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Ruk is ubiquitinated but not degraded by the proteasome Fre ´ de ´ rique Verdier 1 , Taras Valovka 1 , Alexander Zhyvoloup 1,4 , Ludmila B. Drobot 5 , Vladimir Buchman 2,3 , Mike Waterfield 1 and Ivan Gout 1,4 1 Ludwig Institute for Cancer Research, University College of London Medical School, London, UK; 2 Department of Preclinical Veterinary Sciences, the University of Edinburgh, UK; 3 Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia; 4 Institute of Molecular Biology and Genetics, Kyiv, Ukraine; 5 Institute of Cell Biology, Lviv, Ukraine The regulator of ubiquitous kinase (Ruk) protein, also known as CIN85 or SETA, is an adaptor-type protein belonging to the CD2AP/CMS family. It was found in complexes with many signaling proteins, including phos- phoinositol (PtdIns) 3-kinase (EC 2.7.1.137) 1 ,Cbl,GRB2, p130Cas and Crk. Functional analysis of these interactions, implicated Ruk in the regulation of apoptosis, receptor endocytosis and cytoskeletal rearrangements. We have recently demonstrated that overexpression of Ruk induces apoptotic death in neurons, which could be reversed by activated forms of PtdIns 3-kinase and PKB/Akt. Further- more, Ruk was shown to be a negative regulator of PtdIns 3-kinase activity through binding to its P85 regulatory sub- unit [Gout, I., Middleton, G., Adu, J., Ninkina, N. N., Drobot, L. B., Filonenko, V., Matsuka, G., Davies, A. M., Waterfield, M. & Buchman, V. L. (2000) Embo J. 19, 4015–4025]. Here, we report for the first time, that all three isoforms of Ruk (L, M and S) are ubiquitinated. Specific interaction between the E3 ubiquitin ligase Cbl and all three Ruk isoforms was demonstrated by coexpression studies in Hek293 cells. The interaction of Ruk M and S isoforms with Cbl was found to be mediated via heterodimerization with Ruk L. The use of proteosomal and lysosomal inhibitors clearly indicated that ubiquitination of Ruk L does not lead to its degradation. Based on this study, we propose a possible mechanism for the regulation of Ruk function by ubiquiti- nation. Keywords: signal transduction; adaptor protein; ubiquitina- tion; proteasomal degradation. Ubiquitination of proteins plays a major role in the regulation of various cellular processes. The best studied function of ubiquitination is its role in protein degradation, where polyubiquitinated proteins are recognized by the 26S proteasome or, in certain cases, by the lysosomes/vacuole and rapidly degraded. Ubiquitination of target proteins involves a cascade of reactions catalysed by the E1, E2 and E3 enzymes. Ubiquitin (Ub) is first activated by an activating enzyme (E1) to form a high energy thioester bond between Ub and E1 and is then transferred to a conjugating enzyme (E2). The Ub protein ligases or E3s are responsible for specific substrate recognition and for promoting covalent Ub ligation to the target protein. Thus, the E3s provide specificity to the Ub system [1,2]. The Cbl proteins form a family of related proteins harboring several highly conserved domains, such as an N-terminal variant SH2 domain, a RING finger and a C-terminal proline-rich domain containing potential tyro- sine phosphorylation sites. Previous studies have shown that c-Cbl and Cbl-b 2 function as adaptor proteins by interacting with other signaling molecules through their various pro- tein–protein interacting motifs [3]. Biochemical and genetic studies have shown that Cbl family proteins, including those from Drosophila and Caenorhabditis elegans, attenuate intracellular signaling induced by the engagement of cell surface receptors. The mechanism underlying the negative regulation of activated receptors by Cbl proteins has been recently described. Cbl functions as an E3 Ub protein ligase, which mediates the ubiquitination of activated receptor tyrosine kinases [4–8] or nonreceptor tyrosine kinases (e.g. Fyn, Syk [9,10]) and targets them for degradation. We have recently identified a novel adaptor-type protein named Ruk [11]. It has been cloned by other groups and named Cin85 or SETA [12,13]. Based on sequence homology and domain organization, Ruk L was integrated into a new subfamily of adaptor molecules that includes the protein