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[Objective]: Map3k8 (Cot/Tpl2) activates the MKK1/2-ERK1/2, MAPK pathway downstream from interleukin-1R, tumor necrosis factor-αR, NOD-2R (nucleotide-binding oligomerization domain-like 2R), adiponectinR, and Toll-like receptors. Map3k8 plays a key role in innate and adaptive immunity and influences inflammatory processes by modulating the functions of different cell types. However, its role in atherogenesis remains unknown. In this study, we analyzed the role of this kinase in this pathology. [Approach and Results]: We show here that Map3k8 deficiency results in smaller numbers of Ly6C high CD11c low and Ly6C low CD11c high monocytes in ApoE - /- mice fed a high-fat diet (HFD). Map3k8 ApoE monocytes displayed high rates of apoptosis and reduced amounts of Nr4a1, a transcription factor known to modulate apoptosis in Ly6C low CD11c high monocytes. Map3k8 ApoE splenocytes and macrophages showed irregular patterns of cytokine and chemokine expression. Map3k8 deficiency altered cell adhesion and migration in vivo and decreased CCR2 expression, a determinant chemokine receptor for monocyte mobilization, on circulating Ly6C high CD11c low monocytes. Map3k8 ApoE mice fed an HFD showed decreased cellular infiltration in the atherosclerotic plaque, with low lipid content. Lesions had similar size after Map3k8 +/+ ApoE bone marrow transplant into Map3k8 ApoE and Map3k8 +/+ ApoE mice fed an HFD, whereas smaller plaques were observed after the transplantation of bone marrow lacking both ApoE and Map3k8. [Conclusions]: Map3k8 decreases apoptosis of monocytes and enhances CCR2 expression on Ly6C high CD11c low monocytes of ApoE mice fed an HFD. These findings explain the smaller aortic lesions in ApoE mice with Map3k8 ApoE bone marrow cells fed an HFD, supporting further studies of Map3k8 as an antiatherosclerotic target.

GATA1 is a hematopoietic transcription factor essential for expression of most genes encoding erythro-megakaryocytic proteins, i.e., globins and platelet glycoproteins. A role for GATA1 as a cell proliferation regulator has been proposed, as some of its bona fide targets comprise global regulators, such as c-KIT or c-MYC, or cell cycle factors, i.e., CYCLIN D or p21CIP1. In this study, we describe that GATA1 directly regulates the expression of replication licensing factor CDC6. Using reporter transactivation, electrophoretic mobility shift and chromatin immunoprecipitation assays, we show that GATA1 stimulates CDC6 transcription by binding to a canonical binding site located within a 166bp enhancer region upstream CDC6 promoter. This evolutionary conserved GATA binding site conforms to recently described chromatin occupancy rules, i.e., preferred bases within core WGATAR (TGATAA), 5′ and 3′ flanking bases (GGTGATAAGG) and distance to the transcription initiation site. We also found adjacent conserved binding sites for ubiquitously expressed transcription factor CP2, needed for GATA activity on CDC6 enhancer. Our results add to the growing evidence for GATA1 acting as a direct transcriptional regulator of the cell cycle machinery, thus linking cell proliferation control and specific gene expression programs during lineage differentiation. © 2012 Landes Bioscience., This work and L.P. were supported by grant BFU2008–03386/BMC from former Ministerio de Ciencia e Innovación (MICINN) of Spain. B.F-M was recipient of a fellowship from the “Formación de Personal Investigador” Program, also from MICINN.

