Cbl-b, a RING-type E3 Ubiquitin Ligase, Targets Phosphatidylinositol 3-Kinase for Ubiquitination in T Cells*

Cbl-b is implicated in setting the threshold of T lymphocyte activation. In Cbl-b-deficient T cells, the activation of Vav, a guanine nucleotide exchange factor, is significantly enhanced. The molecular mechanism underlying Cbl-b-regulated Vav activation was unclear. Here it is shown that Cbl-b interacts with and induces ubiquitin conjugation to the p85 regulatory subunit of phosphatidylinositol 3-kinase, an upstream regulator of Vav. A functional RING finger of Cbl-b was essential for p85 ubiquitination. However, a loss of function mutation at the well-conserved amino-terminal variant src homology (SH) 2 domain of Cbl-b did not affect its ligase activity. A distal carboxyl-terminal proline-rich region in Cbl-b was mapped to contain the primary binding sequences for the SH3 domain of p85. Deletion of either the distal proline-rich region in Cbl-b or the SH3 domain of p85 severely reduced ubiquitin conjugation to p85. The data suggest a molecular link for Cbl-b-mediated negative regulation of Vav, with phosphatidylinositol 3-kinase as a direct target for Cbl-b E3 ubiquitin ligase.

Cbl-b and its close mammalian homologue, Cbl, consist of an amino-terminal variant SH2 1 domain, a RING finger, and a carboxyl-terminal proline-rich domain with potential tyrosine phosphorylation sites (1,2). Previous studies have shown that Cbl-b and Cbl function as adaptor proteins by interacting with othercriticalsignalmolecules,includingthevariantSH2domaindependent interaction with cell surface receptor tyrosine kinases or intracellular protein tyrosine kinases such as Syk and Zap-70 and the carboxyl-terminal region-dependent interaction with Grb2, 14-3-3, phosphatidylinositol 3-kinase (PI3-K), Vav, and Crk-L (3,4). Genetic and biochemical 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. One mechanism for this negative role is Cbl-mediated ubiquitination of receptor tyrosine kinases (5)(6)(7). It is now understood that Cbl functions as an E3 ubiquitin (Ub) ligase whose RING finger recruits a Ub-conjugating enzyme, E2, and whose SH2 domain recognizes activated receptor tyrosine kinases for Ub conjugation (7)(8)(9)(10).
Ubiquitination is an important cellular process that involves ligation of a protein substrate with Ub, thereby marking it for degradation by the 26S proteasome (11)(12)(13), and it involves a cascade of reactions including E1, E2, and E3 enzymes. Ub is first activated by an activating enzyme (E1) to form a high energy thiolester bond between Ub and E1 and is then transferred to a conjugating enzyme (E2). The E3s or Ub protein ligases are the components responsible for specific substrate recognition and for promoting Ub ligation to the target protein.
Therefore, the E3s can provide specificity to the Ub system. Two recent genetic studies using Cbl-b gene-targeted mice showed that Cbl-b deficiency can change the signaling thresholds: Cbl-b deficiency uncouples T-cell proliferation and interleukin 2 production from the costimulation of CD28, and the gene-targeted mice develop spontaneous autoimmunity or become highly susceptible to exogenous antigen-induced autoimmune diseases (14,15). These studies suggest a critical role of Cbl-b in the regulation of T-cell activation thresholds and hence in the maintenance of a balance between immunity and tolerance. In Cbl-b-deficient T cells, the tyrosine phosphorylation and/or activation of Vav, a GDP/GTP exchange factor, are significantly enhanced (14,15), suggesting that Cbl-b negatively regulates T-cell signaling by inhibiting Vav activation. However, the molecular mechanism underlying Cbl-b-mediated negative regulation of Vav remains to be determined.