CD2AP, also named CMS. All members of this family contain three SH3 domains at the N-terminus, followed by a proline-rich region, a PEST 3 region, and a coiled-coil domain towards the C-terminus. A variety of signaling molecules, including Grb2, Crk, Sos, Cbl were shown to interact with Cin85 via these protein–protein interaction domains [12,14]. We previously described the existence of three Ruk isoforms named Ruk L, Ruk M and Ruk S, which are products of alternative splicing and differential promoter usage (Fig. 1A). These isoforms share a common C-terminal region but are truncated at their N-termini. Ruk M possesses only one SH3 domain but all downstream domains are the same as in the Ruk L protein. In contrast, Ruk S retains only the C-terminal coiled-coil region. Although the precise role of this Ruk/CD2AP family is still unknown, they are involved in the control of apoptosis and the regulation of cytoskeletal architecture [11,13,15–18]. Correspondence to F. Verdier, De ´ partement d’He ´ matologie, Institut Cochin, 27 Rue du Faubourg Saint Jacques, 75014 Paris, France. Fax:+33140516510,Tel.:+33140516501, E-mail: verdier@cochin.inserm.fr Abbreviations: Ub, ubiquitin; Ruk, regulator of ubiquitous kinase; PtdIns 3-kinase, phosphoinositol 3-kinase; HA, hemaglutinin (Received 13 March 2002, revised 14 May 2002, accepted 30 May 2002) Eur. J. Biochem. 269, 3402–3408 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03031.x We found that Ruk L binds to PtdIns-3 kinase via the P85 regulatory subunit of the enzyme and inhibits its catalytic activity. Furthermore, Ruk L overexpression in primary neurons induces apoptosis, which could be rescued by coexpression of constitutively activated forms of PtdIns-3 Kinase or its downstream effector PKB/Akt [11]. In agreement with these findings, overexpression of SETA was shown to trigger apoptosis in astrocytes [13]. Specific associations between SETA and proteins involved in apoptotic processes, such as AIP1, Alg2, Sb-1 further confirm its importance in maintaining cellular homeostasis [13,18]. Cin85 has been also implicated in the regulation of cytoskeletal architecture since it interacts with p130 Cas and colocalizes with actin cytoskeleton in epithelial cells [18] and more recently Ruk L has been involved in the regulation of receptor-mediated endocytosis [19]. Because Ruk L contains PEST sequences that are usually found in proteins with short half lives [20], and because it associates with the E3 ubiquitin ligase Cbl, we wondered whether Ruk proteins are modified by ubiquitination. In this report, we demonstrate that all three Ruk isoforms are ubiquitinated. Co-expression studies in Hek293 cells allowed us to detect specific association between the E3 Ub ligase Cbl and all three Ruk isoforms. Moreover, binding of c-Cbl to Ruk M and Ruk S was found to be dependent on heterodimerization with Ruk L, via its coiled-coil domain. Detailed analysis of the stability of Ruk indicated that ubiquitination does not trigger its degradation by proteo- somes. MATERIALS AND METHODS Reagents and antibodies Protease inhibitors N-Ac-Leu-Leu-norLeucinal (ALLN) and lactacystin were purchased from Sigma and Calbio- chem, respectively. Rabbit polyclonal anti-hemagglutinin (HA) 4 Ig and anti-Cbl Ig were purchased from Santa Cruz. Mouse monoclonal anti-EE 5 Ig were kindly provided by L. Stephens (AFRC Babraham Institute, Cambridge). Mouse monoclonal anti-(b-actin) Ig were obtained from Sigma, and mouse anti-(b catenine) Ig from Transduction Laboratories. Rabbit polyclonal anti-Ruk antibodies, directed against the C-terminal peptide were produced as described previously [11]. Expression constructs The full-length coding sequences corresponding to all three splicing forms of Ruk (Ruk L, Ruk M and Ruk S) were amplified by PCR using rat cDNAs as templates. Amplified cDNA fragments were then cloned into pRc/CMV2 vector (Invitrogen, Life Technologies) in-frame with the N-ter- minal EE-tag epitope (MEFMPME). Generated constructs were verified by restriction enzyme digestion and DNA sequencing. pcDNA3/Cbl plasmid was a generous gift from Y. Yarden (The Weizmann Institute of Science, Rehovot, Israel). Mammalian expression vector encoding Ub–HA was a