Polycomb chromatin modifiers regulate hematopoietic pluripotent stem and progenitor cell self-renewal and expansion. Polycomb complex redundancy and biochemical heterogeneity complicate the unraveling of the functional contributions of distinct components. We have studied the hematopoietic activity of RYBP, a direct interactor and proposed modulator of RING1A/RING1B-dependent histone H2A monoubiquitylation (H2AUb). Using a mouse model to conditionally inactivate Rybp in adult hematopoiesis, we have found that RYBP deletion results in a reversion of B-1-to-B-2 B-cell progenitor ratios, i.e., of the innate (predominantly fetal) to acquired (mostly adult) immunity precursors. Increased numbers of B-1 progenitors correlated with a loss of pre-proB cells, the B-2 progenitors. RYBP-deficient stem and progenitor cell populations (LKS) and isolated common lymphoid progenitors (CLP) gave rise to increased numbers of B-1 progenitors in vitro. Rybp inactivation, however, did not result in changes of global H2AUb and did not interact genetically with Ring1A or Ring1B deletions. These results show that a sustained regulation of the B-1-to-B-2 switch is needed throughout adult life and that RYBP plays an important role in keeping B-2 dominance, most likely independently of its Polycomb affiliation.

The functions of polycomb products extend beyond their well-known activity as transcriptional regulators to include genome duplication processes. Polycomb activities during DNA replication and DNA damage repair are unclear, particularly without induced replicative stress.We have used a cellularmodel of conditionally inactive polycomb E3 ligases (RING1A and RING1B), which monoubiquitylate lysine 119 of histone H2A (H2AK119Ub), to examine DNA replication in unperturbed cells. We identify slow elongation and fork stalling during DNA replication that is associated with the accumulation of mid and late S-phase cells. Signs of replicative stress and colocalisation of double-strand breaks with chromocenters, the sites of coalesced pericentromeric heterocromatic (PCH) domains, were enriched in cells at mid S-phase, the stage at which PCH is replicated. Altered replication was rescued by targeted monoubiquitylation of PCH through methyl- CpG binding domain protein 1. The acute senescence associated with the depletion of RING1 proteins, which is mediated by p21 (also known as CDKN1A) upregulation, could be uncoupled from a response to DNA damage. These findings link cell proliferation and the polycomb proteins RING1A and RING1B to S-phase progression through a specific function in PCH replication, This work was supported by the Ministerio de Economı́a y Competitividad (MINECO) [grant numbers BFU2010-18146, SAF2013-47997 to M.V.]; the CAM OncoCycle Programme [grant number S2010/BMD-2470 to M.V.]; and by a MINECO grant to A.A. [grant number BFU2013-42918-P]. M.B. and F.N. are recipients of MINECO and Campus de Excelencia Internacional Universidad Autónoma de Madrid + Consejo SUperior de Investigaciones Cientı́ficas (CEI UAMCSIC) fellowships, respectively; K.S. is supported by FP7-PEOPLE-2011-ITN [grant number 289611].

Cord blood (CB) has successfully been used as a stem cell source for haemopoietic reconstitution. However, a significant delay in platelet engraftment is consistently found in CB versus adult peripheral blood (PB) or bone marrow transplants. We sought to determine whether or not CB megakaryocytes have reached terminal maturation and, hence, full thrombopoietic potential. A comparative analysis of megakaryocytes cultured from either CB or PB progenitors in the presence of thrombopoietin (TPO) showed a similar differentiation response, although proliferation was 2.4 times higher in CB than in PB cells. Importantly, the TPO-induced ploidy level was notably different: whereas 82.7% of CB megakaryocytes remained diploid (2N) at the end of the culture, more than 50% of PB megakaryocytes had reached a DNA content equal to or higher than 4N. Western blot and flow cytometry analyses revealed that only polyploid PB megakaryocytes expressed cyclins E, A and B, whereas cyclin D3 was detected in both fetal and adult megakaryocytic nuclei. These data suggest that establishment of endomitotic cycles is impaired in CB megakaryocytes, associated with a differential regulation of G1/S cell cycle factors. We believe that the relative immaturity of fetal megakaryocytes could be a contributing factor to the delayed platelet engraftment in cord blood transplantation.

