PKD phosphorylation and COP9/Signalosome modulate intracellular Spry2 protein stability.

Spry2 is a molecular modulator of tyrosine kinase receptor signaling pathways that has cancer-type-specific effects. Mammalian Spry2 protein undergoes tyrosine and serine phosphorylation in response to growth factor stimulation. Spry2 expression is distinctly altered in various cancer types. Inhibition of the proteasome functionality results in reduced intracellular Spry2 degradation. Using in vitro and in vivo assays, we show that protein kinase D (PKD) phosphorylates Spry2 at serine 112 and interacts in vivo with the C-terminal half of this protein. Importantly, missense mutation of Ser112 decreases the rate of Spry2 intracellular protein degradation. Either knocking down the expression of all three mammalian PKD isoforms or blocking their kinase activity with a specific inhibitor contributes to the stabilization of Spry2 wild-type protein. Downregulation of CSN3, a component of the COP9/Signalosome that binds PKD, significantly increases the half-life of Spry2 wild-type protein but does not affect the stability of a Spry2 after mutating Ser112 to the non-phosphorylatable residue alanine. Our data demonstrate that both PKD and the COP9/Signalosome play a significant role in control of Spry2 intracellular stability and support the consideration of the PKD/COP9 complex as a potential therapeutic target in tumors where Spry2 expression is reduced.

CCHD Screening Implementation Efforts in Latin American Countries by the Ibero American Society of Neonatology (SIBEN).

Congenital heart disease (CHD) is among the four most common causes of infant mortality in Latin America. Pulse oximetry screening (POS) is useful for early diagnosis and improved outcomes of critical CHD. Here, we describe POS implementation efforts in Latin American countries guided and/or coordinated by the Ibero American Society of Neonatology (SIBEN), as well as the unique challenges that are faced for universal implementation. SIBEN collaborates to improve the neonatal quality of care and outcomes. A few years ago, a Clinical Consensus on POS was finalized. Since then, we have participated in 12 Latin American countries to educate neonatal nurses and neonatologists on POS and to help with its implementation. The findings reveal that despite wide disparities in care that exist between and within countries, and the difficulties and challenges in implementing POS, significant progress has been made. We conclude that universal POS is not easy to implement in Latin America but, when executed, has not only been of significant value for babies with CHD, but also for many with other hypoxemic conditions. The successful and universal implementation of POS in the future is essential for reducing the mortality associated with CHD and other hypoxemic conditions and will ultimately lead to the survival of many more Latin American babies. POS saves newborns' lives in Latin America.

The CSN3 subunit of the COP9 signalosome interacts with the HD region of Sos1 regulating stability of this GEF protein.

Sos1 is an universal, widely expressed Ras guanine nucleotide-exchange factor (RasGEF) in eukaryotic cells. Its N-terminal HD motif is known to be involved in allosteric regulation of Sos1 GEF activity through intramolecular interaction with the neighboring PH domain. Here, we searched for other cellular proteins also able to interact productively with the Sos1 HD domain. Using a yeast two-hybrid system, we identified the interaction between the Sos1 HD region and CSN3, the third component of the COP9 signalosome, a conserved, multi-subunit protein complex that functions in the ubiquitin-proteasome pathway to control degradation of many cellular proteins. The interaction of CSN3 with the HD of Sos1 was confirmed in vitro by GST pull-down assays using truncated mutants and reproduced in vivo by co-immunoprecipitation with the endogenous, full-length cellular Sos1 protein. In vitro kinase assays showed that PKD, a COP9 signalosome-associated-kinase, is able to phosphorylate Sos1. The intracellular levels of Sos1 protein were clearly diminished following CSN3 or PKD knockdown. A sizable fraction of the endogenous Sos1 protein was found ubiquitinated in different mammalian cell types. A significant reduction of RasGTP formation upon growth factor stimulation was also observed in CSN3-silenced as compared with control cells. Our data suggest that the interaction of Sos1 with the COP9 signalosome and PKD plays a significant role in maintenance of cellular Sos1 protein stability and homeostasis under physiological conditions and raises the possibility of considering the CSN/PKD complex as a potential target for design of novel therapeutic drugs.