gift from D. Bohmann (EMBL, Heidelberg, Germany). Cell culturing and transient transfection Human embryonic kidney cells (HEK293) were cultured at 37 °Cand5%CO 2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Life Technologies, Inc.), 2 m ML -glutamine, 50 UÆmL )1 penicil- lin and 50 lgÆmL )1 streptomycin. Transient transfections were carried out by using LipofectAMINE according to the manufacturer’s recommendations (Life Technologies, Inc.). Twenty-four hours post-transfection cells were treated with 500 l M cycloheximide, 50 l M ALLN, 50 l M lactacystin, 20 m M NH 4 Cl, 200 l M chloroquine or vehicle alone for indicated time. Fig. 1. Ruk L, Ruk M and Ruk S ubiquitination. (A) Schematic representations of the domain organization of the three Ruk proteins. SH3, Src homology 3 domain; Pro, proline rich region; PEST, sequences enriched in proline, glutamic acid, serine and threonine; CC, Coiled-Coil domain. (B) Hek293 cells were transfected with 1.5 lg of plasmid containing EE-tagged Ruk L cDNA, EE-tagged Ruk M cDNA or EE-tagged Ruk S in the absence or presence of HA-tagged Ub plasmid (1.5 lg). As a control, Hek293 cells were transfected with an empty vector. Cell lysates were immunoprecipitated with anti-EE Ig. The immunoprecipitates were subjected to immunoblotting with anti-HA antibody (top panel). The positions of polyubiquitinated Ruk species [(Ub)n-Ruk] are indicated. The arrowheads mark the locations of the unmodified forms of Ruk L, M, S. The membrane was reprobed with anti-EE antibody (bottom panel). Ó FEBS 2002 Ruk is ubiquitinated but not degraded by the proteasome (Eur. J. Biochem. 269) 3403 Immunoprecipitation and Western blot analysis Hek293 cells were washed with ice-cold NaCl/P i and lysed with buffer containing 1% Brij 98, 10 m M Tris, 150 m M NaCl, 5 m M EDTA, 5 m M EGTA, 20 m M NaF, 20 l M 2-glycero-phosphate, 1 m M sodium pyrophosphate, 1 m M vanadate, 10% glycerol, 1 m M phenylmethanesulfonyl fluoride and a cocktail of protease inhibitors (Roche) at 4 °C for 20 min. Lysates were cleared by centrifugation for 30 min at 27 000 g and supernatants used for further experiments. Immunoprecipitating antibodies were incuba- ted with solubilized cell extracts for 1 h at 4 °C before the addition of protein G–Sepharose beads, prewashed in lysis buffer. After a 2-h incubation on the wheel, the beads were washed three times with lysis buffer and twice with buffer containing 0.1% Brij 98 6 . Immune complexes were removed from beads by boiling in Laemmli sample buffer and separated by SDS/PAGE. Resolved proteins were trans- ferred onto a poly(vinylidene difluoride) membrane, which was incubated for 1 h with blocking solution (5% milk in Tris/NaCl/0.1%Tween) followed by specific antibody over- night at 4 °C. After extensive washing with NaCl/Tris/0.1% Tween, the membrane was incubated for 1 h with horse- radish peroxidase-conjugated secondary antibody. The antigen–antibody complexes were detected using enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech). When immunoblots had to be reprobed, the membranes were initially stripped and reblocked prior to incubation with another type of primary antibody. Detection of ubiquitinated proteins Ub is highly conserved among eukaryotes, as only three of 76 amino acids differ between the human and yeast proteins. Therefore, Ub is not an optimal antigen and anti-Ub Ig rarely possess good affinity. Taking this into account and that anti-Ruk Ig are not very efficient in immunoprecipi- tation experiments, we decided to cotransfect Hek293 cells with plasmids encoding HA-tagged ubiquitin and EE- tagged versions of Ruk isoforms. (EE–Ruk L, EE–Ruk M and EE–Ruk S). Transiently expressed Ruk isoforms were immunoprecipitated with anti-EE Ig and resolved by SDS/ PAGE. Modification of Ruk by Ub was determined by immunoblotting with anti-HA Ig. RESULTS All three Ruk isoforms are ubiquitinated in vivo Sequence analysis of Ruk L indicated the presence of multiple PEST motives located between a stretch of proline- rich sequences and the C-terminal coiled-coil domain. The appearance of PEST motifs in protein sequences is often associated with reduced protein stability and a short half-life [20]. Taking this into account and the