Megakaryocytes become polyploid by entering a truncated cell cycle, consisting of alternate S phases and abortive mitoses. We have investigated the regulation of the G1/S transition by comparing two megakaryoblastic cell lines, HEL and K562, which respectively do or do not become polyploid in response to phorbol esters. A pronounced downregulation of cyclin A, and to a lesser extent of cyclin E, occurred in K562 cells during the first 24 h after TPA treatment, in contrast with re-replicating HEL cells, in which both cyclins were present in individual G2/M cells. Transactivation experiments suggested that the absence of cyclin A in differentiated K562 cells could be due to a TPA-mediated inhibition of its transcription. To investigate the potential role of cyclin E in the establishment of re-replication cycles, we isolated K562 clones constitutively expressing cyclin E. The resulting clones, and also K562 cells transiently expressing cyclin E, entered re-replication cycles when treated with TPA. The transcriptional activity of the cyclin A promoter was not inhibited after TPA treatment, and although the levels of cyclin A fluctuated during further re-replication cycles, they never decreased below S phase levels. We conclude that the presence of cyclin E in megakaryoblastic G2/M cells determines cyclin A expression and allows the entrance into an extra S phase.

11 pages, 7 figures.-- et al., Polycomb group (PcG) proteins act as positive regulators of cell proliferation. Ring1B is a PcG gene essential for embryonic development, but its contribution to cell turnover in regenerating tissues in not known. Here, we have generated a conditional mouse mutant line to study the Ring1B role in adult hematopoiesis. Mutant mice developed a hypocellular bone marrow that paradoxically contained an enlarged, hyperproliferating compartment of immature cells, with an intact differentiation potential. These alterations were associated with differential upregulation of cyclin D2, which occurred in all mutant bone marrow cells, and of p16(Ink4a), observed only in the differentiated compartment. Concurrent inactivation of Ink4a rescued the defective proliferation of maturing cells but did not affect the hyperproliferative activity of progenitors and resulted in a shortening of the onset of lymphomas induced by Ink4a inactivation. These data show that Ring1B restricts the progenitors' proliferation and promotes the proliferation of their maturing progeny by selectively altering the expression pattern of cell cycle regulators along hematopoietic differentiation. The novel antiproliferative role of Ring1B's downregulation of a cell cycle activator may play an important role in the tight control of hematopoietic cell turnover., T.M. and M.R-T. were recipients of FPI and FPU fellowships, respectively, from the Ministerio de Educación y Ciencia. This work was supported by grants from the Fundación Médica Mutua Madrileña (C.C.), Plan Nacional de Investigacion Científica BFU2005-03651 (C.C.) and SAF2004-06952-CO2-01 (M.V.), the OncoCycle program from the Comunidad de Madrid (M.V.), and in part by a grant of the Genome Network Project from the Ministry of Education Culture, Sports, Science and Technology of the Japanese Government (H.K.).

Summary The 3q21q26 syndrome leukaemias are characterised by dystrophic megakaryocytes, elevated platelet counts, ectopic EVI1 protein production and poor prognosis. To investigate the molecular basis of this disease, we developed a model system to examine the biological activity of EVI1 in a megakaryocyte progenitor cell line. For this purpose, Evi1 was conditionally expressed in human erythroleukaemia cells (HEL) that progress along the megakaryocyte lineage in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA-stimulated HEL cells normally undergo: (1) growth arrest; (2) altered morphology; (3) endomitosis and (4) characteristic changes in gene expression, including reduction of the erythroid-specific glycophoryn A and elevation of the specific glycoproteins GPIIIa and GPVI. Enforced Evi1 expression alone had no effect upon HEL cell proliferation or differentiation but a phenotype was manifest upon stimulation to differentiate. Evi1expressing, TPA-treated HEL cells still showed growth arrest, had reduced and enhanced glycophoryn A and GPIIIa mRNA’s, respectively, but failed to significantly elevate GPVI mRNA. This was accompanied by inhibition of endomitosis and altered cell morphology. Sustained CDK2 catalytic activity, typically associated with megakaryocyte endomitosis, was dramatically decreased in TPA-stimulated Evi1-expressing HEL cells because of significantly reduced levels of cyclin A. Therefore, enforced Evi1 expression could inhibit megakaryocyte differentiation although retention of some characteristic molecular changes, in combination with a block in endomitosis and altered morphology, suggest a defect in lineage progression. These results suggest that ectopic Evi1 expression contributes to a defective megakaryocyte differentiation programme and is likely to contribute to the phenotype observed in 3q21q26 syndrome leukaemias.