Regulation of CBP and Tip60 coordinates histone acetylation at local and global levels during Ras-induced transformation.

Cell transformation is clearly linked to epigenetic changes. However, the role of the histone-modifying enzymes in this process is still poorly understood. In this study, we investigated the contribution of the histone acetyltransferase (HAT) enzymes to Ras-mediated transformation. Our results demonstrated that lysine acetyltransferase 5, also known as Tip60, facilitates histone acetylation of bulk chromatin in Ras-transformed cells. As a consequence, global H4 acetylation (H4K8ac and H4K12ac) increases in Ras-transformed cells, rendering a more decompacted chromatin than in parental cells. Furthermore, low levels of CREB-binding protein (CBP) lead to hypoacetylation of retinoblastoma 1 (Rb1) and cyclin-dependent kinase inhibitor 1B (Cdkn1b or p27Kip1) tumour suppressor gene promoters to facilitate Ras-mediated transformation. In agreement with these data, overexpression of Cbp counteracts Ras transforming capability in a HAT-dependent manner. Altogether our results indicate that CBP and Tip60 coordinate histone acetylation at both local and global levels to facilitate Ras-induced transformation.

Mammalian son of sevenless Guanine nucleotide exchange factors: old concepts and new perspectives.

The Son of Sevenless (Sos) factors were originally discovered 2 decades ago as specialized Ras activators in signaling pathways controlling the process of R7 cell development in the eye of Drosophila melanogaster. The 2 known members of the mammalian Sos family (Sos1 and Sos2) code for ubiquitously expressed, highly homologous (69% overall) proteins involved in coupling signals originated by cell surface receptor tyrosine kinases (RTKs) to downstream, Ras-dependent mitogenic signaling pathways. Mechanistically, the Sos proteins function as enzymatic factors interacting with Ras proteins in response to upstream stimuli to promote guanine nucleotide exchange (GDP/GTP) and subsequent formation of the active Ras-GTP complex. In this review, we summarize current knowledge on structural, regulatory, and functional aspects of the Sos family, focusing on specific aspects of Sos biology such as structure-function relationship, crosstalk with different signaling pathways, and in vivo functional significance as deduced from phenotypic characterization of Sos knockout mice and human genetic syndromes caused by germline hSos1 mutations.

Sprouty2 and Spred1-2 proteins inhibit the activation of the ERK pathway elicited by cyclopentenone prostanoids.

Sprouty and Spred proteins have been widely implicated in the negative regulation of the fibroblast growth factor receptor-extracellular regulated kinase (ERK) pathway. In considering the functional role of these proteins, we explored their effects on ERK activation induced by cyclopentenone prostanoids, which bind to and activate Ras proteins. We therefore found that ectopic overexpression in HeLa cells of human Sprouty2, or human Spred1 or 2, inhibits ERK1/2 and Elk-1 activation triggered by the cyclopentenone prostanoids PGA(1) and 15d-PGJ(2). Furthermore, we found that in HT cells that do not express Sprouty2 due to hypermethylation of its gene-promoter, PGA(1)-provoked ERK activation was more intense and sustained compared to other hematopoietic cell lines with unaltered Sprouty2 expression. Cyclopentenone prostanoids did not induce Sprouty2 tyrosine phosphorylation, in agreement with its incapability to activate tyrosine-kinase receptors. However, Sprouty2 Y55F, which acts as a defective mutant upon tyrosine-kinase receptor stimulation, did not inhibit cyclopentenone prostanoids-elicited ERK pathway activation. In addition, Sprouty2 did not affect the Ras-GTP levels promoted by cyclopentenone prostanoids. These results unveil both common and differential features in the activation of Ras-dependent pathways by cyclopentenone prostanoids and growth factors. Moreover, they provide the first evidence that Sprouty and Spred proteins are negative regulators of the ERK/Elk-1 pathway activation induced not only by growth-factors, but also by reactive lipidic mediators.

