| It is well established that IGFs display a variety of bioactivities, includinginduction of growth promotion, differentiation, and metabolic function. IGFs bind tothe extracellular subunits of their respective receptors and induce intramolecularconformational changes resulting in the activation of the subunit intrinsic tyrosinekinase activity. Activated receptor kinases phosphorylate insulin receptor substrates(IRSs). It is well known that IRS acts as a mediator for signal transduction ofinsulin-like growth factors and several cytokines. Thus the identification ofIRS-associated proteins is an essential prerequisite for understanding the alteration ofIGF signals.The p53binding protein, termed53BP2, also known as ASPP2, has beenidentified as a haplo-insufficient tumor suppressor in mice and belongs to the ASPPfamily of proteins, which consists of three members: ASPP1, ASPP2, and iASPP. TheASPP family of proteins is characterized by a common C-terminal sequence: ankyrinrepeats, SH3domain, and proline-rich region containing protein. The best knownfunction of ASPP2is its binding to p53, p63, and p73, and the stimulation of theirapoptotic functions by selectively enhancing their DNA binding and transactivationactivities on pro-apoptotic genes such as BAX and PIG3. In the past decade, ASPP2has also been identified as a binding partner of a number of other proteins. Hakuno etal.(2007) found53BP2s can interact with IRS in the use of yeast two-hybridscreening. But the mechanism of53bp2cross-talking with IGF singnal pathway isstill uncleared. Here, we use zebrafish as a model animal to investigate the function of53bp2in vivo in early developmental stages.We cloned53bp2gene and analysised the temporal and spatial expression patternusing RT-PCR and in-situ hybridization. There were two53bp2genes and oneisoform in zebrafish. Expression of53bp2a and53bp2b were observed in all tissues in early embryonic developmental stages and adult. In contrast,53bp2bi which had lostSH3domain was not a matermal gene and only expressed in nerve tissue and heart.When forced expression of53bp2a and53bp2b mRNA, no major patterningabnormalities were detected in zebrafish embryos, but the full length of embryos wasreduced whereas the somite number was normal. Taken together, these data indicatedthat53bp2may reduce the length of sarcomere.IGF signaling pathway can activate PI3k-Akt cascade and Ras-MAPK cascade,respectively. It has become clear that activation of Akt and Erk can promote cellproliferation, differentiation and tumor cell survival. In our study, Akt (serine473)phosphorylation was enhanced by overexpression of zebrafish53bp2, whereas Erkphosphorylation was not affected. Overexpression of myr-Akt1could rescue thephenotypes caused by overexpression of53bp2. These results indicated that53bp2could reduce the embryo`s length by inhibiting the Akt phosphorylation.Because the N terminus of m53BP2and IRS-1interact in vitro, we hypothesizedthat zebrafish53bp2may bind and antagonize Irs-1’s function in IGF signals. Hence,we examined the capacity of53bp2in interacting with Irs-1in HEK293T cells, and aninteraction between53bp2and Irs-1was detected. Ankyrin repeats and SH3domainare much conserved in ASPP family, and they play an important role in the interactionwith p53.53bp2mutants which lost ankyrin repeats and SH3domain couldn’t reducefull length of embryos and couldn’t inhibit the Akt phosphorylation, indicated that theinteraction between53bp2and Irs-1also depended on ankyrin repeats and SH3domain.In conclusion, zebrafish53bp2has functions beyond its well-characterizedability to enhance p53-mediated apoptosis. The function of53bp2to negativeregulates IGF signals by reducing the phosphorylation of Akt indicates a newmechanism, and our findings give rise to the notion that ASPP2might function as atumor suppressor. |
PDF Full Text Request