Cdc6 is a key regulator of the strict alternation of S and M phases during the mitotic cell cycle. In mammalian and plant cells that physiologically become polyploid, cdc6 is transcriptionally and post-translationally regulated. We have recently reported that Cdc6 levels are maintained in megakaryoblastic HEL cells, but severely downregulated by ectopic expression of transcriptional repressor Drosophila melanogaster escargot. Here, we show that cdc6 promoter activity is upregulated during megakaryocytic differentiation of HEL endoreplicating cells, and that Escargot interferes with such activation. Transactivation experiments showed that a 1.7 kb region located at 2800 upstream cdc6 transcription initiation site behaved as a potent enhancer in endoreplicating cells only. This activity was mainly dependent on a novel cis-regulatory element composed by an E2 box overlapping a GATA motif. Ectopic Escargot could bind this regulatory element in vitro and endogenous GATA-1 and E2A formed specific complexes in megakaryoblastic cells as well as in primary megakaryocytes. Chromatin Immunoprecipitation analysis revealed that both transcription factors were occupying the E2 box/GATA site in vivo. Altogether, these data suggest that cdc6 expression could be actively maintained during megakaryocytic differentiation through transcriptional mechanisms involving specific cis- and trans-regulatory elements. © Oxford University Press 2004; all rights reserved., This work was supported by grants PM98-0046 from the Spanish Ministry of Science and Technology, CAM 08.3/0037/2001 from the ‘Comunidad Autónoma de Madrid’ and FIS 01/0090-02 from ‘Fondo de Investigaciones Sanitarias’ to C.C. N.V. was partly supported by a Postdoctoral Fellowship from ‘Comunidad Autónoma de Madrid’ (Spain).

12 pages, 7 figures., Endomitosis is the process by which mammalian megakaryocytes become polyploid during terminal differentiation. As in other endoreplicating cells, cyclin-cdk complexes are distinctly regulated, probably to overcome the strict mechanisms that prevent rereplication in most somatic cells. We have asked whether key factors involved in the assembly and licensing of replication origins are equally regulated during endomitosis. Cdc6, cdt1, and geminin expression was analyzed during differentiation of two human megakaryoblastic cell lines, HEL and K562, which respectively do and do not establish endoreplication cycles. Geminin was downregulated, whereas cdt1 levels were maintained upon differentiation of both cell lines, independently of whether cells entered extra S-phases. In contrast, cdc6 was present and remained nuclear only in differentiated endoreplicating cells. Interestingly, cdc6 protein expression was reestablished in K562 cells that underwent endomitosis after transient or stable cyclin E overexpression. The high levels of cyclin E reached in these cells appeared to influence the stabilization of cdc6 protein rather than its RNA transcription rate. Finally, cdc6 overexpression drove HEL cells into endoreplication cycles in the absence of differentiation stimuli. Our results show that both cdt1 and cdc6 are differentially regulated during megakaryocytic differentiation and suggest an active role of cdc6 in endomitosis., This work was supported by a grant from the Ministry of Education to C.C. (PM98–0046). N.V. was supported by a Postdoctoral Fellowship from “Comunidad Autónoma de Madrid” (Spain).

