Jaekyoon Shin

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 and the sequestosome, a novel mechanism for protein metabolism

In addition to selecting proteins for degradation by the 26S proteasome, ubiqitination appears to serve other regulatory functions, including for endosomal/lysosomal targeting, protein translocation, and enzyme modification. Currently, little is known how multiubiquitin chains are recognized by these cellular mechanisms. Within the 26S proteasome, one subunit (Mcb1/S5a) has been identified that has affinity for multiubiquitin chains and may function as a ubiquitin receptor. We recently found that a non-proteasomal protein p62 also preferentially binds multiubiquitin chains and forms a novel cytoplasmic structure “sequestosome” which serves as a storage place for ubiquitinated proteins. In the present manuscript, the role and regulation of p62 in relation to the sequestosomal function will be reviewed.

Genomic structure and promoter analysis of the p62 gene encoding a non-proteasomal multiubiquitin chain binding protein

p62 is a novel immediate early response gene encoding a ubiquitin chain binding protein. To investigate the mechanism of p62 gene expression, we isolated and characterized the 20 kb long human p62 gene. The p62 gene contains seven introns and eight exons. The splice sites conformed to the GT/AG rule, except introns 6 and 7 which used the unusual GC dinucleotides. The p62 promoter is TATA‐less, and 357 nucleotides of the 5′‐flanking region contain basic machineries for transcription. A reporter gene linked to 1800 nucleotides of the 5′‐flanking region was rapidly activated by various extracellular signals. The presence of a CpG island as well as multiple binding sites for SP‐1, AP‐1, NF‐κB, and Ets‐1 family in the promoter region supports the regulated activation of the p62 gene.

Immediate early response of the p62 gene encoding a non-proteasomal multiubiquitin chain binding protein.

p62 is a cytoplasmic ubiquitin chain binding protein. Upon a variety of extracellular signals, both transcript and protein levels of p62 were rapidly increased. These signals include phorbol 12‐myristate 13‐acetate (PMA) and calcium ionomycin for peripheral blood mononuclear cells, serum or PDGF for serum‐starved NIH3T3 cells, IL‐3 for the G1 arrested pre‐B cell line Ba/F3, and PMA for a human promyelocyte line U937. The elevation of p62 transcript level is due to temporal stabilization of mRNA and rapid activation of the p62 gene. Cycloheximide‐induced enhanced transcription suggests the immediate early response of the p62 gene. The rapid induction of p62 indicates the presence of a novel ubiquitination‐mediated regulatory mechanism during cell proliferation and differentiation.

Mutational Analysis of the Structure and Function of the CD4 Protein

The CD4 and CD8 glyeoproteins are T-eell specific surfaee proteins whieh are members of the immunoglobulin gene superfamily (Williams & Barclay 1988). In mature T cells, they are expressed on functionally distinct populations of T lymphocytes. Whereas the CD4 protein is predominantly expressed on T-helper eells which are restricted to class II major histocompatibility complex (MHC) antigens, the CD8 protein is typically found on cytotoxie T lymphocytes which are restricted to class I MHC antigens on target eells (Reinherz & Schlossman 1980, Engleman et al. 1981, Biddison et al. 1984, Krensky et al. 1982, Meuer et al. 1982). Antibody inhibition studies imply that these accessory molecules act to increase the avidity of effector-target cell interactions (Wilde et al. 1983, Rogozinski et al. 1984, Swain et al. 1984). Transfection of CD4 and CD8 has been shown to augment the T-eell response to antigen mediated by the T-eell reeeptor and MHC-bearing target cells (Dembic et al. 1987, Gay et al. 1987). These observations have led to the speculation that CD4 and CD8 bind to monomorphic determinants on the appropriate MHC molecule, thereby augmenting low-afTmity T-eell reeeptor-antigen interaetions (Marraek et al. 1983, Greenstein et al. 1984, 1985, Gay et al. 1988). In order to address this issue, we have used an SV40-derived vector system to express a cDNA eneoding the CD4 protein in simian fibroblasts (Doyle & Strominger 1987). The high expression level achieved using the SV40 system has allowed the observation ofa direct interaction between CD4, expressed on CVl cells, and class II molecules expressed on the surface of B lymphocytes. Notably, this interaction occurs in the absence of the T-cell receptor or any other lymphocyte adhesion molecules. Similar studies have recently demonstrated a similar