fact that Cin85/SETA binds the E3 ligase c-Cbl [12,18], we decided to investigate ubiquitination of Ruk isoforms in vivo. In this experiment, EE-tagged versions of all three isoforms of Ruk were cotransfected into Hek293 cells together with HA-tagged Ub. Two days after transfection, Ruk isoforms were immunoprecipitated using anti-EE antibodies, separated by electrophoresis and analysed by immunoblotting using anti-HA Ig. The results presented in Fig. 1B clearly demonstrate the appearance of multiple high molecular mass bands detected by anti-HA antibody. No signal was observed when Hek293 cells were transfected only with EE-tagged isoforms of Ruk, indicating the specificity of anti-HA Ig. Reprobing of the membrane with anti-Ruk Ig, which is specific for all three isoforms, confirmed that equal amounts of EE-tag fusions were coimmunoprecipitated from transfected cells (Fig. 1B, lower panel). The appear- ance of multiple bands on the anti-HA Western blot, clearly demonstrate that Ruk isoforms are polyubiquitinated. Specific interaction between Ruk isoforms and the E3 ubiquitin ligase Cbl Specific interaction between c-Cbl and Cin85/SETA, which corresponds to the Ruk L isoform, has been recently reported [12,18]. These findings and the results presented above prompted us to investigate whether all Ruk isoforms can interact with Cbl. To assess these interactions, EE-tagged versions of Ruk L, M or S were cotransfected into Hek293 cells with c-Cbl or vector alone. Immune complexes were precipitated with anti-EE Ig and analysed by Western blotting using anti-Cbl Ig. Figure 2A clearly demonstrates that all three isoforms of Ruk interact with exogenously expressed Cbl. Moreover, we also observed coimmunoprecipitation of endogenous Cbl with anti-EE Ig from cells, which were transfected with EE-tagged isoforms of Ruk. Observed interactions were also detected in reciprocal experiments, when anti-Cbl immunoprecipitates were probed in Western blotting with anti-EE Ig. Ruk S is detected only on a much longer exposure. (Fig. 2B). We have also noted that the longest isoform of Ruk (Ruk L) exhibits the strongest association with endogenous and exogenously expressed Cbl. The association of Ruk M and Ruk S with c-Cbl was quite unexpected, as these isoforms do not possess the two N-terminal SH3 domains, found previously to mediate the interaction with c-Cbl [12,18]. Heterodimerization of Ruk L with Ruk M and Ruk S All Ruk isoforms share a common C-terminal region which possesses a coiled-coil domain. Numerous studies have implicated coiled-coil domains in mediating protein–protein interaction via homodimerizations and heterodimerizations [21]. Taking this into account, we speculated that associ- ation of Ruk M and Ruk S with Cbl is not direct, but could be mediated via heterodimerization with the longest isoform, Ruk L. In order to test this hypothesis, we cotransfected nontagged Ruk L together with each EE-tagged Ruk isoforms. Following anti-EE Ig immuno- precipitations, immune complexes were subjected to West- ern blot analysis with the C-terminal anti-Ruk Ig, which recognizes all three isoforms. As shown in Fig. 3A, untagged Ruk L specifically coimmunoprecipitates with all three EE-tagged isoforms. The expression of EE-tagged Ruk isoforms in transfected cells was confirmed by immuno- blotting of total cell lysates with anti-EE antibodies (Fig. 3B). These data suggest that Ruk isoforms have the potential to form homodimers and heterodimers and that the coiled-coil domain mediates the formation of these complexes. Moreover, specific interaction between Cbl and two shorter isoforms of Ruk (Ruk M and Ruk S) was found to be mediated by heterodimerization with Ruk L. 3404 F. Verdier et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Ruk L is ubiquitinated but not degraded by proteasomes Ubiquitination, in most cases, targets modified proteins for degradation by 26S proteasomes. In order to determine whether ubiquitination of Ruk isoforms would induce their degradation, a panel of proteosome- and lysosome-specific inhibitors has been used. It is well established that lactacys- tine and the peptide aldehyde ALLN inhibit proteasome- mediated proteolysis, causing an accumulation of