Inhibition of protein tyrosine phosphatase 1B (PTP1B) has been suggested as an attractive target to improve insulin sensitivity in different cell types. In the present work, we have investigated the effect of PTP1B deficiency on the response of human and murine macrophages. Using in vitro and in vivo approaches in mice and silencing PTP1B in human macrophages with specific siRNAs, we have demonstrated that PTP1B deficiency increases the effects of pro-inflammatory stimuli in both human and rodent macrophages at the time that decreases the response to alternative stimulation. Moreover, the absence of PTP1B induces a loss of viability in resting macrophages and mainly after activation through the classic pathway. Analysis of early gene expression in macrophages treated with pro-inflammatory stimuli confirmed this exacerbated inflammatory response in PTP1Bdeficient macrophages. Microarray analysis in samples from wild-type and PTP1B-deficient macrophages obtained after 24 h of pro-inflammatory stimulation showed an activation of the p53 pathway, including the excision base repair pathway and the insulin signaling pathway in the absence of PTP1B. In animal models of lipopolysaccharide (LPS) and D-galactosamine challenge as a way to reveal in vivo inflammatory responses, animals lacking PTP1B exhibited a higher rate of death. Moreover, these animals showed an enhanced response to irradiation, in agreement with the data obtained in the microarray analysis. In summary, these results indicate that, although inhibition of PTP1B has potential benefits for the treatment of diabetes, it accentuates pro-inflammatory responses compromising at least macrophage viability., This work was supported by grants BFU2011-24760 and SAF2012-33283 from MINECO, S2010/BMD-2378 and S2010/BMD-2423 from Comunidad de Madrid and FIS-RIC RD06/0014/0025 and EFSD and Amylin Paul Langerhans Grant. Ciberdem and Ciberehd are funded by the Instituto de Salud Carlos III.

Certain cell types escape the strict mechanisms imposed on the majority of somatic cells to ensure the faithful inheritance of parental DNA content. This is the case in many embryonic tissues and certain adult cells such as mammalian hepatocytes and megakaryocytes. Megakaryocytic endomitosis is characterized by repeated S phases followed by abortive mitoses, resulting in mononucleated polyploid cells. Several cell cycle regulators have been proposed to play an active role in megakaryocytic polyploidization; however, little is known about upstream factors that could control endomitosis. Here we show that ectopic expression of the transcriptional repressor escargot interferes with the establishment of megakaryocytic endomitosis. Phorbol ester-induced polyploidization was inhibited in stably transfected megakaryoblastic HEL cells constitutively expressing escargot. Analysis of the expression and activity of different cell cycle factors revealed that Escargot affects the G(1)/S transition by influencing Cdk2 activity and cyclin A transcription. Nuclear proteins that specifically bind the Escargot-binding element were detected in endomitotic and non-endomitotic megakaryoblastic cells, but down-regulation occurred only during differentiation of cells that become polyploid. As Escargot was originally implicated in ploidy maintenance of Drosophila embryonic and larval cells, our results suggest that polyploidization in megakaryocytes might respond to mechanisms conserved from early development to adult cells that need to escape normal control of the diploid state.

11 pages, 7 figures., In the present study, the molecular cloning and characterization of a 49-kDa form of casein kinase (CK)I from Dictyostelium discoideum is reported. The predicted amino acid sequence shares 70% identity with the catalytic domain of the mammalian delta and epsilon isoforms, Drosophila CKIepsilon and Schizosaccharomyces pombe Hhp1, and 63% identity with Hrr25, a 57-kDa form of yeast CK involved in DNA repair. D. discoideum CKI (DdCKI) was expressed in vegetative asynchronous cells as well as in differentiated cells, as detected by Northern-blot analysis. The level of DdCKI expression did not change during the cell cycle. Antibodies raised against a truncated version of the protein recognized a 49-kDa protein from D. discoideum extracts. Protein expression paralleled the pattern found for the RNA. The expression of DdCKI in Escherichia coli resulted in an active enzyme that autophosphorylated and phosphorylated casein. Immunofluorescence assays showed that DdCKI was localized in the cytoplasm and nuclei of Dictyostelium cells. The lack of disruptants of the CKI gene suggests that this protein is essential for the vegetative growth of D. discoideum. Overexpression of DdCKI resulted in cells with increased resistance to hydroxyurea, suggesting a potential role for this kinase in DNA repair., G. M.-B. was the beneficiary of a fellowship from the Universidad Autónoma de Madrid. This work has been supported by grants from the D.G.C.Y.T.