Previous studies have shown that PI3-K, which phosphorylates PI lipid at the D3 position of the inositol ring to form active lipid second messengers, regulates the exchange activity of Vav through the binding of the active lipid products to the pleckstrin homology (PH) domain of Vav (16,17). We have investigated whether Cbl-b acts as an E3 Ub ligase to promote ubiquitination of PI3-K. Here we show that Cbl-b binds p85, the regulatory subunit of PI3-K, and induces its ubiquitination. We propose a model by which Cbl-b indirectly regulates Vav activation by inducing ubiquitination of PI3-K.

EXPERIMENTAL PROCEDURES
Antibodies-The polyclonal anti-Cbl-b and anti-Fyn antibodies and anti-c-Myc, anti-HA, and anti-Vav monoclonal antibodies (mAbs) were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-p85 antibody was from UBI (Lake Placid, NY). Anti-Xpress mAb was from Invitrogen (Carlsbad, CA). Anti-human CD3 mAb OKT3 was purified from ascites using a protein G-Sepharose affinity column.
Plasmids-The Cbl-b cDNAs encoding full-length Cbl-b, a Cbl-b amino-terminal region (a.a. 1-349; Cbl-b 1-349), and Cbl-b G298E, with a HA or Xpress epitope in an elongation factor promoter-driven mammalian vector (pEFneo), were as described previously (18,19). The Cbl-b truncated constructs containing the RING finger and the carboxylterminal proline-rich sequences (a.a. 341-982), the entire carboxylterminal proline-rich sequences alone (a.a. 443-982), the distal prolinerich sequences (a.a. 595-982), the amino-terminal variant SH2 domain and RING finger (a.a. , or an addition of the proximal prolinerich domains to Cbl-b 1-481 (a.a. 1-595) were amplified by polymerase chain reaction, tagged with a Xpress or HA epitope, and subcloned into the pEFneo plasmid. Point mutations, which replaced cysteine 372 with alanine (C372A) and tryptophan 400 with alanine (W400A) in the RING finger of the full-length Cbl-b, were made by site-directed mutagenesis (QuickChange; Stragagene, La Jolla, CA). The Myc-tagged Ub cDNA from a HA-tagged Ub plasmid (20) was generated by polymerase chain reaction and subcloned into pEFneo. The p85␣ plasmid with or without a HA tag, the p85␣ SH3 domain with a HA tag, and the HA-tagged p110␣ plasmid of PI3-K in pEFneo were provided by T. Mustelin (La Jolla Institute for Allergy and Immunology, San Diego, CA). An SH3 domain-deficient p85 (p85⌬SH3) was constructed by subcloning the BglII and XbaI fragment of p85 into pEFneo with a HA tag. The Vav plasmid in pEFneo was from A. Altman (La Jolla Institute for Allergy and Immunology, San Diego, CA).
Immunoprecipitation and Immunoblotting-For immunoprecipitation, lysates (ϳ1 ϫ 10 7 cells) were mixed with antibodies (1 g) for 2 h, followed by the addition of 30 l of protein G-Sepharose beads (Santa Cruz Biotechnology) for an additional 2 h at 4°C. Immunoprecipitates were washed four times with 1ϫ Nonidet P-40 lysis buffer and boiled in 20 l of 2ϫ Laemmli's buffer. Samples were subjected to 8% or 10% SDS-polyacrylamide gel electrophoresis analysis and electrotransferred onto polyvinylidene difluoride membranes (Millipore). Membranes were probed with the indicated primary antibodies (usually 1 g/ml), followed by horseradish peroxidase-conjugated secondary antibodies. Membranes were then washed and visualized with an enhanced chemiluminescence detection system (ECL; Amersham Pharmacia Biotech). When necessary, membranes were stripped by incubation in stripping buffer (62.5 mM Tris-HCl, pH 5.7, 100 mM 2-mercaptoethanol, and 2% SDS) for 1 h at 70°C with constant agitation, washed, and then reprobed with other antibodies as indicated.

