The Wilms' tumor suppressor gene wt1 encodes a zinc-finger transcription factor that plays an important role in the development of the mammalian genitourinary system. Mutations in WT1 in humans lead to anomalies of kidney and gonad development and cause Wilms' tumor, a pediatric kidney cancer. The inactivation of both wt1 alleles in mice gives rise to multiple organ defects, among them agenesis of kidney, spleen, and gonads. In zebrafish, an ortholog of wt1 has been described that is expressed in the pronephric field and is later restricted to the podocytes. Here, we report the existence of a second wt1 gene in zebrafish, which we have named wt1b (we named the initial gene wt1a). The overall sequence identity of the two Wt1 proteins is 70% and 92% between the zinc-finger regions, respectively. In contrast to wt1a, wt1b is expressed from the earliest stages of development onward, albeit at low levels. Both wt1a and wt1b are expressed in the intermediate mesoderm, with wt1b being restricted to a smaller area lying at the caudal end of the wt1a expression domain. In adult fish, high expression levels for both genes can be found in gonads, kidney, heart, spleen, and muscle. Developmental Dynamics 235:554 -561, 2006.
Heterotrimeric G proteins in physiological and pathological processes have been extensively studied so far. However, little is known about mechanisms regulating the cellular content and compartmentalization of G proteins. Here, we show that the association of nucleoside diphosphate kinase B (NDPK B) with the G protein ␥ dimer (G␥) is required for G protein function in vivo. In zebrafish embryos, morpholino-mediated knockdown of zebrafish NDPK B, but not NDPK A, results in a severe decrease in cardiac contractility. The depletion of NDPK B is associated with a drastic reduction in G 1␥2 dimer expression. Moreover, the protein levels of the adenylyl cyclase (AC)-regulating G␣ s and G␣i subunits as well as the caveolae scaffold proteins caveolin-1 and -3 are strongly reduced. In addition, the knockdown of the zebrafish G 1 orthologs, G1 and G1like, causes a cardiac phenotype very similar to that of NDPK B morphants. The loss of G 1/G1like is associated with a down-regulation in caveolins, AC-regulating G␣-subunits, and most important, NDPK B. A comparison of embryonic fibroblasts from wild-type and NDPK A/B knockout mice demonstrate a similar reduction of G protein, caveolin-1 and basal cAMP content in mammalian cells that can be rescued by re-expression of human NDPK B. Thus, our results suggest a role for the interaction of NDPK B with G␥ dimers and caveolins in regulating membranous G protein content and maintaining normal G protein function in vivo.cAMP ͉ cardiac contractility ͉ G proteins ͉ NDPK ͉ zebrafish S ignaling through the activation of G proteins represents the most widely used signaling pathway in mammalian biology (1). A variety of G protein-coupled receptors (GPCRs) mediate extracellular signals via heterotrimeric G proteins, which are composed of a guanine nucleotide binding ␣-subunit (G␣), as well as a -subunit (G) and a ␥-subunit (G␥). Upon GPCR activation, the bound GDP in G␣ is exchanged for GTP and both the GTP-liganded G␣ and the stable dimer G␥ regulate downstream effectors (2).Nucleoside diphosphate kinases (NDPKs), which catalyze the transfer of ␥-phosphate between NTPs and NDPs, represent a family of multifunctional proteins encoded by nine human nm23 genes. The two major isoforms, NDPK A and B (17-21 kDa), play crucial roles in a wide array of cellular processes [for review see (3)]. Despite their high sequence homology and the well known formation of heterohexamers to perform their house keeping enzyme activity (4), NDPK A and B have distinct cellular functions, which are based on the possibility of both isoforms contributing to multimeric protein complexes like the SET complex (5) and a complex formed with Ca 2ϩ -activated potassium channel KCa3.1 (6). In such complexes, NDPK not only supplies NTPs but also acts as protein kinase (6). We have previously shown that NDPK B, but not NDPK A, forms a complex with G␥ (7, 8) and acts as a histidine kinase for G. The high energetic phosphate on G can be specifically transferred to GDP and the GTP that is formed, induces G protei...
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