p62, a phosphotyrosine-independent ligand of the SH2 domain of p56lck, belongs to a new class of ubiquitin-binding proteins.

p62 is a novel cellular protein which was initially identified as a phosphotyrosine-independent ligand of the SH2 domain of p56lck. In the yeast two-hybrid system, p62 specifically interacted with ubiquitin in vivo. Furthermore, p62 bound to ubiquitin-conjugated Sepharose beads in vitro and was efficiently competed by soluble ubiquitin. The interaction was independent of ATP hydrolysis, and its dissociation did not require a reducing agent. Thus, p62 binds to ubiquitin noncovalently. Further analysis showed that the C-terminal 80 amino acids of p62 were indispensable for its interaction with ubiquitin. However, p62 has homology neither with ubiquitin C-terminal hydrolases nor with the S5a subunit of the 26 S proteasome complex, the only proteins known to bind to ubiquitin noncovalently. These results suggest that p62 belongs to a new class of ubiquitin-binding proteins and that p62 affects signal transduction at least partly through ubiquitination-mediated protein degradation.

p62 is a novel cellular protein which was initially identified as a phosphotyrosine-independent ligand of the SH2 domain of p56 lck . In the yeast two-hybrid system, p62 specifically interacted with ubiquitin in vivo. Furthermore, p62 bound to ubiquitin-conjugated Sepharose beads in vitro and was efficiently competed by soluble ubiquitin. The interaction was independent of ATP hydrolysis, and its dissociation did not require a reducing agent. Thus, p62 binds to ubiquitin noncovalently. Further analysis showed that the C-terminal 80 amino acids of p62 were indispensable for its interaction with ubiquitin. However, p62 has homology neither with ubiquitin C-terminal hydrolases nor with the S5a subunit of the 26 S proteasome complex, the only proteins known to bind to ubiquitin noncovalently. These results suggest that p62 belongs to a new class of ubiquitinbinding proteins and that p62 affects signal transduction at least partly through ubiquitination-mediated protein degradation.
The lymphoid-specific Src family tyrosine kinase p56 lck has a Src homology 2 (SH2) domain and a Src homology 3 (SH3) domain that mediate protein-protein interactions between signaling proteins through direct binding to regions containing phosphotyrosine and proline-rich sequences, respectively (1,2). In addition to the well established protein phosphotyrosinedependent SH2 domain binding, interaction of SH2 domains with non-phosphotyrosyl proteins are also recognized (3,4). Recently, a specific phosphotyrosine-independent interaction of the Lck SH2 domain with a novel cytosolic 62-kDa protein (p62) has been identified (5) and its cDNA cloned (6). p62 expression in all human tissues tested (6) suggests that the enzymatic and/or regulatory functions of p62 are not restricted to the lymphoid-specific p56 lck SH2 domain binding. Rather, p62 may serve as a common signal transducer in cells and may use the Lck SH2 domain binding for T cell-specific signal transduction.
Recently, ubiquitination of cellular proteins has emerged as a crucial feature in regulation of signal transduction and cell cycle progression through ubiquitination-dependent prote-asomal degradation of important cellular proteins, including cell surface receptors such as platelet-derived growth factor receptor and T cell receptor chain, mitotic cyclins, Cdk inhibitor p27 kip , oncoproteins, transcriptional regulators such as IB, NF-B, c-Jun, and c-Fos, and the tumor suppressor p53 (reviewed in Ref. 7).
Proteins binding to ubiquitin can be grouped into three different categories based on their binding modes. (i) Proteins forming isopeptide bonds with ubiquitin: most proteasomal substrates are conjugated to multiubiquitins through isopeptide bond formation between the C-terminal glycine of ubiquitin and an ⑀-amino group of a lysine residue of the substrate (8). (ii) Proteins forming a thioester bond with ubiquitin: only the ubiquitination enzymes E1, E2, and E3 are known to form a thioester with ubiquitin (9). (iii) Proteins binding noncovalently to ubiquitin: all C-terminal hydrolases (deubiquitinating enzymes) and an S5 subunit of the 26 S proteasome bind noncovalently to ubiquitin (10 -12). Some studies suggest that E2 and E3 may also bind to ubiquitin noncovalently in addition to the thioester bond formation (13).