10 pages, 6 figures., Endoglin is a transmembrane glycoprotein 633 residues in length expressed at the surface of endothelial cells as a disulphide-linked homodimer; the specific cysteine residues involved in endoglin dimerization are unknown. Mutations in the coding region of the endoglin gene are responsible for hereditary haemorrhagic telangiectasia type 1 (HHT1), a dominantly inherited vascular disorder. Many of these mutations, if translated, would lead to truncated forms of the protein. It is therefore of interest to assess the protein expression of different truncated forms of endoglin. Infections in vitro or in vivo with recombinant vaccinia virus, as well as transient transfections with expression vectors, were used to express normal and truncated forms of endoglin. Truncated mutants could be classified into three different groups: (1) those that did not produce stable transcripts; (2) those that produced stable transcripts but did not secrete the protein; and (3) those that secreted a soluble dimeric protein. This is the first time that a recombinant truncated form of endoglin has been found to be expressed in a soluble form. Because a chimaeric construct encoding the N-terminal sequence of platelet/endothelial cell adhesion molecule (PECAM-1) antigen fused to residues Ile281–Ala658 of endoglin also yielded a dimeric surface protein, these results suggest that cysteine residues contained within the fragment Cys330–Cys412 are involved in disulphide bond formation. Infection with vaccinia recombinants encoding an HHT1 mutation did not affect the expression of the normal endoglin, and did not reveal an association of the recombinant soluble form with the transmembrane endoglin, supporting a haploinsufficiency model for HHT1., This work has been supported by grants from Comisión Interministerial de Ciencia y Tecnología (CICYT-SAF97-0034 and CICYT-BIO94-0118), Comunidad Autónoma de Madrid (CAM) and Biomed Program of the European Community (BMH4-T95-0995).

LIMP II is a glycoprotein expressed in the membrane of lysosomes and secretory granules with lysosomal properties. Sequence analysis of a CNBr-cleaved peptide allowed the synthesis of a 47-mer oligonucleotide that was used to screen a rat liver cDNA library in lambda gt11. This resulted in isolation of a 2-kilobase cDNA containing 1,434 bases encoding the entire protein. The deduced amino acid sequence indicates that LIMP II consists of 478 amino acid residues. The segment spanning residues 4-6 to 26 constitute an uncleavable signal peptide. LIMP II possesses a hydrophobic amino acid segment near the carboxyl end, that together with the uncleaved signal peptide may anchor the protein to the membrane through two distant segments. The major portion of the protein resides on the luminal side and displays 11 potential N-glycosylation sites and 5 cysteine residues. Two short cytoplasmic tails, 2-4 and 20-21 amino acids long, correspond to the NH2- and COOH-terminal ends of the protein, respectively. Transfection of COS cells with the cDNA of LIMP II resulted in expression of the protein and its transport to lysosomes. Comparison of the entire sequence to various data bases of known proteins revealed extensive homology between LIMP II and the cell surface protein CD36 involved in cell adhesion. No significant homology was detected with the two families of lysosomal membrane proteins A and B, recently described.

Initiation factor 2 from adult rat brain was isolated from salt-washed microsomes using a three-step purification process consisting of heparin-Sepharose, phosphocellulose and diethylaminoethyl-cellulose (DEAE-cellulose) column chromatographics. The initiation factor 2 (eIF-2) was phosphorylated in subunits α and β by the endogenous protein kinase activity present in the purified preparation. This protein kinase activity proved to be mostly a casein kinase, although the possible presence of a very specific α kinase activity cannot be dismissed.

Developmental changes at the level of initiation step of translation in the rat brain were studied. The level of deacylated tRNAimet in rat brain was measured at two stages of postnatal development. Although the amount of tRNA was slightly lower in adult than in young (4 day old) rats, the charging capacity of initiator tRNAimet in vitro was similar at both ages. No differences during development were found in methionyl-tRNA synthetase activity, which throws doubt on its possible participation in regulation of the initiation step. When assayed in the ribosomal salt wash protein fractions, initiation factor 2 activity decreased during brain development, and increased activities were detected in the supernatant of the microsomal fractions. The decrease in eIF-2 activity paralleled the observed decrease in the rat of overall protein synthesis or initiation activity in vitro, suggesting that the regulation of the initiation step of translation during brain development may be tightly linked to changes in initiation factor 2 activity in brain tissue.

