Identification and characterization of the gene encoding human cytoplasmic polyadenylation element binding protein.

The maturation of human oocytes occurs in the absence of gene transcription. In model organisms, such as Drosophila, Xenopus, and the mouse, oocyte maturation and early pattern formation is mediated through the regulated translation of maternally derived mRNAs. The maturation-dependent stimulation of maternal mRNA translation is correlated with increases in poly(A) tail length, controlled through a process termed cytoplasmic polyadenylation. However, this mechanism of mRNA translational control has not been characterized in humans. In this study we report the cloning of a human cytoplasmic polyadenylation element binding (hCPEB) protein with sequence-specific RNA binding activity. Our data demonstrate that alternative splicing generates hCPEB mRNAs that encode proteins with a conserved C-terminal RNA binding domain but with different N-terminal regulatory domains. The hCPEB mRNA is expressed in the brain and heart as well as in immature oocytes, consistent with the hypothesis that cytoplasmic polyadenylation may regulate the translation of human mRNAs in both oocytes and somatic cells.

The temporal control of Wee1 mRNA translation during Xenopus oocyte maturation is regulated by cytoplasmic polyadenylation elements within the 3'-untranslated region.

The Wee1 protein tyrosine kinase is a key regulator of cell cycle progression. Wee1 activity is necessary for the control of the first embryonic cell cycle following the fertilization of meiotically mature Xenopus oocytes. Wee1 mRNA is present in immature oocytes, but Wee1 protein does not accumulate in immature oocytes or during the early stages of progesterone-stimulated maturation. This delay in Wee1 translation is critical since premature Wee1 protein accumulation has been shown to inhibit oocyte maturation. In this study we provide evidence that Wee1 protein accumulation is regulated at the level of mRNA translation. This translational control is directed by sequences within the Wee1 mRNA 3'-untranslated region (3' UTR). Specifically, cytoplasmic polyadenylation element (CPE) sequences within the Wee1 3' UTR are necessary for full translational repression in immature oocytes. Our data further indicate that while CPE-independent mechanisms may regulate the levels of Wee1 protein accumulation during progesterone-stimulated oocyte maturation, the timing of Wee1 mRNA translational induction is directed through a CPE-dependent mechanism.

Disruption of the 14-3-3 binding site within the B-Raf kinase domain uncouples catalytic activity from PC12 cell differentiation.

A number of Raf-associated proteins have recently been identified, including members of the 14-3-3 family of phosphoserine-binding proteins. Although both positive and negative regulatory functions have been ascribed for 14-3-3 interactions with Raf-1, the mechanisms by which 14-3-3 binding modulates Raf activity have not been fully established. We report that mutational disruption of 14-3-3 binding to the B-Raf catalytic domain inhibits B-Raf biological activity. Expression of the isolated B-Raf catalytic domain (B-Rafcat) induces PC12 cell differentiation in the absence of nerve growth factor. By contrast, the B-Rafcat 14-3-3 binding mutant, B-Rafcat S728A, was severely compromised for the induction of PC12 cell differentiation. Interestingly, the B-Rafcat 14-3-3 binding mutant retained significant in vitro catalytic activity. In Xenopus oocytes, the analogous full-length B-Raf 14-3-3 binding mutant blocked progesterone-stimulated maturation and the activation of endogenous mitogen-activated protein kinase kinase and mitogen-activated protein kinase. Similarly, the full-length B-Raf 14-3-3 binding mutant inhibited nerve growth factor-stimulated PC12 cell differentiation. We conclude that 14-3-3 interaction with the catalytic domain is not required for kinase activity per se but is essential to couple B-Raf catalytic activity to downstream effector activation.

Functional conservation of the wingless-engrailed interaction as shown by a widely applicable baculovirus misexpression system.