proteins that are usually degraded by this pathway [22]. In contrast to lactacystine, which is a highly specific proteasomal inhibitor [23], ALLN inhibits also nonproteasomal proteases, such as calpains and cathepsins. Initially, we tested the stability of EE-tagged Ruk L transiently overexpressed in Hek293 cells. Two days after transfection, cells were treated with various inhibitors or with the vehicle alone. Equal amounts of total cell lysates Fig. 3. Heterodimerization of Ruk L with Ruk M and Ruk S. Hek293 cells were transfected with 1.5 lg of plasmid containing nontagged version of Ruk L cDNA, and cotransfected with the same quantity of either EE-tagged Ruk L, EE-tagged Ruk M, or EE-tagged Ruk S cDNA. As a control, Hek293 cells were transfected with an empty vector (NT). Cell lysates were immunoprecipitated with anti-EE antibody. The immunoprecipitates were analysed by Western Blot using anti-Ruk antibody raised against the last 17 C-terminal amino acids and thereby able to recognize all Ruk isoforms (Ruk L, EE-tagged Ruk L, EE-tagged Ruk M and EE-tagged Ruk S). Ruk L and EE-tagged Ruk L can be separated by SDS/PAGE, since their molecular masses differ from approximatly one kDa (A). An aliquot of each cell lysate was immunoblotted with anti-EE Ig to check the expression of EE-Ruk isoforms (B). The blot in the lower panel of (B) was exposed 10 times longer than the upper panel. Fig. 2. Ruk L, Ruk M and Ruk S coprecipitate with Cbl. Hek293 cells were transfected with 1.5 lg of plasmid containing EE-tagged RukL cDNA, EE-tagged Ruk M cDNA or EE-tag- ged Ruk S cDNA in the absence or presence of Cbl plasmid (1.5 lg). As a control, Hek293 cells were transfected with an empty vector (NT).Celllysatesweresplitintwoandwere immunoprecipitated either with anti-EE Ig (A) or with anti-Cbl Ig (B). The immunoprecipi- tates were subjected to immunoblotting with anti-Cbl Ig (A) or with anti-EE Ig (B). An aliquot of each cell lysate was immuno- blotted with anti-EE antibody to check expression of Ruk isoforms (C). Ó FEBS 2002 Ruk is ubiquitinated but not degraded by the proteasome (Eur. J. Biochem. 269) 3405 were separated by SDS/PAGE and immunoblotted with anti-EE Ig. No changes in the level of exogenously expressed Ruk L was detected, when the activities of proteosomal and lysosomal proteases were blocked by specific inhibitors (Fig. 4A). We reprobed the membrane with anti-(b-actin) Ig to confirm equal loading of proteins in each lane (Fig. 4A, lower panel). One can argue that only ubiquitinated form of Ruk L would be targeted for degradation and if this fraction is small, it might be difficult to detect the changes in the level of total Ruk L protein. To overcome this problem, we coexpressed EE-tagged Ruk L with HA-Ub in Hek293 cells. Then, cells were treated with proteosomal inhibitor ALLN or vehicle alone. As shown in Fig. 4B, no accumu- lation of ubiquitinated EE-Ruk L is detected in the presence of ALLN. We then measured the half-life of endogenous Ruk L, which is expressed at high level in Hek293 cells. In most cases, the half-life of ubiquitinated proteins is short due to their rapid degradation by the proteasome. Time-course treatment of cells with a protein synthesis inhibitor cyclo- heximide showed no variations in the level of endogenous Ruk L, suggesting a long half-life for the protein (Fig. 4C). Furthermore, we examined the effect of selected inhibitors of protein degradation on the level of endogenous Ruk L. As can be seen in Fig. 4D, neither ALLN nor lactacystine induce any accumulation of endogenous Ruk L in Hek293 cells, even 6 h after treatment. These results were also confirmed in human monocytic cell line U937 (data not shown). To verify that proteolytic activities of proteasomes were effectively inhibited by the use of indicated inhibitors, we checked the expression level of b-catenin, which is degraded via the Ub-proteasome pathway [24,25]. Re-probing of the membrane with anti-(b-catenin) Ig clearly demonstrated the accumulation of ubiquitinated forms of b-catenin upon ALLN and lactacystine treatment (Fig. 4D, lower panel). No effect on the stability of Ruk L protein was also observed when cells were treated with lysosomal inhibitors: NH 4 Cl or chloroquine 