No abstract

One of the mechanisms that regulate cell death is the reversible phosphorylation of proteins. ERK/MAPK phosphorylates caspase-9 at Thr125, and this phosphorylation is crucial for caspase-9 inhibition. Until now, the phosphatase responsible for Thr125 dephosphorylation has not been described. Here, we demonstrate that in IL-2-proliferating cells, phosphorylated serine/threonine phosphatase type 1α (PP1α) associates with phosphorylated caspase-9. IL-2 deprivation induces PP1α dephosphorylation, which leads to its activation and, as a consequence, dephosphorylation and activation of caspase-9 and subsequent dissociation of both molecules. In cell-free systems supplemented with ATP caspase-9 activation is induced by addition of cytochrome c and we show that in this process PP1α is indispensable for triggering caspase-9 as well as caspase-3 cleavage and activation. Moreover, PP1α associates with caspase-9 in vitro and in vivo, suggesting that it is the phosphatase responsible for caspase-9 dephosphorylation and activation. Finally, we describe two novel phosphatase-binding sites different from the previously described PP1α consensus motifs, and we demonstrate that these novel sites mediate the interaction of PP1α with caspase-9.

The Aiolos transcription factor, member of the Ikaros family of zinc finger proteins, plays an important role in the control of mature B lymphocyte differentiation and proliferation, and its function appears to be modulated through alternative splicing. IntroductionMature B-cell neoplasms, including chronic lymphocytic leukemia (CLL), represent frequent malignancies of mature B cells whose cell derivation and pathogenesis are unknown. 1,2 Here we focused on Aiolos, a hematopoietic chromatin remodeler and transcription factor of the Ikaros family that plays critical roles in B-cell differentiation, 3-6 and we examined whether a deregulation of its expression was an integral part of malignant transformation in CLL and other B-cell lymphomas. Methods Subjects and B-cell isolationCryopreserved peripheral blood mononuclear cell (PBMC) samples were collected and informed consent obtained in accordance with the Declaration of Helsinki from CLL or other B-cell lymphoma patients followed at the Hematological Department of Pitié-Salpêtrière Hospital, according to a protocol approved by the institutional ethic committee. Diagnosis was assessed according to the recent World Health Organization classification. In all cases, blood sample collection was performed at the time of diagnosis. Fresh blood from healthy donors was collected by the Etablissement Francais du Sang (France), and PBMCs were frozen after density gradient isolation. Total RNA extraction was done after B cells isolation by anti-CD19 magnetic beads (Dynal Biotech, Oslo, Norway). Reverse-transcribed quantitative polymerase chain reaction analysisRNA (500 ng) was reversed transcribed and amplified, either by Titanium one-step RT-PCR (Clontech, Mountain View, CA) or by Superscript II (Invitrogen, Carlsbad, CA) and quantitative polymerase chain reaction (PCR) done using predeveloped TaqMan gene expression assays (Applied Biosystems, Foster City, CA). Chromatin immunoprecipitationAnti-CD19 Dynal beads were detached by DETACHaBEAD before proceeding to chromatin cross-link. Chromatin immunoprecipitation (ChIP) assays were carried out as previously described 7 with primers spanning a 2-kb region upstream and downstream of the Aiolos translation initiation site. Western blot analysisWestern blot analysis was carried out as previously described. 8 Results and discussion The hAio1 transcript represents more than 80% of the Aiolos isoforms in B cellsWe first investigated the expression pattern of different Aiolos isoforms in PBMCs of patients diagnosed with mature B-cell malignancies (17 CLL, 8 mantle zone, and 2 marginal zone lymphomas; Figure 1A; Table 1). Analysis of healthy donors revealed the presence of 6 Aiolos variants. Five corresponded to hAio1, hAio2, hAio3/4, and hAio5 as previously described. 9 A new shorter variant was observed and identified by direct sequencing of the PCR products as hAio-del(4,5,6), lacking the 4 N-terminal zinc fingers. No major differences in the distribution of Aiolos isoforms were detected between healthy donors and CLL/other B-cell...