BACKGROUND: The expression patterns of the segment polarity genes wingless and engrailed are conserved during segmentation in a variety of arthropods, suggesting that the regulatory interactions between these two genes are also evolutionarily conserved. Hypotheses derived from such comparisons of gene expression patterns are difficult to test experimentally as genetic manipulation is currently possible for only a few model organisms. RESULTS: We have developed a system, using recombinant baculoviruses, that can be applied to a wide variety of organisms to study the effects of ectopic expression of genes. As a first step, we studied the range and type of infection of several reporter viruses in the embryos of two arthropod and one vertebrate species. Using this system to express wingless, we were able to induce expression of engrailed in the anterior half of each parasegment in embryos of the fruit fly Drosophila melanogaster. Virus-mediated wingless expression also caused ectopic naked ventral cuticle formation in wild-type Drosophila larvae. In the flour beetle, Tribolium castaneum, ectopic wingless also induced engrailed expression. As in Drosophila, this expression was only detectable in the anterior half of the parasegment. CONCLUSIONS: The functional interaction between wingless and engrailed, and the establishment of cells competent to express engrailed, appears to be conserved between Drosophila and Tribolium. The data on the establishment of an engrailed-competent domain also support the idea that prepatterning by pair-rule genes is conserved between these two insects. The recombinant baculovirus technology reported here may help answer other long-standing comparative evolutionary questions.

Nerve growth factor-stimulated B-Raf catalytic activity is refractory to inhibition by cAMP-dependent protein kinase.

The cAMP-dependent protein kinase (PKA) exhibits both inhibitory and stimulatory effects upon growth factor signaling mediated by the mitogen-activated protein kinase signaling pathway. PKA has been demonstrated to inhibit Raf-1-mediated cellular proliferation. PKA can both prevent Ras-dependent Raf-1 activation and directly inhibit Raf-1 catalytic activity. In contrast to the inhibitory effect of PKA on Raf-1-dependent processes, PKA potentiates nerve growth factor-stimulated PC12 cell differentiation, a B-Raf mediated process. This potentiation, rather than inhibition, of PC12 cell differentiation is curious in light of the ability of PKA to inhibit Raf-1 catalytic activity. The kinase domains of Raf-1 and B-Raf are highly conserved, and it has been predicted that B-Raf catalytic activity would also be inhibited by PKA. In this study we examined the ability of PKA to regulate the kinase activity of the B-raf proto-oncogene. We report that nerve growth factor-stimulated B-Raf activity is not inhibited by PKA. By contrast, an N-terminally truncated, constitutively active form of B-Raf is inhibited by PKA both in vitro and in transfected PC12 cells. These results suggest that the N-terminal regulatory domain interferes with the ability of PKA to modulate B-Raf catalytic activity and provide an explanation for the observed resistance of B-Raf-dependent processes to PKA inhibition.

The mitogen-activated protein kinase signaling pathway stimulates mos mRNA cytoplasmic polyadenylation during Xenopus oocyte maturation.

The Mos protein kinase is a key regulator of vertebrate oocyte maturation. Oocyte-specific Mos protein expression is subject to translational control. In the frog Xenopus, the translation of Mos protein requires the progesterone-induced polyadenylation of the maternal Mos mRNA, which is present in the oocyte cytoplasm. Both the Xenopus p42 mitogen-activated protein kinase (MAPK) and maturation-promoting factor (MPF) signaling pathways have been proposed to mediate progesterone-stimulated oocyte maturation. In this study, we have determined the relative contributions of the MAPK and MPF signaling pathways to Mos mRNA polyadenylation. We report that progesterone-induced Mos mRNA polyadenylation was attenuated in oocytes expressing the MAPK phosphatase rVH6. Moreover, inhibition of MAPK signaling blocked progesterone-induced Mos protein accumulation. Activation of the MAPK pathway by injection of RNA encoding Mos was sufficient to induce both the polyadenylation of synthetic Mos mRNA substrates and the accumulation of endogenous Mos protein in the absence of MPF signaling. Activation of MPF, by injection of cyclin B1 RNA or purified cyclin B1 protein, also induced both Mos protein accumulation and Mos mRNA polyadenylation. However, this action of MPF required MAPK activity. By contrast, the cytoplasmic polyadenylation of maternal cyclin B1 mRNA was stimulated by MPF in a MAPK-independent manner, thus revealing a differential regulation of maternal mRNA polyadenylation by the MAPK and MPF signaling pathways. We propose that MAPK-stimulated Mos mRNA cytoplasmic polyadenylation is a key component of the positive-feedback loop, which contributes to the all-or-none process of oocyte maturation.

Ambulatory blood pressure in hypertensive patients with autosomal dominant polycystic kidney disease.

