Space flight alters bacterial gene expression and virulence and reveals a role for global regulator Hfq.

A comprehensive analysis of both the molecular genetic and phenotypic responses of any organism to the space flight environment has never been accomplished because of significant technological and logistical hurdles. Moreover, the effects of space flight on microbial pathogenicity and associated infectious disease risks have not been studied. The bacterial pathogen Salmonella typhimurium was grown aboard Space Shuttle mission STS-115 and compared with identical ground control cultures. Global microarray and proteomic analyses revealed that 167 transcripts and 73 proteins changed expression with the conserved RNA-binding protein Hfq identified as a likely global regulator involved in the response to this environment. Hfq involvement was confirmed with a ground-based microgravity culture model. Space flight samples exhibited enhanced virulence in a murine infection model and extracellular matrix accumulation consistent with a biofilm. Strategies to target Hfq and related regulators could potentially decrease infectious disease risks during space flight missions and provide novel therapeutic options on Earth.

Overexpression of Hp95 induces G1 phase arrest in confluent HeLa cells.

Xp95, a protein recently identified in Xenopus laevis, is potentially involved in progesterone-induced Xenopus oocyte maturation. In this study, we cloned a human homologue of Xp95, designated Hp95, and examined the effect of its overexpression on the growth properties of human malignant HeLa cells which have lost the contact inhibition of cell proliferation. We observed that although HeLa cells did not undergo G1 phase arrest at any stage after confluence, they were able to downregulate their G1 phase CDK activities in response to confluence. When Hp95 was overexpressed in HeLa cells by transfection with a constitutive or an inducible expression vector containing a full-length Hp95 transgene, HeLa cells became able to undergo G1 phase arrest and form a monolayer culture after confluence. However, the G1 phase CDK activities in these Hp95 overexpressing cells were not inhibited further as compared to control cells after confluence. These results indicate that the defects in HeLa cells that cause the loss of contact inhibition of cell proliferation are in components downstream of the G1 phase CDKs and that overexpression of Hp95 counteracts some of these defects.

Identification and cloning of xp95, a putative signal transduction protein in Xenopus oocytes.

A 95-kDa protein in Xenopus oocytes, Xp95, was shown to be phosphorylated from the first through the second meiotic divisions during progesterone-induced oocyte maturation. Xp95 was purified and cloned. The Xp95 protein sequence exhibited homology to mouse Rhophilin, budding yeast Bro1, and Aspergillus PalA, all of which are implicated in signal transduction. It also contained three conserved features including seven conserved tyrosines, a phosphorylation consensus sequence for the Src family of tyrosine kinases, and a proline-rich domain near the C terminus that contains multiple SH3 domain-binding motifs. We showed the following: 1) that both Xp95 isolated from Xenopus oocytes and a synthetic peptide containing the Src phosphorylation consensus sequence of Xp95 were phosphorylated in vitro by Src kinase and to a lesser extent by Fyn kinase; 2) Xp95 from Xenopus oocytes or eggs was recognized by an anti-phosphotyrosine antibody, and the relative abundance of tyrosine-phosphorylated Xp95 increased during oocyte maturation; and 3) microinjection of deregulated Src mRNA into Xenopus oocytes increased the abundance of tyrosine-phosphorylated Xp95. These results suggest that Xp95 is an element in a tyrosine kinase signaling pathway that may be involved in progesterone-induced Xenopus oocyte maturation.

A phosphatase activity in Xenopus oocyte extracts preferentially dephosphorylates the MPM-2 epitope.