In the present study, a novel cellular protein, p62, has been shown to bind to ubiquitin noncovalently, in addition to its association with p56 lck (5,6). However, p62 has no homology to any known ubiquitin-binding protein. Thus, p62 may represent a new ubiquitin-binding protein that regulates signal-mediated ubiquitination and/or proteasomal degradation of cellular proteins.

EXPERIMENTAL PROCEDURES
Yeast Two-hybrid System-p62 fusion to the Gal4 binding domain (Gal4B-p62) was constructed using EcoRI and XhoI sites of pGBT9 vector (Clontech) and internal restriction sites (StuI-XhoI) of the p62 cDNA in which the EcoRI and StuI sites were fused by blunt end ligation. A HeLa cDNA library fused to the Gal4 activation domain (Gal4A) in pGADGH vector (Clontech) was introduced into Gal4B-p62 transformed yeast strain HF7c (MATa, his3 3-200, trp 1-901, leu 2-3, 112, LYS2::Gal1-His3, URA3::1(Gal4 17mers) 3 -CYC-lacZ). Approximately 10 6 transformants were plated on his Ϫ plates, and his ϩ colonies were isolated and tested for ␤-galactosidase activity using the filter assay (14). Plasmids were isolated from his ϩ lacZ ϩ colonies and transformed into an Escherichia coli Leu Ϫ strain of HB101 in order to rescue the plasmids of library origin. Specific interaction of p62 with the isolated plasmids was confirmed in a different yeast strain SFY 526 (MATa, his 3-200, trp 1-901, leu 2-3, 112, URA3::Gal1-lacZ) in which LacZ is under control of the Gal1 promoter which is different from the promoter used in HF7c strain. ␤-Galactosidase assay was performed both in SFY526 and HF7c strains after cotransformation of Gal4B-p62 with either the isolated plasmid or a negative control, Gal4A-Lamin. cDNA inserts of the positive plasmids were characterized by sequencing using a Sequenase Kit (U. S. Biochemical Corp.).
Ubiquitin Binding Assay-Bovine ubiquitin (Sigma) was conjugated to activated CH-Sepharose (Pharmacia Biotech Inc.) by following the manufacturer's protocol. Confluent HeLa cells in a 150-mm dish were washed with phosphate-buffered saline, lysed in 1 ml of lysis buffer (25 mM Tris, pH 7.5, 100 mM NaCl, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride). A ubiquitin-free and p62-enriched fraction (Fraction 2) was prepared by one-step ion-exchange chromatography using a DE52 column and an elution buffer (25 mM Tris, pH 7.5, 500 mM KCl, Ref. 15). Fraction 2 was diluted 1:4 with Tris buffer (25 mM Tris, pH 7.5, 5 mM MgCl 2 , and 1 mM ATP) and used for further assay. ATP was omitted in some experiments as indicated in the figure legends. For the ubiquitin binding assay, ubiquitin-coupled Sepharose (Ub 1 -Sepharose) beads (25 l) were added to the diluted fraction 2 and incubated for an hour at 4°C. After washing the beads 4 times with lysis buffer, bound proteins were eluted either with SDS buffer or with other buffers as indicated in figure legends. Presence of p62 in the eluates was analyzed by immunoblotting using a polyclonal anti-p62 antiserum (6) and horseradish peroxidase-conjugated goat anti-rabbit IgG.
Mutation and Expression of p62-Construction of wild type and deletion mutants of T7 epitope-tagged p62 (p62-T7) in pRSET vector were described elsewhere (6). These constructs were transfected into HeLa cells using Lipofectin (Life Technologies, Inc.), and expression of encoding proteins was induced by infection of vaccinia virus vTF7-3. After 24 h of infection, cells were washed with phosphate-buffered saline, and lysates were prepared as described.

