Expression of a new isoform of the tumor susceptibility TSG101 protein lacking a leucine zipper domain in Burkitt lymphoma cell lines

Abstract

The tumor susceptibility gene, TSG101, has been identi®ed as a candidate tumor suppressor gene. We have examined the expression of TSG101 in Burkitt lymphoma cell lines. Several aberrant messages were detected in all cell lines. Aberrant splice donor sites are located within exon 1 at positions 132, 154, 172 and 284. Splice acceptors are located at positions 847 and 1054 within exon 5. The aberrant messages are coexpressed with a normal message and could be the result of additional splicing reactions of the mature message that behaves as an intermediate. The normal message codes for 46 kDa protein (TSG101A). One aberrant message joins in frame nucleotides 283 ± 1055 and codes for a protein isoform of 17 kDa (TSG101B), as demonstrated by in vitro translation assays. The TSG101B isoform lacks the leucine zipper near the C-terminus, a transcriptional repressor domain, and retains most of the N-terminal region which has homology to E2 ubiquitin regulatory enzymes and the CROC-1 transcriptional regulator. The TSG101B isoform was detected in sixteen out of twenty-two (72%) BL cell lines, but not in normal lymphoid populations. The presence of two TSG101 isoforms with di erent dimerization potential opens up a new level of regulation of the TSG101 proteins possibly a ecting cell cycle regulation.

TSG101 interaction with HRS mediates endosomal trafficking and receptor down-regulation

“…However, excessive production of TSG101 also can promote abnormal cell growth (10), consistent with evidence for a cellular imperative to maintain TSG101 levels within a narrow range (19). Aberrant splicing of, partial deletions in, or overproduction of TSG101 protein have been reported to occur in human cancers (45)(46)(47)(48). Consistent with a crucial role of the intracellular level of TSG101 in determining its actions is evidence that cells that totally lack TSG101 cannot be propagated (8,9), whereas a lesser deficiency of TSG101 allows cell survival while still yielding prominent effects on endosomal trafficking and other biological processes (10,17,23,24).…”

The human vaccinia-related kinase 1 (VRK1) phosphorylates threonine-18 within the mdm-2 binding site of the p53 tumour suppressor protein

“…The synthesis of cDNA has been previously reported (Ferrer et al, 1999). From this cDNA 1 ml was used as substrate to amplify by PCR dierent fragments of the cDNA in order to make dierent constructions of fusion proteins with dierent types of expression vectors.…”

Loss of the TSG101 leucine zipper domain in aggressive non-Hodgkin's lymphomas

“…The different types of messages were determined following the same methodological strategy previously described in Burkitt lymphomas. 6 The message coding for isoform B, when present, is readily detected (Figure 1). Nucleotide sequencing of this new B message confirmed that it joins nucleotide 283, in exon 3, to nucleotide 1055, within exon 9, using a cryptic splice acceptor site, and thus with a different carboxy terminus.…”

“…We have recently detected, in 77% of Burkitt's lymphoma cell lines, the presence of a new alternatively spliced message that codes for a 17-kDa protein (isoform B) that lacks the coiled-coil (leucine zipper) domain, but that partially retains the amino terminus with the ubiquitination inhibitor domain. 6 This message has not been detected in normal cells despite the fact that a subpopulation of normal cells is capable of forming alternatively spliced non-coding messages. This isoform B, coded by the new transcript, might have a different function due to the loss of specific proteinprotein interactions mediated by the coiled-coil domain (leucine zipper) in the carboxy terminus, and consequent loss of its transcriptional regulation role.…”

“…Non-coding messages were observed in 10-25% of the normal lymphoid cell samples, and the isoform B was not detected in these cases. 6 However, noncoding transcripts were found in 42-100% of the tumors (Table 1) (P Ͻ 0.0001, using the likelihood ratio chi-square method to compare with the distribution in normal lymphoid cells). These transcripts were constantly expressed in primary large B cell lymphomas (100%), but only in 42-55% of chronic lymphocytic leukemias (CLL) and follicular lymphomas (FL) (P Ͻ 0.001) ( Table 1).…”