Cbl-b Induces p85
Ubiquitination-Classical PI3-K is composed of a p85 regulatory subunit and a p110 catalytic subunit. The p85 subunit contains an amino-terminal SH3 domain and two carboxyl-terminal SH2 domains to mediate protein-protein interactions and an interdomain between the two SH2 domains for association with the p110 subunit (21). Previous studies have shown that Cbl-b associates with PI3-K (14,18). The recent discovery that Cbl functions as a RING-type E3 ligase for receptor tyrosine kinases prompted us to investigate whether PI3-K could be a target protein for Cbl-b-induced ubiquitination. As the first step, we examined whether p85 is ubiquitinated in vivo by transient overexpression of p85 and Myc epitope-tagged Ub in Jurkat T cells. As shown in Fig. 1A, transient overexpression of p85 with Myc-Ub but not p85 alone resulted in the formation of a slowly migrating species of p85 that was recognized by anti-Myc antibody. Treatment of T cells with pervanadate did not enhance Ub conjugation to p85. However, the ubiquitinated forms of p85 were enhanced by further addition of cells with MG132, a proteasome inhibitor (22).
We then examined whether Cbl-b induces Ub conjugation to p85. Cbl-b was coexpressed with p85 together with Myc-Ub in Jurkat T cells, and the transfected cells were immunoprecipitated with anti-p85 antibody. The immunoprecipitates were analyzed using an anti-Myc antibody to detect ubiquitinated p85. Coexpression with Cbl-b markedly enhanced ubiquitinated forms of p85 (Fig. 1B), which were further enhanced by pretreatment with MG132. To rule out the possibility that the ubiquitinated species results from coimmunoprecipitated proteins other than p85, we added 0.1% SDS to the lysis buffer to reduce nonspecific protein-protein interaction. An additional control was included by using normal rabbit serum for immunoprecipitation, and it did not precipitate slowly migrating species (data not shown). The data indicate that the p85 subunit of PI3-K is polyubiquitinated and that Cbl-b can act as an E3 ligase for p85.
Cbl-b RING Finger but not the Variant SH2 Domain Is Essential for Its Ligase Activity-Recent studies including our own (7-9) have shown that Cbl RING finger recruits a Ubconjugating enzyme E2 and that an intact RING finger is required for E2 binding and its E3 ligase activity. Specifically, mutation of the conserved cysteine 381 to alanine (Cbl C381A) disrupts its E2 binding and ligase activity (8). We then examined whether mutation of the conserved cysteine 372 to alanine in Cbl-b (Cbl-b C372A), which is equivalent to the Cbl C381A mutation, affected its ligase activity toward p85 ubiquitination. Coexpression of Cbl-b C372A completely abolished its ability to promote p85 ubiquitination ( Fig. 2A). Analysis of the cell lysates showed comparable levels of protein expression between wild-type Cbl-b and the C381A mutant.
Previous studies have documented the importance of the Cbl amino-terminal variant SH2 domain and RING finger in mediating ubiquitination of receptor tyrosine kinases (7,8,23), thus supporting a model in which the amino-terminal variant SH2 domain specifically targets the substrates. Consistent with this, a loss of function mutation (G306E) at this domain, which disrupts the association of Cbl with the tyrosine kinases, can also abrogate Cbl-induced ubiquitination of the receptor tyrosine kinases (7,23). Next, we examined the functional role of the variant SH2 domain of Cbl-b in p85 ubiquitination. A loss of function mutation of glycine 298 to glutamic acid in Cbl-b (Cbl-b G298E), which is equivalent to the Cbl G306E mutation, was previously shown to disrupt the interaction with Zap-70 (19). However, the same mutation did not affect its ability to induce p85 ubiquitination because both the wild-type Cbl-b and Cbl-b G298E induced the formation of slowly migrating p85 species to a similar degree (Fig. 2B). Analysis of the cell lysates showed equivalent amounts of protein expression between the wild-type Cbl-b and the G298E mutant. The result indicates that the evolutionarily conserved variant SH2 domain in Cbl-b is not essential for p85 ubiquitination.
