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Activity and stability of the proto-oncogene c-Myb are regulated by post-translational modifications, though the molecular mechanisms underlying such control are only partially understood. Here we describe the functional interaction of c-Myb with Pin1, an isomerase that binds to phosphorylated Ser/Thr-Pro motifs. We found that co-expression of c-Myb and Pin1 led to a net increase of c-Myb transactivation activity, both on reporter constructs as well as on an endogenous target gene. DNA-binding studies revealed that Pin1 did not increase the association of c-Myb with its response element in DNA. The increase of c-Myb transactivation activity was strictly dependent on the presence of an active catalytic center in Pin1. We provide evidence that c-Myb and Pin1 physically interacted, both upon ectopic expression of the proteins in HEK-293 cells as well as in the more physiological setting of HL60 cells, where c-Myb and Pin1 are resident proteins. By point mutating each individual Ser/Thr-Pro motif in c-Myb as well as by using deletion mutants we show that S528 in the EVES-motif was the docking site for Pin1. Mass spectrometry confirmed that S528 is phosphorylated in vivo. Finally, functional studies showed that mutation of S528 to alanine almost abolished the increase of transactivation activity by Pin1. This study reveals a new paradigm by which phosphorylation controls c-Myb function.

The c-myb gene is the progenitor of the v-myb oncogene, which causes avian myelomonocytic leukemia. Dysregulated c-myb gene expression is linked to the development of myeloid leukemia in mice and is predictive of poor prognosis in human colorectal cancer. Among the variety of post-translational modifications controlling the c-Myb protein, phosphorylation was shown to affect the transactivation activity and the rate of protein degradation. In this work we show that phosphorylation of c-Myb in response to stress led to rapid protein degradation, which occurred via a proteasome-dependent pathway. The kinases principally involved in this response were p38MAPK delta and, to a lesser extent, p38MAPK gamma. To assess whether c-Myb degradation was driven by changes in the overall level of phosphorylation or rather by phosphorylation at specific sites we systematically mutated potential sites of phosphorylation fulfilling the consensus for recognition by MAPKs (Ser/Thr-Pro). Among the point mutants examined, residues located downstream to the transactivation domain appeared to be essential for c-Myb stability. Particularly, mutation of Thr(354), Thr(486), Ser(556) and Thr(572) to Alanine conferred resistance to stress-induced degradation. The implications of c-Myb downregulation during inflammatory responses are discussed.

Nous avons affine la structure cristalline de la cobaltarthurite decouverte pres de Pastrana, dans le sud-est de l'Espagne [monoclinique, a 10.2694(4), b 9.6790(3), c 5.5723(2) A, β 94.277(2)°, P2 1 /c] selon la methode de Rietveld utilisee avec des donnees en diffraction X, jusqu'a des indices de concordance R w p et R B de 7.7 and 1.7%, respectivement. La cobaltarthurite est un nouveau membre du groupe de l'arthurite, un arseniate a dominance de cobalte, dont la formule ideale serait Co 2 + Fe 3 + 2 (AsO 4 ) 2 (OH) 2 .4H 2 O. Les resultats de l'affinement confirment que la cobaltarthurite est isostructurale avec les autres membres du groupe. Les analyses a la microsonde electronique montrent que la formule possede la stoechiometrie ideale, avec une incorporation mineure de Mg, Mn, Ni, Cu et Ca pour le Co, et des traces de P et de S en substitution pour As.

Diabetes impairs endothelial function and reparative neovascularization. The p75 receptor of neurotrophins (p75 NTR ), which is scarcely present in healthy endothelial cells (ECs), becomes strongly expressed by capillary ECs after induction of peripheral ischemia in type-1 diabetic mice. Here, we show that gene transfer-induced p75 NTR expression impairs the survival, proliferation, migration, and adhesion capacities of cultured ECs and endothelial progenitor cells (EPCs) and inhibits angiogenesis in vitro. Moreover, intramuscular p75 NTR gene delivery impairs neovascularization and blood flow recovery in a mouse model of limb ischemia. These disturbed functions are associated with suppression of signaling mechanisms implicated in EC survival and angiogenesis. In fact, p75 NTR depresses the VEGF-A/Akt/eNOS/NO pathway and additionally reduces the mRNA levels of ITGB1 [beta (1) integrin] , BIRC5 (survivin ), PTTG 1 ( securin ) and VEZF1 . Diabetic mice, which typically show impaired postischemic muscular neovascularization and blood perfusion recovery, have these defects corrected by intramuscular gene transfer of a dominant negative mutant form of p75 NTR . Collectively, our data newly demonstrate the antiangiogenic action of p75 NTR and open new avenues for the therapeutic use of p75 NTR inhibition to combat diabetes-induced microvascular liabilities.