BACKGROUND: Ambulatory blood pressure is more closely correlated with various indices of hypertensive target-organ damage, and is a better prognostic predictor of cardiovascular morbidity and mortality than conventional methods of blood pressure measurement. Autosomal dominant polycystic kidney disease (ADPKD) is complicated by hypertension, progressive renal failure, and an increased risk of cardiovascular mortality. This study investigated the 24-h ambulatory blood pressure profile in patients with ADPKD in view of the sparsity of such data in these patients and the possibility that abnormal diurnal blood pressure variations may have prognostic consequences. METHODS: Ambulatory blood pressure was measured over a 24-h period by the oscillometric method with an automatic non-invasive recorder (SpaceLabs 90207 system) in matched groups of 25 hypertensive patients with ADPKD and 25 patients with essential hypertension. RESULTS: Both groups showed a nocturnal decrease in blood pressure, but this was significantly smaller in patients with ADPKD. There was no evidence of enhanced lability of blood pressure in ADPKD. CONCLUSIONS: The nocturnal fall in blood pressure was attenuated in patients with ADPKD. Further studies are required to assess the importance of this finding and its possible contribution to the progression of renal failure or increased cardiovascular mortality in these patients.

pXen, a utility vector for the expression of GST-fusion proteins in Xenopus laevis oocytes and embryos.

We describe a plasmid, pXen, designed for the optimized expression of proteins fused to glutathione-S-transferase (GST) in Xenopus laevis oocytes and embryos. The Xenopus model system permits the biochemical analysis of signaling pathways and analysis of embryo phenotype in response to manipulation of proto-oncogene expression. pXen is a modified pSP64T vector which contains an SP6 RNA polymerase promoter followed by the translational initiation sequence of Xenopus beta-globin and the glutathione binding domain of GST. The Xenopus 3' beta-globin untranslated region and polyadenylation site immediately follow the multiple cloning site to permit the efficient translation of in vitro transcribed RNA in oocytes and embryos. The utility of pXen is demonstrated by cloning the catalytic domain of the serine/threonine kinase proto-oncogene Raf-1 into this vector and injecting the corresponding in vitro transcribed RNA into oocytes. Catalytically active GST-vRaf fusion protein was expressed in the injected oocytes and induced oocyte maturation. Moreover, the GST-vRaf fusion protein could be readily purified from Xenopus extracts using glutathione Sepharose. We demonstrate that the Raf-1 catalytic domain retains activity when fused with the N-terminal GST moiety and is subject to negative regulation by the cyclic AMP-dependent protein kinase (PKA). The pXen vector will be useful for an in vivo analysis of the physiological role and regulation of a wide variety of signaling molecules when expressed in Xenopus oocytes and embryos.

Regulation of Raf-1-dependent signaling during early Xenopus development.

The Raf-1 gene product is activated in response to cellular stimulation by a variety of growth factors and hormones. Raf-1 activity has been implicated in both cellular differentiation and proliferation. We have examined the regulation of the Raf-1/MEK/MAP kinase (MAPK) pathway during embryonic development in the frog Xenopus laevis. We report that Raf-1, MEK, and MAPK activities are turned off following fertilization and remain undetectable up until blastula stages (stage 8), some 4 h later. Tight regulation of the Raf-1/MEK/MAPK pathway following fertilization is crucial for embryonic cell cycle progression. Inappropriate reactivation of MAPK activity by microinjection of oncogenic Raf-1 RNA results in metaphase cell cycle arrest and, consequently, embryonic lethality. Our findings demonstrate an absolute requirement, in vivo, for inactivation of the MAPK signaling pathway to allow normal cell cycle progression during the period of synchronous cell divisions which occur following fertilization. Further, we show that cytostatic factor effects are mediated through MEK and MAPK.

Haplotype analysis in autosomal dominant polycystic kidney disease.

Haplotype analysis was performed in 35 autosomal dominant polycystic kidney disease (ADPKD) families typed with 13 markers close to the PKD1 locus. The identification of recombinants close to the PKD1 gene on chromosome 16p indicates that PKD1 lies between CMM65 distally and 26-6 proximally. In addition, three unlinked (PKD2) families and two families with potential new mutation were identified.

Activation of Raf-1 by 14-3-3 proteins.