RESULTS AND DISCUSSION
The yeast two-hybrid system was used to identify proteins that interact with p62. p62 cDNA fusion to the Gal4 DNA binding domain (Gal4B-p62) was used to screen a HeLa cDNA library fused to the GAL4 activation domain (Gal4A) using two reporter genes (his and lacZ) under the control of GAL4 upstream activating sequence. In a yeast strain HF7c, interaction of Gal4B-p62 with any protein fused to the Gal4 activation domain will activate transcription of the reporter genes. Initial screening of 10 6 transformants for activation of his and lacZ genes yielded 96 positive clones. Each plasmid from these positive clones was individually examined for its specific interaction with p62 in another yeast strain SFY526 that carries either Gal4B-p62 or a negative control Gal4B-Lamin. When transfected, 46 plasmids from the 96 his ϩ and lacZ ϩ colonies activated transcription of reporter genes in the cells carrying Gal4B-p62 but not in the cells carrying Gal4B-Lamin. Sequencing of the cDNA plasmids isolated from the 46 final positive clones revealed that the majority of them (43 of 46) belonged to the ubiquitin gene family.
These 43 ubiquitin genes whose products interacted with p62 consisted of three distinct groups; cDNAs encoding (i) diubiquitin (dUb), (ii) polyubiquitin (pUb), and (iii) a ubiquitin-conjugated ribosomal protein UBA52 (rUb) (16). The Gal4A fused with any member of these three groups of cDNAs (Gal4A-Ub) when coexpressed with Gal4B-p62 conferred the His ϩ phenotype, while Gal4B-p62 or any of ubiquitin genes alone did not induce the His ϩ phenotype (Fig. 1). A control bait Gal4B-Lamin, however, when coexpressed with any of those ubiquitin fusion genes, failed to induce the His ϩ phenotype. Furthermore, compared to various controls, coexpression of Gal4B-p62 and Gal4A-Ub in yeast specifically induced activation of ␤-galactosidase that results in blue colony formation (Table I). These results suggest that p62 interacted specifically with ubiquitin in any context, either in a monomeric or in an oligomeric form.
Interaction of p62 with ubiquitin was further confirmed by in vitro binding studies using ubiquitin-conjugated Sepharose beads (Ub-Sepharose). Proteins bound to Ub-Sepharose were precipitated and analyzed by Western blotting using anti-p62 antibody. Ub-Sepharose but not Sepharose beads alone precip itated p62 from HeLa cell lysates ( Fig. 2A). Furthermore, more than a half of p62 binding to Ub-Sepharose, which had been coupled in 10 M ubiquitin solution, was inhibited by the presence of 25 M soluble ubiquitin in the reaction mixture. These results show that p62 interacts with ubiquitin in vitro as well as in vivo in the yeast two-hybrid screen.
Binding mode of p62 to ubiquitin was then analyzed. p62 interacted with Ub-Sepharose in the absence of ATP equally as well as in its presence (Fig. 2B, lanes 2-5). Furthermore, p62 bound to Ub-Sepharose was eluted completely either by SDS sample buffer or by 1 M KCl in Tris buffer, pH 9.0, both under nonreducing conditions (Fig. 2B, lanes 6 -8). These results suggest that p62 binds noncovalently to ubiquitin, rather than forming a thioester or an isopeptide bond.
However, p62 has no homology with C-terminal ubiquitin hydrolases, with the E2 and E3 enzymes, or with the S5a FIG. 1. Interaction of p62 with ubiquitin in the yeast twohybrid system. Yeast strain HF7c was transformed with plasmids carrying Gal4B-p62, Gal4B-Lamin, or one of the Gal4A-ubiquitins either alone or in combination. Colonies containing different combinations of plasmids were streaked on leu Ϫ trp Ϫ or leu Ϫ trp Ϫ his Ϫ plates and incubated for at 30°C for 2 days. Lanes 1, 2, and 3 represent Gal4Adiubiquitin (dUb), Gal4A-polyubiquitin (pUb), and Gal4A-UBA52 (rUb) cDNA fusions, respectively, that were used for transformation.

FIG. 2. Interaction and binding mode of p62 with ubiquitin in vitro.
