A new AQP1 null allele identified in a Gypsy woman who developed an anti-CO3 during her first pregnancy.

BACKGROUND AND OBJECTIVES: The Colton blood group antigens are carried by the AQP1 water channel. AQP1(-/-) individuals, also known as Colton-null since they express no Colton antigens, do not suffer any apparent clinical consequence but may develop a clinically significant alloantibody (anti-CO3) induced by transfusion or pregnancy. Identification and transfusion support of Colton-null patients are highly challenging, not only due to the extreme rarity of this phenotype, the lack of appropriate reagents in most laboratories, as well as the possibility of confusing it with the recently described CO:-1,-2,3,-4 phenotype where AQP1 is present. This study investigated a new Colton-null case and evaluated three commercially available anti-AQP1s to identify Colton-null red blood cell samples. METHODS: The Colton-null phenotype was investigated by standard serological techniques, AQP1 sequencing, immunoblot and flow cytometry analyses. RESULTS: We identified and characterized the Colton-null phenotype in a Gypsy woman who developed an anti-CO3 during her first pregnancy. After developing a simple and robust method to sequence AQP1, we showed that she was apparently homozygous for a new AQP1 null allele, AQP1 601delG, whose product is not expressed in her red blood cells. We also established the Colton specificity of three commercially available anti-AQP1s in immunoblot and/or flow cytometry analyses. CONCLUSION: This Gypsy woman represents the sixth Colton-null case characterized at the serological, genetic and biochemical levels. The validation here of new reagents and methods should facilitate the identification of Colton-null individuals.

Rsk1 mediates a MEK-MAP kinase cell survival signal.

BACKGROUND: Growth factors activate an array of cell survival signaling pathways. Mitogen-activated protein (MAP) kinases transduce signals emanating from their upstream activators MAP kinase kinases (MEKs). The MEK-MAP kinase signaling cassette is a key regulatory pathway promoting cell survival. The downstream effectors of the mammalian MEK-MAP kinase cell survival signal have not been previously described. RESULTS: We identify here a pro-survival role for the serine/threonine kinase Rsk1, a downstream target of the MEK-MAP kinase signaling pathway. In cells that are dependent on interleukin-3 (IL-3) for survival, pharmacological inhibition of MEKs antagonized the IL-3 survival signal. In the absence of IL-3, a kinase-dead Rsk1 mutant eliminated the survival effect afforded by activated MEK. Conversely, a novel constitutively active Rsk1 allele restored the MEK-MAP kinase survival signal. Experiments in vitro and in vivo demonstrated that Rsk1 directly phosphorylated the pro-apoptotic protein Bad at the serine residues that, when phosphorylated, abrogate Bad's pro-apoptotic function. Constitutively active Rsk1 caused constitutive Bad phosphorylation and protection from Bad-modulated cell death. Kinase-inactive Rsk1 mutants antagonize Bad phosphorylation. Bad mutations that prevented phosphorylation by Rsk1 also inhibited Rsk1-mediated cell survival. CONCLUSIONS: These data support a model in which Rsk1 transduces the mammalian MEK-MAP kinase signal in part by phosphorylating Bad.

Disruption of 3-phosphoinositide-dependent kinase 1 (PDK1) signaling by the anti-tumorigenic and anti-proliferative agent n-alpha-tosyl-l-phenylalanyl chloromethyl ketone.

The anti-tumorigenic and anti-proliferative effects of N-alpha-tosyl-l-phenylalanyl chloromethyl ketone (TPCK) have been known for more than three decades. Yet little is known about the discrete cellular targets of TPCK controlling these effects. Previous work from our laboratory showed TPCK, like the immunosuppressant rapamycin, to be a potent inhibitor of the 70-kilodalton ribosomal S6 kinase 1 (S6K1), which mediates events involved in cell growth and proliferation. We show here that rapamycin and TPCK display distinct inhibitory mechanisms on S6K1 as a rapamycin-resistant form of S6K1 was TPCK-sensitive. Additionally, we show that TPCK inhibited the activation of the related kinase and proto-oncogene Akt. Upstream regulators of S6K1 and Akt include phosphoinositide 3-kinase (PI 3-K) and 3-phosphoinositide-dependent kinase 1 (PDK1). Whereas TPCK had no effect on either mitogen-regulated PI 3-K activity or total cellular PDK1 activity, TPCK prevented phosphorylation of the PDK1 regulatory sites in S6K1 and Akt. Furthermore, whereas both PDK1 and the mitogen-activated protein kinase (MAPK) are required for full activation of the 90-kilodalton ribosomal S6 kinase (RSK), TPCK inhibited RSK activation without inhibiting MAPK activation. Consistent with the capacity of RSK and Akt to mediate a cell survival signal, in part through phosphorylation of the pro-apoptotic protein BAD, TPCK reduced BAD phosphorylation and led to cell death in interleukin-3-dependent 32D cells. Finally, in agreement with results seen in embryonic stem cells lacking PDK1, protein kinase A activation was not inhibited by TPCK showing TPCK specificity for mitogen-regulated PDK1 signaling. TPCK inhibition of PDK1 signaling thus disables central kinase cascades governing diverse cellular processes including proliferation and survival and provides an explanation for its striking biological effects.

Interaction of cyclooxygenases with an apoptosis- and autoimmunity-associated protein.

Cyclooxygenases (COXs) 1 and 2 are 72-kDa, intralumenal residents of the endoplasmic reticulum (ER) and nuclear envelope, where they catalyze the rate-limiting steps in the conversion of arachidonate to the physiologically dynamic prostanoids. Recent studies, including the generation of knockout mice, show COX-1 and COX-2 to have biologically distinct roles within cells and organisms. Also apparent is that arachidonate substrate is selectably metabolized by COX-2 after mitogen stimulation in many cells that contain both isoforms. Because COX-1 and COX-2 are highly conserved in all residues needed for catalysis and in their purified forms have almost identical kinetic properties, we have searched for COX-interacting ER proteins that might mediate these unique isoenzymic properties. Using COXs as bait in the yeast two-hybrid system, we identified autoimmunity- and apoptosis-associated nucleobindin (Nuc) as a protein that specifically interacts with both isoenzymes. COX-Nuc binding was substantiated by immunoprecipitation experiments, which showed that COX-1 and, to a lesser extent, COX-2 form complexes with Nuc in vitro. When overexpressed in COS-1 cells, Nuc was found to be extracellularly released. However, when Nuc was co-overexpressed with COX-1 or COX-2, its release was reduced by >80%. This finding suggests that COX isoenzymes participate in the retention of Nuc within the lumen of the ER, where COX may regulate the release of Nuc from the cell. It also identifies Nuc as a potential regulator of COXs through this interaction.