The protein Raf-1, a key mediator of mitogenesis and differentiation, associates with p21ras (refs 1-3). However, the regulation of the serine/threonine kinase activity of Raf-1 is still not understood. Using the yeast two-hybrid system, we identified two structurally related proteins that interact with the aminoterminal region of Raf-1. These proteins, 14-3-3 zeta (PLA2) and 14-3-3 beta (HS1), are members of the 14-3-3 family of proteins. Expression of 14-3-3 proteins in Xenopus oocytes enhanced Raf-1 activity and promoted Raf-1-dependent oocyte maturation. A dominant negative mutant of Raf-1 blocked the effects of 14-3-3 protein.

Signaling molecules that mediate the actions of FGF.

Signaling by fibroblast growth factor (FGF) is critical for cellular growth and differentiation. We have studied the role of FGF and its receptor in development and also the signaling pathways utilized by these molecules. Transgenic mice expressing a dominant negative FGF receptor specifically in lung tissue fail to develop lungs. These findings suggest that FGF signaling is important for airway branching and alveolar formation. In Xenopus embryos, FGF is involved in the development of posterior structures. Induction of mesoderm by FGF is mediated by the serine-threonine kinase RAF. The expression of a dominant negative RAF selectively blocks the mesoderm-inducing action of FGF. Expression of activated RAF accentuated the development of posterior structures Other intracellular molecules that are involved with FGF signaling include RAS, a low-molecular weight GTP-binding protein. We have identified a novel gene (RIP, for RAS-interacting protein) that contains a RAS-binding domain. This domain is found in at least one other protein, ralGDS. Both RIP and ralGDS bind to activated RAS suggesting that these proteins are likely candidates for mediators of RAS function. These findings help elucidate the role of FGF signaling in development.

Raf-1 protein kinase is important for progesterone-induced Xenopus oocyte maturation and acts downstream of mos.

In somatic cells, the Raf-1 serine/threonine protein kinase is activated by several polypeptide growth factors. We investigated the role of Raf-1 in progesterone-induced meiotic maturation of Xenopus laevis oocytes. Raf-1 enzymatic activity and phosphorylation (reflected by a mobility shift on sodium dodecyl sulfate gels) were increased in oocytes following progesterone stimulation. The increase in Raf-1 activity was concurrent with an elevation in the activity of mitogen-activated protein (MAP) kinase. When RNA encoding an oncogenic form of Raf-1 (v-Raf) was injected into immature oocytes, MAP kinase mobility shift, germinal vesicle breakdown, and histone H1 phosphorylation increased markedly. When RNA encoding a dominant-negative version of Raf-1 was injected, progesterone-induced oocyte maturation was blocked. When RNA encoding Xenopus mos (mosxe) was injected into oocytes, Raf-1 and MAP kinase mobility shifts were observed after several hours. Also, when antisense mosxe oligonucleotides were injected into oocytes, progesterone-induced Raf-1 and MAP kinase mobility shifts were blocked. Finally, when antisense mosxe oligonucleotides were coinjected with v-Raf RNA into oocytes, histone H1 kinase activation, germinal vesicle breakdown, and MAP kinase mobility shift occurred. These findings suggest that Raf-1 activity is required for progesterone-induced oocyte maturation and that Raf-1 is downstream of mosxe activity.

Raf-1 kinase is essential for early Xenopus development and mediates the induction of mesoderm by FGF.

Animal cap explants from Xenopus embryos injected with a dominant negative Raf-1 mutant, termed NAF (not a functional Raf), demonstrated a complete block to basic fibroblast growth factor (FGF)-stimulated mesoderm induction. Activin induction of mesoderm was normal in embryos that expressed NAF. Injection of NAF RNA into 2-cell stage embryos blocked normal development during neurula stages and caused severe posterior truncations in tadpoles. The phenotype induced by NAF could be rescued by coinjection of wild-type raf-1 RNA. The NAF mutant functioned by specifically blocking the activation of endogenous Raf kinase activity. These findings suggest that Raf-1 mediates FGF, but not activin, receptor signaling during mesoderm induction and implicate Raf-1 as a key signaling molecule in the development of posterior structure.

A study of genetic linkage heterogeneity in 35 adult-onset polycystic kidney disease families.