A, Fraction 2 of HeLa cell lysate was prepared as described under "Experimental Procedures" and incubated with Sepharose (Sph) or Ub-Sepharose beads (Ub-Sph). Proteins bound to the beads were analyzed by immunoblotting with anti-p62 antiserum. For competition assays, Fraction 2 was incubated with Ub-Sepharose beads in the presence of 0, 25, and 100 M soluble ubiquitin, respectively. B, Fraction 2 prepared from HeLa lysates was incubated with Sepharose (lane 1) or Ub-Sepharose beads (lanes 2-8), either in the presence (lanes 2 and 3) or absence of ATP (lanes 4 and 5). Bound proteins were eluted either with SDS sample buffer containing 100 mM DTT (lanes 1-3) or SDS sample buffer without DTT (lanes 4 and 5). The presence of p62 in the complex was analyzed by immunoblotting using anti-p62 antibody.  reporter gene Specificity of the interaction of p62 with ubiquitin in yeast was assayed using the 5-bromo-4-chloro-3-indolyl ␤-D-galactoside filter method as described under "Experimental Procedures." B, blue color colony; W, white color colony; dUb, ubiquitin dimer; pUb, ubiquitin polymer; rUb, ubiqutin ribosomal fusion.

Gal4B
Gal4B-p62 Gal4B-Lamin subunit of the 26 S proteasome. Furthermore, p62 did not have ubiquitin C-terminal hydrolase activity (data not shown). Thus, p62 belongs to a novel class of ubiquitin-binding proteins. Its function may be exerted through binding to ubiquitin, but it may not be directly involved as an enzyme in the ubiq-uitination and/or deubiquitination process. In order to delineate the region of p62 which interacts with ubiquitin, a series of p62 deletion mutants containing C-terminal T7 epitope tag (p62-T7) have been constructed, expressed in HeLa cells, and examined for their ability to bind to Ub-Sepharose. The C-terminal 182-amino acid deletion from Asp 258 to Leu 440 (d258 -440) completely abolished the binding of p62 to ubiquitin, while deletions from Met 1 to Lys 187 (d1-187) and from Glu 32 to Pro 322 (d32-322) had little or no effect on binding (Fig. 3A). Further analysis showed that the C-terminal 80 amino acids (Ser 361 to Leu 440 ) are essential for p62 to associate with ubiquitin (Fig. 3B). Interestingly, partial deletions in this 80-amino acid region, from Asp 368 to Asp 391 (d368 -391) and from Asp 391 to Leu 440 (d391-440), had partial inhibitory effects on the p62-ubiquitin interaction. This result suggests either that two regions of p62 may interact with one ubiquitin molecule to provide maximal binding affinity or that more than one ubiquitin binding site is present.
As all known ubiquitin-binding proteins are involved in ubiquitination-dependent proteasomal proteolysis, the biological function of p62 may also be related to this protein modification/ degradation pathway. Since, the N-terminal 50 amino acids of p62 are critical for interaction with the SH2 domain of p56 lck (6), p62 has at least two separate domains, one for binding to an SH2 domain and the other for binding to ubiquitin. Furthermore, p62 either has or is associated with a Ser/Thr kinase activity and also has a cysteine-rich zinc finger-like region and a region homologous to Cdc24, both of which are potential protein-protein interaction sites (6). Thus, a physiological role for p62 may involve connection of mitogenic signals to the ubiquitination-mediated specific protein degradation pathway. Alternatively, p62 may have a still uncharacterized enzymatic activity relating to the ubiquitination-mediated proteasomal degradation pathway that is regulated by binding to such signaling molecules.
FIG. 3. Mapping of ubiquitin binding region of p62. A series of deletion mutants of p62 were constructed with a T7 tag at the C terminus. Lysates of HeLa cells expressing various mutants of p62 were incubated with Ub-Sepharose, and binding of mutant p62 to Ub-Sepharose was analyzed by immunoblotting with anti-T7 monoclonal antibody (upper panels in A and B). Expression levels of the various mutants were measured in total lysates. Diagrammatic representation of each mutant is shown in the bottom panels.