Guanine nucleotide exchange factor (GEF) activity in ribosomal high salt wash and cytosolic fractions from suckling (4–10-day-old) and adult (60-day-old) rats was assayed by two different methods, by measuring: (i) its ability to promote binding of [3H]Met-tRNAi to eukaryotic initiation factor-2 (eIF-2) preparations that are partially or wholly in the form of eIF-2-GDP complexes (at Mg2+ concentrations near the optimum for protein synthesis), and (ii) under similar conditions, its ability to catalyze the displacement of [3H]GDP, previously bound to eIF-2, by unlabelled GDP. A purified eIF-2 (GEF-free) from brain was used as the source of eIF-2 activity. GEF activity in ribosomal fractions is higher in the brain of suckling than adults rats, and a direct correlation therefore exists between ribosomal GEF activity and the previously observed age-related decrease in eIF-2 activity in ribosomal high salt wash protein fractions. On the other hand GEF activity in the postmicrosomal supernatant is lower in the brain of suckling than adult rats. These findings further support the hypothesis that the progressive decrease in protein synthesis during brain development is controlled through regulation of the initiation step, by modulation of eIF-2/GEF activities.

The functional behavior of initiation factor 2 was studied in purified preparations from the brains of suckling (4-12-day-old) and adult (60-day-old) rats. Adult eIF2 has lower GDP and GTP affinity than suckling eIF2, even in the presence of a large excess of GTP, whereas suckling eIF2 has a lower capacity to bind GTP. Since these two factors are free of guanine nucleotide exchange factor (GEF), and ribosomal fractions show an age-dependent difference in GEF activity, the observed functional heterogeneity may be due to a different ratio in eIF2 species (eIF2-GDP, eIF2(αP)).Developing brainProtein synthesisInitiation factor 2Guanine nucleotide exchange factor

About 30% of human tumours contain a mutation in one of the three ras genes leading to the production of p21ras oncoproteins that are thought to make a major contribution to the transformed phenotype of the tumour1,2. The biochemical mode of action of the ras proteins is unknown but as they bind GTP and GDP and have an intrinsic GTPase activity, they may function like regulatory G proteins3–7 and control cell proliferation by regulating signal transduction pathways at the plasma membrane8–10. It is assumed that an external signal is detected by a membrane molecule (or detector) that stimulates the conversion of p21.GDP to p21.GTP which then interacts with a target molecule (or effector) to generate an internal signal. Recently a cytoplasmic protein, GAP, has been identified that interacts with the ras proteins, dramatically increasing the GTPase activity of normal p21 but not of the oncoproteins11. We report here that GAP appears to interact with p21ras at a site previously identified as the 'effector' site12,13, strongly implicating GAP as the biological target for regulation by p21.

A plasmid has been constructed which contains the normal human N-ras proto-oncogene under the transcriptional control of the steroid-sensitive promoter of the mouse mammary tumor virus long terminal repeat. This plasmid has been introduced into NIH-3T3 cells producing a clone of cells, T15, which is phenotypically normal in the absence of the transcription inducer, dexamethasone, and transformed when treated with high levels of the inducer. At lower levels of dexamethasone, both morphological transformation and stimulation of DNA synthesis are titratable functions of p21N-ras levels. T15 cells have been used to demonstrate that: (i) a 20- to 50-fold over-expression of normal p21ras is required for complete cellular transformation, (ii) p21N-ras expression induces DNA synthesis and the effect can be amplified by epidermal growth factor, (iii) moderate increases in normal p21ras expression can influence cell behaviour.

Eukaryotic initiation factor 2 (eIF-2) was isolated from salt-washed microsomes of 4-day-old rat brain which show a high rate of protein synthesis. A three-step purification scheme was employed, including heparin-Sepharose, phosphocellulose, and DEAE-cellulose column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isolated factor revealed three polypeptides with molecular weights of 43,000, 54,000, and 59,000 and 90% purity. The rat brain eIF-2 forms ternary complexes with [3H]methionyl-tRNAi and GTP. In terms of specific activity, the purification does not correspond to that revealed by electrophoretic analysis. During purification there is an apparent loss of additional factors that modulates the activity of eIF-2 and explains the high rate of activity of the crude fraction.