A genetic heterogeneity analysis of 35 kindreds with adult-onset polycystic kidney disease (ADPKD) was carried out using the D16S85, D16S84, D16S125 and D16S94 loci that are closely linked to the PKD1 locus on chromosome 16. The results show that the likelihood of two ADPKD loci is 2,514.9 times greater than for a single locus (P < 0.0001). The maximum likelihood lod score is 27.38 under heterogeneity with PKD1 lying 4.9 cM proximal to D16S85 (in males). At least 3% of kindreds are unlinked to PKD1, since the 95% confidence limits of alpha, the proportion of families linked to PKD1, are 0.54-0.97. Only 2 out of 35 kindreds (5.7%) show statistically significant evidence of non-linkage to PKD1, with conditional probabilities of 0.987 and 0.993 that the disease locus is unlinked. This confirms the existence of a small subgroup of ADPKD kindreds that are unlinked to PKD1 and provides a firm basis for genetic counselling of this population on the basis of DNA probes.

Multipoint mapping of adult onset polycystic kidney disease (PKD1) on chromosome 16.

Analysis of genetic linkage data in 33 adult onset polycystic kidney (ADPKD) families was carried out using probes for the D16S85, D16S84, and D16S94 loci. The data set of 33 families shows no evidence of genetic heterogeneity since one unlinked family was previously excluded. Two point linkage analysis showed maximum likelihood values of the recombination fraction of 0.07 for ADPKD and D16S85 (lod score 18.78), 0.02 for ADPKD and D16S84 (lod score 7.55), and 0.00 for ADPKD and D16S94 (lod score 6.73). Multipoint analysis showed a maximum likelihood order of tel-D16S85-0.06-D16S84-0.02-(PKD1, D16S94)-cen with a multipoint lod score of 32.16. Analysis of rare recombinants lying close to PKD1 gave results consistent with this order.

Effects of angiotensin converting enzyme inhibition in adult polycystic kidney disease.

The pathogenesis of hypertension in autosomal-dominant polycystic kidney disease (ADPKD) is unclear, but increased activity of the renin-angiotension system may contribute. The renal and systemic hemodynamic response to lisinopril, an angiotension converting enzyme (ACE) inhibitor, in patients with ADPKD without renal failure was compared with the response in matched unaffected family members. Mean blood pressure and renal vascular resistance decreased in the affected group after lisinopril, with no significant change in the unaffected group. Glomerular filtration rate (GFR) was unchanged and therefore filtration fraction fell significantly. Changes in urinary excretion of 6-keto-PGF1 alpha and kallikrein suggested that increased renal synthesis of PGI2 or activation of the renal kallikrein-kinin system were not likely to be responsible for the hemodynamic effects. The acute decrease in renal vascular resistance without change in GFR suggests that ACE inhibition may have a particular value in the treatment of hypertension associated with ADPKD which should be assessed by further long-term studies.

Renal, cardiovascular and hormonal characteristics of young adults with autosomal dominant polycystic kidney disease.

We studied young adults with autosomal dominant polycystic kidney disease (ADPKD) to determine the characteristics that precede renal impairment. Nineteen affected (A) and 20 unaffected (U) offspring from families with ADPKD showed no significant differences in basal glomerular filtration rate (A: mean 97, SD 19; U: 100, SD 23 ml/min/1.73 m2) or renal functional reserve, but effective renal plasma flow was significantly lower in affected offspring (A: 532, SD 86; U: 605, SD 118 ml/min/1.73 m2, P less than 0.01). Plasma renin activity [A: median 26 (95% CI: 15 to 37); U: 14 (11 to 27) microU/ml, P less than 0.05, one-tailed test] and aldosterone [A: 2.5 (2.0 to 3.0), U: 1.0 (1.5 to 2.0) micrograms/100 ml, P less than 0.04, one-tailed test] were increased in affected offspring despite the higher systolic blood pressure (A: mean 123, SD 5; U: 115, SD 3 mm Hg, P less than 0.02) and significant expansion of total exchangeable sodium (A: 40.8, SD 2.3; U: 38.0, SD 3.5 mmol/kg, P less than 0.01). The ouabain-sensitive component of red cell sodium efflux was less in affected offspring (A: 0.258; SD 0.040; U: 0.288, SD 0.042 hr-1, P less than 0.04) and in both groups was correlated inversely with total exchangeable sodium. Echocardiography revealed no difference in left ventricular mass index nor prevalence of mitral valve prolapse. Potential cyst growth factors such as the glucocorticoids and somatomedin C were similar in both affected and unaffected groups.(ABSTRACT TRUNCATED AT 250 WORDS)