was not detected. These partially differing results may be explained by certain peculiarities of carrying out GST pull-down experiments. As it can be seen from Fig 2, all the five studied Ruk proteins were pulled down effectively by GST-Ruk,, con firming the results of previous studies, which sug-gested the role of coiled-coil region in the di(oligo)merisation of Ruk proteins [22,23]. One should note a significant increase of Ruk^j binding to GST-Ruk, compared to the extent of Ruk^ RULF^, Ruk h and Ruk APro interactions.
The above-mentioned observations allow us to draw the following conclusions. First, intramolecular interactions mediated by Ruk SH3A and B domains are characterized by a cooperativity. Second, the SH3C domain, although not involved in intramo lecular interactions, may play a regulatory role at the level of whole conformation of Ruk protein providing correct SH3A/B -proline-rich motifs recognition. Possibly, the SH3C domain also exerts conformatio nal constraints imposed on coiled-coil region, restric ting the level of oligomerisation.
The obtained data suggest that Ruk may be self-regulated by a physical interaction between its SH3 domains and the SH3 binding sites the exact nature of which is to be determined. Besides, we can conclude that SH3 domains of Ruk serve both as the regulatory domains and as the recruiting units.
While Ruk/CIN85/SETA represents an example of SH3-mediated autoregulation of adaptor protein, the well studied tyrosine kinases of Src family [24][25][26][27] and a serine/threonine mixed lineage kinase 3 (MLK3) [28 ] as well as NADPH oxidase [29 ] offer some interesting parallels. It was shown that the SH3 binding sequence in Src, and also in its relative Fyn, as well as in MLK3 harbors only a single proline residue, although their SHS's preferred ligands were found to be classical proline-rich sequences [24,25,28 ]. These data suggest that the SH3-binding sequen ces in Src kinases and MLK3 represent the suboptimal ligands for their SH3 domains. Although the GAPPR seguence in p47 phox cytosolic component of the NADPH oxidase is not the canonical SH3 binding sequence, it can adopt PPII helix conformation that was determined from the X-ray crystal structure data [29]. There are no PXXP motifs or hydrophobic residues that confer specificity toward the SH3 do mains. It is interesting to note that this PPII helix is recognized by the tandem SH3 domains of NADPH oxidase using different sides of a triangular prism as the basal planes, with the plus and minus orien tations, respectively. Thus, the authors proposed that in this respect GAPPR does not bind tightly to each SH3 domain, but at the same time its specificity is still maintained by simultaneous binding to both SH3 domains [29].
Additionally we could draw a parallel connected with the presence of the cluster of four arginine residues in the cleft between two SH3 domains in NADPH oxidase, which appear to constitute the interaction core [29]. Similar arginine clusters are also present in Ruk/CIN85/SETA structure [4][5][6]. Thus, the evolutionary selection may have given rise to relatively low affinity intramolecular ligands for SH3 domains so that they might be outcompeted by the presentation, in response to an appropriate phy siological signal, of a high affinity proline-rich ligand on another signalling molecule [30,31 ]. Signalling molecules that overcome SH3-mediated autoregulation of Ruk/CIN85/SETA have yet to be identified. One potential candidate is the regulatory subunit of PI 3-kinase that contains SH3 domain and proline-rich sequences through which it interacts with Ruk in a concerted manner [17]. Another example represents members of Cbl ubiquitin ligase family which interact with CIN85 via their SH3 domains, and this asso ciation is enhanced by growth factor-induced tyrosine phosphorylation of Cbl [21 ].
There are data consistent with the idea that phosphorylation of specific serine residues could de stabilize additionally the autoinhibited conformation of signalling proteins to form the unmasked state as it was shown for MLK3 and NADPH oxidase [28,29 ]. Potential serine phosphorylation sites that reside C-terminally to the proline-rich region in Ruk/CIN-85/SETA structure might play such functions.
An unresolved issue is also a possibility that intramolecular interactions mediated by domains and motifs within Ruk molecule take place in a homodimeric manner and have a more complex character. For example, studies on Bruton's tyrosine kinase (Btk) demonstrated that both proline-rich sequences in the Tec homology (TH) region located immediately N-terminally to the SH3 domain stabilize intermolecular interactions, whereas the related Itk kinase containing one proline-rich motif can fold onto and bind its SH3 domain in an intramolecular association [32].
The next question which we decided to elucidate is whether the system of Ruk-dependent intramo lecular interactions undergoes changes during the biological responses of cells. We used leukemic U937 cells induced to apoptosis with TNFa as a model. Lysates of control and induced U937 cells were subjected to GST in vitro binding assay using as bait GST-3SH3 fragment. DNA ladder assay was perfor-B D Fig. 3

TNFa-treated cells at 2 nd , 4 lh and 6 lh hours of induction); B -expression of Ruk, protein in control and TNFa-treated U937 cells; C, Dinteraction of endogenous Ruk| protein with GST-3SH3 fragment in vitro using lysates of control and TNFa-treated U937 cells
med to monitor the course of apoptosis. As can be seen from Fig. 3, A, incubation of U937 cells in the presence of TNFa (10 ng/ml) for 6 hours is followed by oligo-and mononucleosomal DNA fragmentation that is a characteristic feature of apoptosis. Under such conditions, the level of expression of endogenous full-length size Ruk[ isoform with molecular weight of 85 kDa decreases insignificantly already after two hours of induction in comparison with control cells and does not change during the next studied time intervals up to the 6 1h hour (Fig. 3, B). Western blotting of GST precipitates using polyclonal anti-Ruk, antibodies had shown that the quantity of Ruk, bound to GST-3SH3 fragment is not supported at the constant level in the lysates of non-induced cells. The decrease of binding is observed at the 4 th hour followed by a small increase at the 6 lh hour of cell culturing (Fig. 3, C, and D). On the contrary, the dramatic decrease of Ruk, -GST-3SH3 interaction is detected in lysates of TNFa-treated U937 cells at the 2 nd hour followed by a significant increase during the 4 th -6 lh hour period of induction, in comparison with intact cells, which coincides with the accumulation of nucleosomal DNA fragments (Fig. 3, C, D).
The obtained data allow us to conclude that several Ruk conformational states with different bin ding capabilities may co-exist at equilibrium. Due to the concerted nature of Ruk-mediated intra-and intermolecular interactions, disruption of any of them, to a greater or lesser extent, shifts the equilibrium from the self-regulated (closed) conformation to the biologically active (exposed) conformation that enab les interaction with exogenous target sequences in other signalling molecules. To clarify all these issues, further structural studies are necessary. , В. P. Дрель, Г. Ю. Шуваєва, С. В. Гаврило»,  Я. Я. Федиишн, О. М. Маєвська, Н. 1. Ігуменцева, I. Т