Neurotrophins (NTs) control neuron survival and regeneration. Recent research showed that NTs possess cardiovascular actions. In this study, we investigated the hypothesis that the NT nerve growth factor (NGF) prevents cardiomyocyte apoptosis. We demonstrated that cultured rat neonatal cardiomyocytes (RNCMs) produce NGF and express its trkA (tropomyosin-related receptor A (NGF high-affinity receptor)) receptor. RNCMs given a neutralizing antibody for NGF or the trkA inhibitor K252a underwent apoptosis, thus suggesting that NGF is an endogenous prosurvival factor for cardiomyocytes. Adenovirus (Ad)-mediated NGF overexpression protected RNCMs from apoptosis induced by either hypoxia/reoxygenation or angiotensin II (AngII). Similarly, recombinant NGF inhibited AngII-induced apoptosis in isolated rat adult cardiomyocytes. Finally, in a rat model of myocardial infarction, NGF gene transfer promoted cardiomyocyte survival. In RNCMs, recombinant NGF induced trkA phosphorylation, followed by Ser473 phosphorylation and nuclear translocation of phospho-protein kinase B (Akt). In response to Akt activation, Forkhead transcription factors Foxo-3a and Foxo-1 were phosphorylated and excluded from the nucleus. The prosurvival effect of adenoviral vector carrying the human NGF gene was inhibited in vitro by K252a, LY294002 (a pan-phosphatidyl inositol 3-kinase – PI3K – inhibitor), an Akt small interfering RNA, and adenoviruses carrying a dominant negative mutant form of Akt (Ad.DN.Akt) or an Akt-resistant Foxo-3a (Ad.AAA-Foxo-3a). These results newly demonstrate the cardiac prosurvival action of NGF and provide mechanistic information on the signaling pathway, which encompasses trkA, PI3K-Akt, and Foxo.

The emergence of resistance to cisplatin is a serious drawback of cancer therapy. To help elucidate the molecular basis of this resistance, we examined matched ovarian cancer cell lines that differ in their DNA mismatch repair (MMR) status and the response to cisplatin. Checkpoint activation by cisplatin was identical in both lines. However, sensitive cells delayed S-phase transition, arrested at G(2)/M and died by apoptosis. The G(2)/M block was characterized by selective disappearance of homologous recombination (HR) proteins, which likely resulted in incomplete repair of the cisplatin adducts. In contrast, resistant cells transiently arrested at G(2)/M, maintained constant levels of HR proteins and ultimately resumed cell cycle progression. The net contribution of MMR to the cisplatin response was examined using matched semi-isogenic (HCT116+/-chr3) or strictly isogenic (293T-Lalpha(-/+)) cell lines. Delayed transition through S-phase in response to cisplatin was also observed in the MMR-proficient HCT116+chr3 cells. Unlike in the ovarian cell lines, however, both HCT116+chr3 and HCT116 permanently arrested at G(2)/M with an intact complement of HR proteins and died by apoptosis. A similar G(2)/M arrest was observed in the strictly isogenic 293T-Lalpha(-/+) cells. This confirmed that although MMR undoubtedly contributes towards the cytotoxicity of cisplatin, it is only one of several pathways that modulate the cellular response to this drug. However, our data highlighted the importance of HR to cisplatin cytotoxicity and suggested that HR status might represent a novel prognostic marker and possibly also a therapeutic target, the inhibition of which would substantially sensitize cells to cisplatin chemotherapy.

The c-myb gene encodes a transcription factor required for proliferation, differentiation, and survival of hematopoietic cells. Expression of c-Myb is often increased in hematological malignancies, but the underlying mechanisms are poorly understood. We show here that c-Myb has a longer half-life (at least 2-fold) in BCR/ABL-expressing than in normal hematopoietic cells. Such enhanced stability was dependent on a phosphatidylinositol 3-kinase (PI-3K)/Akt/GSKIIIbeta pathway(s) as indicated by the suppression of c-Myb expression upon treatment with PI-3K inhibitors or co-expression with dominant negative Akt or constitutively active GSKIIIbeta. Moreover, inhibition of GSKIIIbeta by LiCl enhanced c-Myb expression in parental 32Dcl3 cells. Compared with wild type c-Myb, three mutants (delta(358-452), delta(389-418), and L389A/L396A c-Myb) of the leucine zipper domain had increased stability. However, only expression of delta(358-452) was not affected by inhibition of the PI-3K/Akt pathway and was not enhanced by a proteasome inhibitor, suggesting that leucine zipper-dependent and -independent mechanisms are involved in the regulation of c-Myb stability. Indeed, delta(389-418) carrying four lysine-to-alanine substitutions (delta(389-418) K387A/K428A/K442A/K445A) was as stable as delta(358-452) c-Myb. Compared with full-length c-Myb, constitutive expression of delta(358-452) and delta(389-418) c-Myb in Lin-Sca-1+ mouse marrow cells increased cytokine-dependent primary and secondary colony formation. In K562 cells, expression of delta(358-452), delta(389-418), and L389A/L396A c-Myb led to enhanced proliferation after STI571 treatment. Thus, enhanced stability of c-Myb by activation of PI-3K-dependent pathway(s) might contribute to the higher proliferative potential of BCR/ABL-expressing and, perhaps, other leukemic cells.