Cbl-b Physically Associates with p85-E3 ligases confer specificity to the Ub system by directly interacting with the substrate proteins and help transfer Ub to them (24). An interaction between Cbl-b and PI3-K has been demonstrated in primary T cells and in Jurkat T cells (14,18), and this interaction is not mediated by the two SH2 domains of p85 (18). To further confirm this interaction, we cotransfected HA epitopetagged Cbl-b with p85 in Jurkat T cells. HA-Cbl-b was coimmunoprecipitated with p85 (Fig. 3A) and with Grb2, as de-scribed previously (18). We then examined the effect of RING finger mutation on p85 binding. Jurkat T cells were cotransfected with HA-tagged p85 with Xpress-tagged Cbl-b or the Cbl-b RING finger mutant C372A. Cbl-b C372A showed similar binding to p85 or to Grb2 as wild-type Cbl-b (Fig. 3B), indicating that the observed loss of Ub ligase activity toward p85 in the Cbl-b C372A mutant is not due to the disruption of its interaction with p85. To examine whether the interaction is activation-dependent, transfected cells were then stimulated with anti-CD3 antibody at different intervals, and the cell lysates were immunoprecipitated with anti-HA antibody. p85 was coimmunoprecipitated with Cbl-b, and that did not change much upon stimulation (Fig. 3C), suggesting that the interaction is constitutive. Next, we analyzed whether Cbl-b-induced ubiquitination of p85 is dependent on T-cell receptor engagement. Stimulation of Jurkat T cells with anti-CD3 antibody for different time periods up to 1 h did not increase Ub conjugation to p85 in either the absence or presence of Cbl-b overexpression (Fig. 3C). The data suggest that Cbl-b interacts with p85 and promotes its ubiquitination in an activation-independent manner.

A Distal Proline-rich Region of the Cbl-b Carboxyl-terminal Region Is Required for the Efficient Interaction with p85 and Its
Ub Conjugation-The interaction region in Cbl-b was then determined by coexpressing p85 with either a HA-tagged amino-terminal region (Cbl-b 1-349) or a construct that contains the RING finger and the carboxyl-terminal region (Cbl-b 341-982). Deletion of the RING finger and the carboxyl-terminal region from Cbl-b abrogated its interaction with p85, as shown in the Cbl-b 1-349 mutant (Fig. 4A). However, the Cbl-b 341-982 mutant still retained its ability to associate with p85 to the same degree as full-length Cbl-b (Fig. 4A).
The Cbl-b carboxyl-terminal-dependent interaction with p85 suggests that this region is responsible for the substrate targeting, at least for p85 protein, and is probably responsible for its ubiquitination. We tested this hypothesis by coexpressing wild-type Cbl-b, Cbl-b 1-349, or Cbl-b 341-982 with p85. Although Cbl-b 1-349 did not enhance Ub conjugation to p85 over the basal level, Cbl-b 341-982 and full-length Cbl-b showed similar ability to induce p85 ubiquitination (Fig. 4B). The data collectively suggest that the RING and the carboxyl-terminal region are sufficient for p85 binding and ubiquitination.
We tried further to determine the binding region of p85 in Cbl-b. To this end, we generated two Cbl-b constructs containing Cbl-b amino-terminal variant SH2 domain plus the RING finger (Cbl-b 1-481) or Cbl-b 1-481 plus the proximal prolinerich domain (Cbl-b 1-595), as depicted in Fig. 5A. Consistent with our data that carboxyl-terminal region is required for p85 ubiquitination (Fig. 3C), deletion of the entire carboxyl-terminal region (Cbl-b 1-481) completely abrogated its interaction with p85, and deletion of the distal proline-rich sequences severely reduced its interaction with p85 (Fig. 5B).
We further mapped the p85 binding region in the Cbl-b carboxyl-terminal portion. There are more than 10 proline-rich stretches spanning the Cbl-b carboxyl-terminal region (2). Two cDNA fragments, which encode proteins encompassing the entire proline-rich sequences from a.a. 443 to a.a. 982 or the distal proline-rich region from a.a. 595 to a.a. 982, were generated and tagged with a HA epitope. The interaction of these two proline-rich domains with p85 was analyzed by coimmunoprecipitation. Both fragment proteins bound to p85 to a similar degree (Fig. 5C) under both resting and pervanadate-stimulated conditions, suggesting that the proximal proline-rich sequences are less important for p85 interaction.
