Marcjanna Bartkiewicz

Ligand-induced ubiquitination of the epidermal growth factor receptor involves the interaction of the c-Cbl RING finger and UbcH7

c-Cbl plays a negative regulatory role in tyrosine kinase signaling by an as yet undefined mechanism. We demonstrate here, using the yeast two-hybrid system and an in vitro binding assay, that the c-Cbl RING finger domain interacts with UbcH7, a ubiquitin-conjugating enzyme (E2). UbcH7 interacted with the wild-type c-Cbl RING finger domain but not with a RING finger domain that lacks the amino acids that are deleted in 70Z-Cbl, an oncogenic mutant of c-Cbl. The in vitro interaction was enhanced by sequences on both the N- and C-terminal sides of the RING finger. In vivo and in vitro experiments revealed that c-Cbl and UbcH7 synergistically promote the ligand-induced ubiquitination of the epidermal growth factor receptor (EGFR). In contrast, 70Z-Cbl markedly reduced the ligand-induced, UbcH7-mediated ubiquitination of the EGFR. MG132, a proteasome inhibitor, significantly prolonged the ligand-induced phosphorylation of both the EGFR and c-Cbl. Thus, c-Cbl plays an essential role in the ligand-induced ubiquitination of the EGFR by a mechanism that involves an interaction of the RING finger domain with UbcH7. This mechanism participates in the down-regulation of tyrosine kinase receptors and loss of this function, as occurs in the naturally occurring 70Z-Cbl isoform, probably contributes to oncogenic transformation.

Leucine Zipper-mediated Homodimerization of the Adaptor Protein c-Cbl A ROLE IN c-Cbl's TYROSINE PHOSPHORYLATION AND ITS ASSOCIATION WITH EPIDERMAL GROWTH FACTOR RECEPTOR

The 120-kDa proto-oncogenic protein c-Cbl is a multidomain adaptor protein that is phosphorylated in response to the stimulation of a broad range of cell surface receptors and participates in the assembly of signaling complexes that are formed as a result of the activation of various signal transduction pathways. Several structural features of c-Cbl, including the phosphotyrosine-binding domain, proline-rich domain, and motifs containing phosphotyrosine and phosphoserine residues, mediate the association of c-Cbl with other components of these complexes. In addition to those domains that have been demonstrated to play a role in the binding of c-Cbl to other signaling molecules, c-Cbl also contains a RING finger motif and a putative leucine zipper. In this study, we demonstrate that the previously identified putative leucine zipper mediates the formation of Cbl homodimers. Using the yeast two-hybrid system, we show that deletion of the leucine zipper domain is sufficient to abolish Cbl homodimerization, while Cbl mutants carrying extensive N-terminal truncations retain the ability to dimerize with the full-length Cbl. The requirement of the leucine zipper for the homodimerization of Cbl was confirmed by in vitro binding assays, using deletion variants of the C-terminal half of Cbl with and without the leucine zipper domain, and in cells using Myc and green fluorescent protein (GFP) N-terminal-tagged Cbl variants. In cells, the deletion of the leucine zipper caused a decrease in both the tyrosine phosphorylation of Cbl and its association with the epidermal growth factor receptor following stimulation with epidermal growth factor, thus demonstrating a role for the leucine zipper in c-Cbls signaling functions. Thus, the leucine zipper domain enables c-Cbl to homodimerize, and homodimerization influences Cbls signaling function, modulating the activity of Cbl itself and/or affecting Cbls associations with other signaling proteins in the cell.

Characterization of the osteoclast vacuolar H+-ATPase B-subunit

During bone resorption, osteoclasts acidify the extracellular bone resorbing compartment via a vacuolar H(+)-ATPase (V-ATPase), which resides in the ruffled-border membrane. In an effort to characterize the composition of the osteoclast V-ATPase catalytic domain, we have isolated a cDNA clone that encodes the V-ATPase B-subunit from a cDNA library constructed from highly purified chicken osteoclasts. Comparison of the predicted amino-acid sequence with the published sequences of isoforms of V-ATPase B-subunits from other sources revealed that the chicken osteoclast B-subunit is brain type and not kidney type. Furthermore, only clones encoding the brain type isoform of subunit B could be generated by PCR from a cDNA library prepared from human osteoclastoma osteoclast-like cells. Northern blot analysis revealed that two B-subunit mRNAs, approx. 1.7 and 3.5 kb in length, are expressed in chicken bone marrow mono-nuclear cells, brain and kidney, although the relative amounts of these two transcripts were different in each tissue. In brain, the 3.5-kb mRNA was predominantly expressed. In bone marrow cells, the levels of the 1.7-kb mRNA were higher than in other tissues and expression of this message was increased by 1,25-dihydroxyvitamin D-3, suggesting that this mRNA is specifically upregulated during osteoclast differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Ribonuclease P as a snRNP

Ribonuclease P is an enzyme that cleaves tRNA precursors to generate the 5’ termini of mature tRNA sequences. In E. coli this enzyme consists of a catalytic RNA subunit and a protein cofactor. In the absence of direct cloning of the gene for the RNA component and functional assays of the transcript of that cloned gene, it is possible to perform other biochemical tests to determine if the enzymatic activity has an essential RNA component. These include, for example, in- activation of the enzymatic activity by pretreatment with another ribonuclease and determination of the buoyant density, which may be characteristic of an RNA-protein complex, as well as direct labeling, identification, and functional assay of RNA components of the enzyme.

Acidification and Bone Resorption: The Role and Characteristics of V-ATPases in the Osteoclast

The purpose of this chapter is to briefly describe the mechanisms involved in the resorption of bone, highlighting the role of vacuolar ATPases and pH, both intracellular and extracellular, in this process. The cell responsible for the resorption of bone, i. e. for the degradation of the mineralized extracellular matrix that forms bone tissue, is the osteoclast.

Reassociable dimer subunit of potato nucleotide pyrophosphatase; specificity and stability

Abstract Treatment of the tetramer ( M r 340 000) of nucleotide pyrophosphatase (dinucleotide nucleotidohydrolase, EC 3.6.1.9) from potato tubers with glycine-NaOH buffer (pH 10) for 1–2 h leads to its dissociation to an active dimer with an M r , estimated from sucrose density gradient centrifugation and modified SDS-polyacrylamide gradient gel electrophoresis, of 170 000. In acidic (pH ≈ 5), and to some extent in neutral media the dimer reassociates to the tetramer. The activity of the dimer is considerably less stable than that of the tetramer. The specific activities of the dimer and tetramer towards thymidine 5′- p -nitrophenylphosphate are identical, whereas the rates of cleavage of pyro- and oligophosphates derived from 5′-nucleotides, such as dT(5′)p 2 (5′)dT, Ap 2 A, Ap 3 A and NAD + , are up to 70% lower for the dimer. By contrast, for substrates derived from 3′-nucleotides such as thymidine 3′- p -nitrophenylphosphate, 2′,3′-cAMP and dT(3′)p 2 (3′)dT as well as for bis( p -nitrophenyl)phosphate and p -nitrophenylphenylphosphonate, the dimer subunit is 3–10-fold more active than tetramer. These increases in activity are not accompanied by large changes in K m values. A modified SDS-polyacrylamide gradient gel electrophoresis technique, adapted for the foregoing study, should be equally applicable to determination of M r values of other oligomeric proteins, especially enzymatically active ones.