MPM-2 antigens are a large family of mitotic phosphoproteins that contain similar phosphoepitopes recognized by the anti-phosphoepitope antibody MPM-2 (MPM-2 epitopes). These proteins are phosphorylated during M phase induction and dephosphorylated from the onset of anaphase through interphase. Since biochemical characterization of the MPM-2 epitope phosphatase requires a specific assay for its activity, we tested different methods for measurement of the MPM-2 epitope phosphatase activity in crude cell lysates. First, an ELISA-based assay was designed that measured the phosphatase-induced reduction of the MPM-2 reactivity in crude M phase cell lysates. Using this assay to follow the phosphatase activity during sequential chromatography of Xenopus oocyte extracts, one predominant peak of phosphatase activity was detected which was separated from the majority of PP1 and PP2A activities. This phosphatase activity dephosphorylated the MPM-2 epitope on multiple MPM-2 antigens. The second method measured dephosphorylation of cdc25, a known MPM-2 antigen. Two major peaks of cdc25 dephosphorylating activities were detected during the sequential chromatography, one that copurified with the major peak of MPM-2 epitope phosphatase activity, and the other with the major peak of PP2A activity. Finally, we examined whether GST-MPM2, a fusion protein between glutathione S-transferase and a 19-residue peptide that contained two representative MPM-2 epitope sequences, could be dephosphorylated efficiently and specifically by the major MPM-2 epitope phosphatase activity in Xenopus oocyte extracts. Neither the crude extract nor the partially purified MPM-2 epitope phosphatase activity efficiently dephosphorylated the MPM-2 epitope on GST-MPM2. These results demonstrate that the ELISA-based assay preferentially detects the MPM-2 epitope phosphatase activity in crude cell lysates which may represent a physiological MPM-2 epitope phosphatase.

Mitogen-activated protein kinase and cyclin B/Cdc2 phosphorylate Xenopus nuclear factor 7 (xnf7) in extracts from mature oocytes. Implications for regulation of xnf7 subcellular localization.

Xenopus nuclear factor 7 (xnf7) is a maternally expressed putative transcription factor that exhibits phosphorylation-dependent changes in subcellular localization during early Xenopus development. Xnf7 is localized to the germinal vesicle (nucleus) of immature oocytes in a hypophosphorylated state. Xnf7 is phosphorylated during oocyte maturation and released to the cytoplasm. The protein is retained in the cytoplasm during early embryonic cleavage stages but returns to nuclei at the mid-blastula transition. Xnf7 is phosphorylated at two sites during oocyte maturation, designated P1, consisting of one threonine at position 103, and P2, consisting of three clustered threonines at positions 209, 212, and 218. Phosphorylation of both sites is important in regulating xnf7 localization. The P1 site can be phosphorylated by cyclin B/Cdc2 in vitro. To further understand the mechanisms regulating subcellular localization of xnf7 during early development, kinases capable of catalyzing phosphorylation of the P2 site were purified from mature oocyte extracts. We found that mitogen-activated protein kinase phosphorylated Thr212 and cyclin B/Cdc2 phosphorylated Thr 209 and Thr212. No other kinase in mature oocyte extracts phosphorylated the xnf7 P2 site to a significant extent. These results implicate mitogen-activated protein kinase and cyclin B/Cdc2 in regulating xnf7 localization during oocyte maturation. This also suggests that localization of xnf7 may be regulated by multiple kinase activation pathways.

MPM-2 epitope sequence is not sufficient for recognition and phosphorylation by ME kinase-H.

Monoclonal antibody MPM-2 recognizes a large family of mitotic phosphoproteins in a phosphorylation-dependent manner. The antigenic phosphoepitope, designated the MPM-2 epitope, putatively consists of hydrophobic residue-Thr/Ser-Pro-hydrophobic residue-uncharged/basic residue. In this study, we addressed whether this sequence motif contains all the information necessary for recognition and phosphorylation by the kinase that phosphorylates most MPM-2 antigens. A fusion protein between glutathione S-transferase and a 19-residue peptide that contained two representative MPM-2 epitope sequences overlapping with two potential MAP kinase phosphorylation sites was constructed. Both the MPM-2 epitope sequences in the fusion protein (GST-MPM2) were phosphorylated by Xenopus egg extract, making the fusion protein MPM-2 reactive. However, while MAP kinase phosphorylated both the MPM-2 epitope sequences, neither ME kinase-H, a good candidate for a major MPM-2 epitope kinase, nor mitotic cdc2 kinase, which is known to phosphorylate certain MPM-2 antigens in vitro, phosphorylated GST-MPM2 to any significant extent. Furthermore, depletion of MAP kinase activity removed most, if not all, of the GST-MPM2 phosphorylating activity from crude Xenopus egg extracts. These results suggest that additional or different structural information than that provided by the deduced MPM-2 epitope sequence is required for recognition and phosphorylation by ME kinase-H or other major MPM-2 epitope kinases. They also offer a valid explanation for selective phosphorylation of certain MPM-2 antigens by MAP kinase as well as selective recognition of certain phosphorylated MAP kinase substrates by MPM-2.