To further confirm that the distal proline-rich sequences are the primary binding region for p85, we compared the p85 interaction of the full-length Cbl-b with that of the 595-982 (a.a. 595-982) mutant. The 595-982 mutant and wild-type Cbl-b showed comparable binding to p85 (Fig. 5D). Reprobe of the same membrane showed equivalent amounts of proteins for wild-type Cbl-b and the 595-982 mutant. The data are consistent with the idea that the distal proline-rich region contains the primary binding sequences for p85.
A functional role for the carboxyl-terminal proline-rich region of Cbl-b was assessed in p85 ubiquitination. Removal of the distal proline-rich sequence in Cbl-b (Cbl-b 1-595), which reduced its interaction with p85 (Fig. 5B), severely hampered its ability to promote p85 ubiquitination (Fig. 6A). The slight augmentation of p85 ubiquitination could be due to the residual binding of the 1-595 mutant to p85, as shown in Fig. 5B. Analysis of the cell lysates showed equivalent amounts of protein expression for wild-type Cbl-b and the 1-595 mutant (Fig.  6B). We conclude that the Cbl-b distal proline-rich sequences are required for both efficient p85 binding and its ubiquitination.
The SH3 Domain of p85 Binds to Cbl-b and Is Required for p85 Ubiquitination-The p85 subunit of PI3-K contains an SH3 domain at the amino terminus. The constitutive interaction between p85 and the distal proline-rich region of Cbl-b suggests that the SH3 domain of p85 mediates the interaction with Cbl-b. We then generated a p85 construct with the SH3 domain deleted (p85⌬SH3) and analyzed whether deletion of the SH3 domain of p85 could affect its interaction with Cbl-b. HA-p85 or HA-p85⌬SH3 was coexpressed with Xpress-tagged Cbl-b. The protein-protein interaction was analyzed by coimmunoprecipitation with anti-Xpress antibody. Removal of the SH3 domain from p85 completely abolished its interaction with Cbl-b, although Cbl-b associated with the full-length p85 and Grb2 under the same conditions (Fig. 7A).
The p85 SH3 domain-dependent interaction with the distal proline-rich sequences of Cbl-b and the defect of p85 ubiquitination by the Cbl-b mutant lacking the distal proline-rich domain suggest that the SH3 domain of p85 is required for its ubiquitination. To test this hypothesis, we examined the Cblb-promoted ubiquitination of p85 or p85⌬SH3 in Jurkat T cells. Under a condition that coexpression of Cbl-b with the fulllength p85 induced Ub conjugation to p85, deletion of the SH3 domain in p85 severely reduced its ubiquitination induced by Cbl-b, even with MG132 treatment (Fig. 7B). Taken together, the results indicate that the p85 SH3 domain is required for Cbl-b interaction and for Cbl-b-promoted ubiquitination of p85. DISCUSSION The results in this report demonstrated that Cbl-b acts as an E3 Ub ligase by interacting with the p85 regulatory subunit of PI3-K and promoting its ubiquitination. A functional RING finger is essential for the Ub ligase activity toward p85, whereas the well-conserved amino-terminal variant SH2 domain is dispensable for this event. We further demonstrated that the distal proline-rich sequences in Cbl-b and the SH3 domain of p85 are required for the efficient interaction and subsequent Ub conjugation to p85. Our data suggest a novel role for Cbl-b in regulating T-cell activation by inducing p85 ubiquitination.