Endoglin is an homodimeric membrane antigen with capacity to bind transforming growth factor-beta (TGF-beta) and whose expression is up-regulated on myeloid cells upon differentiation to macrophages. We have isolated full-length cDNA clones from a lambda gt 10 library, prepared from phorbol 12-myristate 13-acetate-differentiated HL60 cells by screening with an endoglin-specific cDNA probe from endothelial cells. Sequencing of the largest clone (3073 bp), revealed that the leader sequence contains 25 residues and that the 586 amino acids of the extracellular and transmembrane domains were identical to those described for endothelial endoglin. However, the cytoplasmic tail encoded by this cDNA clone contains only 14 amino acids as opposed to the 47 residues previously reported, suggesting the existence of two alternative endoglin variants. The expression of these isoforms was demonstrated by polymerase chain reaction analyses on endothelial cells, myelomonocytic cell lines HL-60 and U-937, and placenta. Independent cDNA constructs corresponding to both forms were transfected into mouse fibroblasts leading to the expression of two distinct endoglin molecules. Both forms were shown to bind TGF-beta 1 and, when overexpressed in transfected mouse fibroblasts, to form disulfide-linked homodimers, indicating that the cysteine residues present in the extracellular domain are responsible for the dimerization.

In most eukaryotic cells, a link between S and M phases of the cell cycle must be assured in order to maintain the ploidy of newly divided cells. However, in some cell l/pes, e.g. the precursors of platelets megakaryocytes, extra S-phases can occur in the absence of concomitant mitoses, resulting in polyploidy. We have used two established cell lines with megakaryoblastic characteristics (HEL and MEG-01) to investigate the molecular events that lead these cells to bypass the regular control checkpoints that govern the interdependency of S and M phases. In the presence of the phorbol ester TPA, both cell lines stopped proliferating and displayed additional megakaryocytic features, including polyploidization. Analysis of key cell cycle regulatory factors implicated in the control of G1/S and G2/M transitions revealed a number of differences compared to normally cycling cells. Differentiating megakaryocytes were found to maintain high levels of cdk2, and cyclins E and A. This was accompanied by the appearance of the retinoblastoma protein in the hyperphosphorylated, functionally inactivated form. In addition, TPA-treated cells showed high levels of cyclin B and cdc2 proteins, however no activation of cdc2 was detected. This lack of cdc2 activation which should occur for entry into M phase appeared to be related to the down regulation of cdc25C phosphatase found in both differentiated HEL and MEG-01 cells. Together, our results suggest that in differentiating megakaryoblastic cells endoreplication is accompanied by sustained levels of cyclins A and E, and a lack of cdc2 activation, which is probably mediated through down regulation of cdc25C protein phosphatase.

Time course experiments of the localization of rat LIMP II expressed in COS cells show that the protein is transported directly from the Golgi complex to lysosomes. Substitution of the tyrosine-lacking carboxyl cytoplasmic tail of LIMP II for the native cytoplasmic tails of the plasma membrane proteins CD36 and CD8 resulted in straight transport of both proteins to lysosomes. The synthetic tyrosine-containing heptapeptide, RGTGVYG, did not replace the natural carboxyl cytoplasmic tail of LIMP II in its ability to transport both CD36 and CD8 to lysosomes, and the two constructs were transported to and expressed at the plasma membrane. Substitution of the cytoplasmic tails of either CD36 or CD8 for the carboxyl cytoplasmic tail of LIMP II resulted in transport of the mutants to the plasma membrane where they underwent endocytosis before accumulating into lysosomes. The results indicate that a motif contained in the tyrosine-lacking carboxyl cytoplasmic tail of LIMP II is sufficient to target proteins directly from the Golgi complex to lysosomes.