WD (tryptophan-aspartic acid dipeptide)-repeat proteins play a central role in signal transduction cascades by co-ordinating the interaction of key signalling molecules. We identified a novel propeller-FYVE [domain identified in Fab1p, YOTB, Vac1p and EEA1 (early endosome antigen 1)] protein, ProF, which is expressed in various cell lines and tissues and consists of seven WD-repeats and a FYVE domain. WD-repeat proteins offer a platform for protein-protein interactions by folding into a seven-bladed propeller-like structure, while the FYVE domain binds to phosphatidylinositol 3-phosphate present mainly on intracellular membranes. The ProF protein partially co-localizes with EEA1 on vesicular structures and binds to the protein kinases Akt and PKCzeta/lambda (protein kinase Czeta/lambda) via its WD-repeat propeller. ProF interacts more strongly with the kinases after hormonal stimulation. Endogenously expressed ProF and the two kinases interact in brain and in the preadipocyte cell line 3T3-L1, suggesting a role in secretory vesicular processes. In summary, we describe a new binding partner for kinases, located on vesicular structures in specialized cells, which may play a role for the spatial organization of signalling cascades.

No abstract

We have previously produced transgenic fish from crosses between a wild-type female tilapia (Oreochromis niloticus) and a G transgenic male. This line of growth-enhanced tilapia carries a single copy of a chinook salmon (s) growth hormone (GH) gene spliced to an ocean pout antifreeze promoter (OPA-FPcsGH) co-ligated to a carp beta-actin/lacZ reporter gene construct, integrated into the tilapia genome. Because little is known about the expression sites of transgenes, we have characterised the gene expression patterns of sGH and tilapia (t)GH in transgenic tilapia using a newly established real-time PCR to measure the absolute mRNA amounts of both hormones. The sGH gene, which was expected to be expressed mainly in liver, was also found to be expressed in other organs, such as gills, heart, brain, skeletal muscle, kidney, spleen, intestine and testes. However, in pituitary no sGH mRNA but only tGH mRNA was found. Tilapia GH mRNA in wild-type pituitary amounted to 226 +/- 30 pg/microg total RNA but in transgenics only to 187 +/- 43 pg/microg total RNA. Liver exhibited the highest level of sGH mRNA (8.3 +/- 2.5 pg/microg total RNA) but the extrahepatic sites expressed considerable amounts of sGH mRNA ranging from 4.1 +/- 2.0 pg/microg total RNA in gills to 0.2 +/- 0.08 pg/microg total RNA in kidney. The widespread expression of the sGH gene is assumed to be due to the tissue specificity of the type III AFP gene promoter. It is assumed that our transgenic experiments, which in contrast to some other approaches caused no obvious organ abnormalities, mimick the GH expression during ontogeny. Because sGH mRNA is expressed both in liver and in extrahepatic sites it may not only promote secretion and release of liver-derived (endocrine) IGF-I leading to an overall growth enhancement but also stimulate IGF-I expression within the different organs in a paracrine/autocrine manner and, thus, further promote organ growth.

Several lines of GH-overexpressing fish have been produced and characterized concerning organ integrity, growth, fertility and health but few and contradictory data are available on IGF-I that mediates most effects of GH. Furthermore, nothing is known on IGF-II. Thus, the expression of both IGFs in liver and various extrahepatic sites of adult transgenic (GH-overexpressing) tilapia and age-matched wild-type fish was determined by real-time PCR. Both IGF-I and IGF-II mRNA were found in all organs investigated and were increased in gills, kidney, intestine, heart, testes, skeletal muscle and brain of the transgenics (IGF-I: 1.4-4-fold; IGF-II: 1.7-4.2-fold). Except for liver, brain and testis the increase in IGF-I mRNA was higher than that in IGF-II mRNA. In pituitary, no significant change in IGF-I or IGF-II mRNA was detected. In spleen, however, IGF-I and IGF-II mRNA were both decreased in the transgenics, IGF-I mRNA even by the 19-fold. In agreement, in situ hybridisation revealed a largely reduced number of IGF-I mRNA-containing leukocytes and macrophages when compared to wild-type. These observations may contribute to better understanding the reported impaired health of GH-transgenic fish. Growth enhancement of the transgenics may be due to the increased expression of both IGF-I and IGF-II in extrahepatic sites. It is also reasonable that the markedly enhanced expression of liver IGF-II mRNA that may mimick an early developmental stage is a further reason for increased growth.

Several lines of growth hormone (GH)-overexpressing fish have been produced and analysed for growth and fertility parameters. However, only few data are available on the growth-promoting hormone insulin-like growth factor I (IGF-I) that mediates most effects of GH, and these are contradictory. Using quantitative real-time RT-PCR, radioimmunoassay, in situ hybridization, immunohistochemistry, and radiochromatography we investigated IGF-I and IGF binding proteins (IGFBPs) in an adult (17 months old) transgenic (GH-overexpressing) tilapia (Oreochromis niloticus). The transgenics showed an around 1.5-fold increase in length and an approximately 2.3-fold higher weight than the non-transgenics. Using radioimmunoassay, the serum IGF-I levels were lower (6.22 +/- 0.75 ng/ml) in transgenic than in wild-type (15.01 +/- 1.49 ng/ml) individuals (P = 0.0012). Radioimmunoassayable IGF-I in transgenic liver was 4.2-times higher than in wild-type (16.0 +/- 2.21 vs. 3.83 +/- 0.71 ng/g, P = 0.0017). No hepatocytes in wild-type but numerous hepatocytes in transgenic liver contained IGF-I-immunoreactivity. RT-PCR revealed a 1.4-times higher IGF-I mRNA expression in the liver of the transgenics (10.51 +/- 0.82 vs. 7.3 +/- 0.49 pg/microg total RNA, P = 0.0032). In correspondence, in situ hybridization showed more IGF-I mRNA containing hepatocytes in the transgenics. A twofold elevated IGF-I mRNA expression was determined in the skeletal muscle of transgenics (0.33 +/- 0.02 vs. 0.16 +/- 0.01 pg/microg total RNA, P < 0.0001). Both liver and serum of transgenics showed increased IGF-I binding. The increased IGFBP content in the liver may lead to retention of IGF-I, and/or the release of IGF-I into the circulation may be slower resulting in accumulation of IGF-I in the hepatocytes. Our results indicate that the enhanced growth of the transgenics likely is due to enhanced autocrine/paracrine action of IGF-I in extrahepatic sites, as shown here for skeletal muscle.