Additional complexity in p73: induction by mitogens in lymphoid cells and identification of two new splicing variants ε and ζ

Abstract

Additional complexity in p73: induction by mitogens in lymphoid cells and identification of two new splicing variants e and z Dear Editor, p53 is a sequence specific transcription factor which transactivates several genes important in the apoptotic pathway, such as p21, mdm2, gadd45, bax and caspases. 1,2 Lack of apoptosis, with inappropriate cell proliferation in cancer is correlated with a high frequency of p53 mutations, in some tumours reaching 50%. Perhaps surprisingly, in view of the importance of p53 in regulating cell death and its strong phylogenetic conservation, only recently have homologous genes been identified.These (p63 and p73) show up to 63% aminoacid identity with p53 in the DNA binding, oligomerization and transcription activation domains, suggesting a similar mechanism of action to p53. 3±7 p63 is expressed as six different forms. 4 These use one of the two alternative transcription initiation sites, each transcript then being expressed as one of three alternatively spliced variants (a, b, g). Since the transcripts using the downstream ATG lack the first three exons coding for the transactivation domain, they act as natural dominant negative mutants of full length p63 and of p53. 4 The p73 gene comprises 14 exons and we have shown previously that in addition to the full length a form and the alternatively spliced transcript lacking exon 13 (b) other splice variants, lacking exon 11 (g) and exons 11, 12 and 13 (d) are also produced. 8 Stimulation of the T lymphoblastoid cell line, Jurkat, and human peripheral blood lymphocytes (PBL) with phytoemagglutinin (PHA) causes a 3 and 2.6-fold increase respectively in p73 expression by Northern blotting after 24 h (panel A). This is associated with induction of 34.7% and 21.2% of apoptotic cells respectively. No p53 was detected in Jurkat cells after PHA treatment (not shown).In order to discriminate the differential induction of the four p73 isoforms we performed an RT±PCR using isoformspecific primers on RNA extracted from cells treated under the same conditions. Panel B shows upregulation of a, b, g and d in PHA-treated PBL and Jurkat cells. Densitometric comparison of these with the housekeeping gene GAPDH showed that the increase in expression of each isoform were comparable (not shown).In addition, a new isoform was amplified from normal PBL. Cloning and sequencing of this p73e identified it as a splicing variant lacking exons 11 and 13 (panel C). To confirm the existence of p73e, we screened a panel of normal and tumour cell lines. As also shown in panel B, p73e was also present in the human hepatoma line HepG2, and a sixth isoform z was identified in the MCF7 human breast cancer cell line and in a human skin biopsy. p73 z is a further splice variant which lacks exons 11 and 12, and results in the loss of 96 aminoacids, the sequence continuing with the C-terminus of the a form (panel C). In p73e, loss of exon 11 deletes 50 aminoacids with a frame shift to the reading frame of the g isoform; splicing of exon 13 deletes an additional 31 aminoacids...

Increased proliferation of CD8+ T cells in SAP‐deficient mice is associated with impaired activation‐induced cell death

“…At least six different isoforms (a, b, c, d, e, f) have been identified, depending on alternative promoter usage and alternative mRNA splicing [30][31][32][33][34]. Proapoptotic function is conferred by the p73 isoforms that contain N-terminal transactivation domains, usually named TAp73 [29].…”

Transcriptional activities of p73 splicing variants are regulated by inter-variant association

“…The presence of six isoforms, α, β, γ, δ, ε and ξ, with differing C-terminal structures has been reported [10][11][12]. Among these, only p73α has been found to have a C-terminal portion containing a region with a striking structural similarity with the SAM (sterile α motif ) domain [13,14], which is proposed to be a putative proteinprotein interaction domain [15].…”

Structure, function and regulation of p63 and p73

“…The molecular mechanisms for this cancer instability and the function of these cancer variants is unknown. The experimental conditions are described in De Laurenzi et al 6,7 p63 and p73 M Levrero et al family members can form hetero-oligomers. Physical interactions between p53, p63 and p73 have been investigated by in vitro assays, by the yeast two-hybrid system, or by co-immunoprecipitation in mammalian cells ( Table 1).…”