7 (Fig. 4D). Taken together, these experiments clearly demonstrate that neither exogen- ously expressed nor native Ruk L isoform is degraded via proteasome or lysosome pathways. DISCUSSION Ubiquitination is now recognized as a regulatory protein modification whose functional significance is comparable to that of phosphorylation. Degradation of cellular proteins by the ubiquitin system encompasses two successive steps: (a) covalent attachment of ubiquitin molecules to selected proteins; and (b) degradation of ubiquitin-conjugated Fig. 4. Exogenously expressed EE-Ruk L (A,B) or Endogenous Ruk L (C,D) are not degraded by the proteasome. (A) Hek293 cells were transfected with 2 lg of plasmid containing EE-tagged Ruk L cDNA. After 48 h, cells were incubated for the time indicated with various inhibitors or with vehicle alone [Final concentrations: 50 l M for ALLN, 50 l M for Lactacystin (Lacta), 20 m M for NH 4 Cl and 200 l M for chloroquine (Chloro)]. Cells were lysed and the quantity of protein measured by Bradford assay. 10 lg of total protein from each cell lysate was separated by SDS/PAGE. The level of exogenous EE-Ruk L was determined by Western Blot analysis using anti-EE Ig (upper panel). The membrane was reprobed with anti- (b-actin) Ig to confirm equal loading of protein in each lane (bottom panel). (B) Hek293 cells were cotransfected with 1.5 lg of plasmid containing EE-tagged Ruk L cDNA and 1.5 lg of Ub-HA plasmid. After 48 h, cells were incubated for 3 h with (+) or without (–) ALLN. Cell lysates were immunoprecipitated with anti-EE Ig. The immunoprecipitates were subjected to immunoblotting with anti-HA Ig. The position of polyubiqui- tinated Ruk species [(Ub)n-Ruk] are indicated. The arrowheads mark the locations of unmodified EE-Ruk L. (C) Hek293 cells were incubated with 500 l M Cycloheximide (CHX) for the times indicated. As described in (A), 30 lg of proteins from the total cell lysates was separated by SDS/PAGE electrophoresis, and the expression level of endogenous Ruk L determined by Western blot (WB) using anti-Ruk Ig (upper panel). The membrane wasreprobedwithanti-(b-actin) Ig to confirm equal loading of protein in each lane (lower panel). (D) Hek293 cells were incubated for the time indicated with various inhibitors or with vehicle alone as described for panel A. Cells were lysed and the quantity of proteins measured by Bradford assay. 30 lg of proteins from the total cell lysates was separated by SDS/PAGE electrophoresis. The expression level of endogenous Ruk L was determined by Western blot analysis using anti-Ruk Ig (upper panel). The membrane was reprobed with anti-(b-catenin) Ig (bottom panel). The position of polyubiquitinated b-catenin species [(Ub)n-b-catenin] are indicated. The arrowheads mark the locations of unmodified b-catenin. 3406 F. Verdier et al. (Eur. J. Biochem. 269) Ó FEBS 2002 proteins. Many ubiquitinated proteins are targeted for degradation by 26S proteasomes, but some undergo endocytosis, leading to proteolysis in the lysosome. New findings show that ubiquitination is not always associated with the degradation of modified proteins, but could be also involved in regulation of enzymatic activities and intranu- clear trafficking of tagged proteins [26]. The data presented in this study clearly demonstrate that a recently identified adaptor-type protein Ruk L, also known as Cin85 or SETA, is ubiquitinated in vivo. Furthermore, we showed that shorter splicing variants of Ruk, termed Ruk M and Ruk S, are also modified by covalent attachment of ubiquitin. Ubiquitination of all three isoforms of Ruk indicates that ubiquitin conjugation occurs at the C-terminal region, which is common between them. It is well established that ubiquitin is conjugated to target proteins through lysine residues. Sequence analysis of Ruk isoforms showed that their common C-terminal region contains numerous lysine residues, which could be potential sites for ubiquitination. The identification and characteri- zation of Ruk ubiquitination sites is currently in progress. Recently, specific association between Cbl and Cin85/ SETA was demonstrated [12,18]. This interaction was found to be mediated by the first two SH3 domains of Cin85/ SETA and the