Descripción: A soluble Ca2+-dependent protein kinase (CDPK) was purified to homogeneity in potato (Solanum tuberosum L.) plants. Potato CDPK was strictly dependent on Ca2+ (one-half maximal activation 0.6 μM) and phosphorylated a wide diversity of substrates, in which Syntide 2 was the best phosphate acceptor (Michaelis constant = 30 μM). The kinase was inhibited by Ca2+- chelating agents, phenotiazine derivatives, and N-(6-aminohexyl)-5-chloro-1- naphthalenesulfonamide (one-half maximal inhibition = 0.25 mM). Polyclonal antibodies directed against the regulatory region of the soybean CDPK recognized a 53-kD polypeptide. In an autophosphorylation assay, this same band was strongly labeled with [γ-32P]ATP in the presence of Ca2+. CDPK activity was high in nontuberized plants, but increased 2.5-fold at the onset of tuber development and was reduced to one-half of its original activity when the tuber had completed formation. In the early stages of tuberization, Ca2+-dependent phosphorylation of endogenous targets (specific bands of 68, 51, and 46 kD) was observed. These polypeptides were not labeled in nontuberizing plants or in completely formed tubers, indicating that this phosphorylation is a stage-specific event. In addition, dephosphorylation of specific polypeptides was detected in tuberizing plants, suggesting the involvement of a phosphatase. Preincubation of crude extracts with phosphatase inhibitors rendered a 100% increase in CDPK activity.

Descripción: Hypoxia-Inducible Factor (HIF) prolyl hydroxylase domains (PHDs) have been proposed to act as sensors that have an important role in oxygen homeostasis. In the presence of oxygen, they hydroxylate two specific prolyl residues in HIF-α polypeptides, thereby promoting their proteasomal degradation. So far, however, the developmental consequences of the inactivation of PHDs in higher metazoans have not been reported. Here, we describe novel loss-of-function mutants of fatiga, the gene encoding the Drosophila PHD oxygen sensor, which manifest growth defects and lethality. We also report a null mutation in dHIF-α/sima, which is unable to adapt to hypoxia but is fully viable in normoxic conditions. Strikingly, loss-of-function mutations of sima rescued the developmental defects observed in fatiga mutants and enabled survival to adulthood. These results indicate that the main functions of Fatiga in development, including control of cell size, involve the regulation of dHIF/Sima. © 2005 European Molecular Biology Organization.

Descripción: The tumor suppressor protein p53 is known to be transported to the nucleus along microtubular tracks by cytoplasmic dynein. However, the connection between p53 and the dynein motor protein complex has not been established. Here, we show that hsp90·binding immunophilins link p53·hsp90 complexes to dynein and that prevention of that linkage in vivo inhibits the nuclear movement of p53. First, we show that p53·hsp90 heterocomplexes from DLD-1 human colon cancer cells contain an immunophilin (FKBP52, CyP-40, or PP5) as well as dynein. p53·hsp90·immunophilin·dynein complexes can be formed by incubating immunopurified p53 with rabbit reticulocyte lysate, and we show by peptide competition that the immunophilins link via their tetratricopeptide repeat domains to p53-bound hsp90 and by means of their PPIase domains to the dynein complex. The linkage of immunophilins to the dynein motor is indirect by means of the dynamitin component of the dynein-associated dynactin complex, and we show that purified FKBP52 binds directly by means of its PPIase domain to purified dynamitin. By using a temperature-sensitive mutant of p53 where cytoplasmic-nuclear movement occurs by shift to permissive temperature, we show that p53 movement is impeded when p53 binding to hsp90 is inhibited by the hsp90 inhibitor radicicol. Also, nuclear movement of p53 is inhibited when immunophilin binding to dynein is competed for by expression of a PPIase domain fragment in the same manner as when dynein linkage to cargo is dissociated by expression of dynamitin. This is the first demonstration of the linkage between an hsp90-chaperoned transcription factor and the system for its retrograde movement to the nucleus both in vitro and in vivo.

Descripción: Although the negative regulation of gene expression by insulin has been widely studied, the transcription factors responsible for the insulin effect are still unknown. The purpose of this work was to explore the molecular mechanisms involved in the insulin repression of the 5-aminolevulinate synthase (ALAS) gene. Deletion analysis of the 5′-regulatory region allowed us to identify an insulin-responsive region located at -459 to -354 bp. This fragment contains a highly homologous insulin-responsive (IRE) sequence. By transient transfection assays, we determined that hepatic nuclear factor 3 (HNF3) and nuclear factor 1 (NF1) are necessary for an appropriate expression of the ALAS gene. Insulin overrides the HNF3β or HNF3β plus NF1-mediated stimulation of ALAS transcriptional activity. Electrophoretic mobility shift assay and Southwestern blotting indicate that HNF3 binds to the ALAS promoter. Mutational analysis of this region revealed that IRE disruption abrogates insulin action, whereas mutation of the HNF3 element maintains hormone responsiveness. This dissociation between HNF3 binding and insulin action suggests that HNF3β is not the sole physiologic mediator of insulin-induced transcriptional repression. Furthermore, Southwestern blotting assay shows that at least two polypeptides other than HNF3β can bind to ALAS promoter and that this binding is dependent on the integrity of the IRE. We propose a model in which insulin exerts its negative effect through the disturbance of HNF3β binding or transactivation potential, probably due to specific phosphorylation of this transcription factor by Akt. In this regard, results obtained from transfection experiments using kinase inhibitors support this hypothesis. Due to this event, NF1 would lose accessibility to the promoter. The posttranslational modification of HNF3 would allow the binding of a protein complex that recognizes the core IRE. These results provide a potential mechanism for the insulin-mediated repression of IRE-containing promoters.

Descripción: Substrate-specific degradation of proteins by the ubiquitin-proteasome pathway is a precise mechanism that controls the abundance of key cell regulators. SCF complexes are a family of E3 ubiquitin ligases that target specific proteins for destruction at the 26S-proteasome. These complexes are composed of three constant polypeptides - Skp1, Cullin1/3 and Roc1/Rbx1 - and a fourth variable adapter, the F-box protein. Slimb (Slmb) is a Drosophila F-Box protein that fulfills several roles in development and cell physiology. We analyzed its participation in egg chamber development and found that slmb is required in both the follicle cells and the germline at different stages of oogenesis. We observed that in slmb somatic clones, morphogenesis of the germarium and encapsulation of the cyst were altered, giving rise to egg chambers with extra germline cells and two oocytes. Furthermore, in slmb somatic clones, we observed ectopic Fasciclin 3 expression, suggesting a delay in follicle cell differentiation, which correlated with the occurrence of ectopic polar cells, lack of interfollicular stalks and mislocalization of the oocyte. Later in oogenesis, Slmb was required in somatic cells to specify the position, size and morphology of dorsal appendages. Mild overactivation of the Dpp pathway caused similar phenotypes that could be antagonized by simultaneous overexpression of Slmb, suggesting that Slmb might normally downregulate the Dpp pathway in follicle cells. Indeed, ectopic expression of a dad-LacZ enhancer trap revealed that the Dpp pathway was upregulated in slmb somatic clones and, consistent with this, ectopic accumulation of the co-Smad protein, Medea, was recorded. By analyzing slmb germline clones, we found that loss of Slmb provoked a reduction in E2f2 and Dp levels, which correlated with misregulation of mitotic cycles during cyst formation, abnormal nurse cell endoreplication and impairment of dumping of the nurse cell content into the oocyte.