Recent genetic studies on Cbl-b-deficient T cells showed that the activation of Vav is increased in these cells, which was proposed to be responsible for costimulation-independent cytokine production and cell proliferation (14,15). This finding is supported by a study showing that coexpression of Cbl-b and Vav inhibits the nucleotide exchange activity of Vav (25). The recent findings, including our own, showing that Cbl functions as an E3 ubiquitin ligase (7-9) prompted us to investigate a potential role of Cbl-b on Vav ubiquitination. Our efforts failed to prove that Vav is a direct target for Cbl-b E3 Ub ligase activity, even after repeated experiments (data not shown). A recent study showed that Vav could be ubiquitinated by the suppressor of cytokine signaling 1, a component of elongin Ub ligase complex. Suppressor of cytokine signaling 1 interacts with Vav and induces its ubiquitination and degradation (26). Although it cannot be ruled out that Vav is a target for Cbl-b E3 ligase activity, we favor a model in which suppressor of cytokine signaling 1 is the primary E3 ligase component for Vav, and Cbl-b may indirectly regulate Vav activation.
We then investigated the possibility that Cbl-b regulates the upstream regulators for Vav activation. One of the well-established regulators is PI3-K, whose lipid product can bind to the pleckstrin homology domain of Vav and activates Vav nucleotide exchange activity (16,17). We demonstrated that Cbl-b induces Ub conjugation to p85, the regulatory subunit of PI3-K, which is dependent on its RING finger domain and distal proline-rich sequences, suggesting that Cbl-b acts as an E3 Ub ligase for p85. Besides PI3-K, Src kinases can also regulate the tyrosine phosphorylation of Vav in synergy with PI3-K (16). Although we did not address a potential role for Cbl-b on Src kinases in this study, our data support a model in which Cbl-b indirectly regulates Vav activation by promoting Ub conjugation to p85.
Previous studies on Cbl have focused on the functional role of its amino-terminal variant SH2 domain in targeting the substrate proteins such as cell surface receptor tyrosine kinases (5)(6)(7)(8)(9)23). A loss of function mutation (G306E) at this domain of Cbl, which disrupts its interaction with receptor tyrosine ki-nases, can also abrogate its Ub ligase activity toward these kinases (7,23). However, a corresponding mutation at Cbl-b (G298E) did not affect its activity to promote p85 ubiquitination, suggesting that the amino-terminal variant SH2 domain of Cbl-b is dispensable for Ub conjugation to p85. Rather, we showed that the carboxyl-terminal proline-rich region and, more specifically, the distal proline-rich sequences of Cbl-b are required for efficient p85 ubiquitination. Thus, our results suggest a novel mechanism by which the Cbl-b carboxyl-terminal region can also recruit target proteins and, with the help of the RING finger, induce the transfer of Ub to them.
Our finding that the Cbl-b carboxyl-terminal region can recruit protein substrates is consistent with the recent crystal structural study on Cbl RING finger and an E2, Ub-conjugating enzyme H7 (10). In this complex, the active cysteine of Ub-conjugating enzyme H7, which forms a thiol-ester bond with Ub, is on the opposite side of the complex relative to the binding site for tyrosine kinase binding domain (amino-terminal domain). Therefore, a substrate that associates with the carboxyl-terminal region would presumably allow an easy transfer of Ub to the substrate. To further support our model, we recently found that the Cbl carboxyl-terminal region could also recruit protein substrates such as Stat5 and that the RING finger and the carboxyl-terminal proline-rich region are sufficient for promoting Stat5 ubiquitination. 2 In fact, the Cbl family proteins including those from C. elegans or Drosophila are well conserved in the amino-terminal variant SH2 domain and the RING finger domain (3), whereas the carboxyl-terminal regions between Cbl and Cbl-b are more divergent, and some Cbl family members (Sli-1, D-Cbl, and Cbl-3) lack or contain only a short form of the carboxyl-terminal proline-rich region. Because both Cbl and Cbl-b carboxyl-terminal regions contain several protein interaction motifs, it is conceivable that differential recruitment of protein substrates by the carboxyl-terminal region would provide diversity and specificity to Cbl-and/or Cbl-b-mediated protein ubiquitination.
The SH3 domain of p85 recognizes a consensus proline-rich motif: RXXRPLPPLPP (28), which is also present in p85 itself.