proline-rich region of Cbl. In addition to that, constitutive binding between both proteins was further induced by stimulation of cells with EGF and found to be dependent on tyrosine phosphorylation of the C-terminal region of Cbl ([12] and our data, not shown). The same mode of interaction has been recently reported between Cbl and CMS/CD2AP, which requires the same domains and is also regulated by tyrosine phosphorylation of Cbl [27]. It is believed that tyrosine phosphorylation at the C-terminus of Cbl induces a conformational change of the protein from a closed to an open conformation, thereby unmasking putative SH3-domain motifs. In agreement with these findings, we show, in vivo,an interaction between Cbl and the longest isoform of Ruk, Ruk L. Furthermore, specific association of Cbl with Ruk M and Ruk S was also demonstrated. As neither Ruk M nor Ruk S possess the first two SH3 domains, which are involved in complex formation with Cbl, the mechanism of these interactions has been investigated. We found that the C-terminal region, which contains a coiled- coil domain common to all Ruk isoforms, is responsible for their heterodimerization. These results suggest that binding between Cbl/Ruk M and Cbl/Ruk S are indirect and require heterodimerization with Ruk L. As all isoforms are in complex with Cbl and are ubiquitinated, Cbl is a potential E3 ligase responsible for ubiquitination of Ruk isoforms. This hypothesis is currently under investigation. The discovery of Ruk isoform ubiquitination in cells prompted us to investigate the importance of this modifi- cation, especially in regulating its stability. Using proteo- somal and lysosomal inhibitors, we found that ubiquitination of exogenously expressed and native Ruk L does not induce its degradation via these proteolytic pathways. If ubiquitination of Ruk L is not a signal for its degradation, what is the role of this post-translational modification? Proteolysis-independent regulation by ubiquitination has recently been reported in several systems. Ubiquitination of a number of cell surface receptors in response to ligand binding serves as an internalization signal [28]. Moreover, ubiquitination-dependent processing of precursor proteins [29] and the regulation of multienzyme complex formation have also been described [30]. An interesting paper by Fang et al. demonstrates that like Ruk L, the P85 regulatory subunit of PtdIns-3 kinase is ubiquitinated, but not degraded by the proteasome pathway [31,32]. In addition, Cbl was shown to be the E3 ligase responsible for P85 ubiquitination and to regulate the recruitment of PtdIns-3 kinase to CD28 and T cell antigen receptor complexes, thereby inhibiting PtdIns-3 kinase activation. Two contradictory mechanisms have been proposed for Cbl binding to the P85 subunit: one involves the proline-rich domain of Cbl and the SH3 domain of P85 and while the other implicates a phosphorylated C-terminal tyrosine (Y731) of Cbl and an undefined domain of P85 [32,33]. We previously reported specific association between Ruk L and the P85 regulatory subunit of PtdIns-3 kinase, which is mediated via proline-rich sequences and the SH3 domain, respectively [11]. This interaction is not inducible by growth factor stimulation and has an inhibitory effect on the activity of PtdIns-3 kinase. The mechanism by which PtdIns-3 kinase could be released from the inhibitory complex with Ruk L is still unknown. Ruk L polyubiquiti- nation could induce conformational changes in the molecule which may modify its binding specificity towards the p85 subunit of the PtdIns-3 Kinase. We are currently investi- gating whether ubiquitination of Ruk L and P85 could affect the association between them and the activity of PtdIns-3 kinase. The use of in vitro binding and ubiquiti- nation assays will allow us to better understand the mechanism of the interaction between Ruk L, E3 ligase Cbl and PtdIns-3 kinase. ACKNOWLEDGEMENTS Fre ´ de ´ rique Verdier is supported by an EMBO (European Molecular Biology Organization) Fellowship. We are grateful to Mark Griffin for expert technical assistance and to H. Rebholz and T. Fenton for critical reading of the manuscript. 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