Sprouty2 binds Grb2 at two different proline-rich regions, and the mechanism of ERK inhibition is independent of this interaction.

Sprouty2 has been widely implicated in the negative regulation of the fibroblast growth factor receptor-extracellular regulated kinase (ERK) pathway. Sprouty2 directly interacts with the adapter protein Grb2, member of the receptor tyrosine kinase-induced signaling pathways. In considering the functional role of Grb2, we investigated whether the interaction with this protein was responsible for ERK pathway inhibition. We found that the binding between Sprouty2 and Grb2 is constitutive, independent of Sprouty2 tyrosine phosphorylation, although it is increased when fibroblast growth factor receptor is activated. This connection is mediated by the N-terminal SH3 domain of Grb2 and two Sprouty2 proline-rich stretches (residues 59-64 and 303-307). Most importantly, a double Sprouty2 mutant (hSpry2 P59AP304A), which is unable to bind Grb2, developed at a similar inhibition level of fibroblast growth factor receptor-ERK pathway than that which originated from Sprouty2 wt. These results are evidence that the Sprouty2 mechanism of ERK inhibition is independent of Grb2 binding.

Grb2 is a negative modulator of the intrinsic Ras-GEF activity of hSos1.

hSos1 is a Ras guanine-nucleotide exchange factor. It was suggested that the carboxyl-terminal region of hSos1 down-regulates hSos1 functionality and that the intrinsic guanine-nucleotide exchange activity of this protein may be different before and after stimulation of tyrosine kinase receptors. Using different myristoylated hSos1 full-length and carboxyl-terminal truncated mutants, we show that Grb2 function accounts not only for recruitment of hSos1 to the plasma membrane but also for modulation of hSos1 activity. Our results demonstrate that the first two canonical Grb2 binding sites, inside the carboxyl-terminal region of hSos1, are responsible for this regulation. Following different approaches, such as displacement of Grb2 from the hSos1-Grb2 complex or depletion of Grb2 levels by small interfering RNA, we found that the full-length Grb2 proteins mediate negative regulation of the intrinsic Ras guanine-nucleotide exchange activity of hSos1.

The histone acetyltransferases CBP/p300 are degraded in NIH 3T3 cells by activation of Ras signalling pathway.

The CBP [CREB (cAMP-response-element-binding protein)-binding protein]/p300 acetyltransferases function as transcriptional co-activators and play critical roles in cell differentiation and proliferation. Accumulating evidence shows that alterations of the CBP/p300 protein levels are linked to human tumours. In the present study, we show that the levels of the CBP/p300 co-activators are decreased dramatically by continuous PDGF (platelet-derived growth factor) and Ras signalling pathway activation in NIH 3T3 fibroblasts. This effect occurs by reducing the expression levels of the CBP/p300 genes. In addition, CBP and p300 are degraded by the 26 S proteasome pathway leading to an overall decrease in the levels of the CBP/p300 proteins. Furthermore, we provide evidence that Mdm2 (murine double minute 2), in the presence of active H-Ras or N-Ras, induces CBP/p300 degradation in NIH 3T3 cells. These findings support a novel mechanism for modulating other signalling transduction pathways that require these common co-activators.

PKC isozymes and diacylglycerol-regulated proteins as effectors of growth factor receptors.

Growth factors exert their cellular effects through signal transduction pathways that are initiated by the ligation of growth factors to their cell surface receptors. One of the well-established effectors of growth factor receptors is protein kinase C (PKC), a family of serine-threonine kinases that have been known for years as the main target of the phorbol ester tumor promoters. While there is abundant information regarding downstream PKC effectors and partners, how individual PKC isozymes become activated by growth factors and the regulation of receptor function by PKCs is only partially understood. Moreover, the identification of novel "non-kinase" DAG-binding proteins has added a new level of complexity to the field of DAG signaling.

Phorbol ester-induced G1 phase arrest selectively mediated by protein kinase Cdelta-dependent induction of p21.

Although protein kinase C (PKC) has been widely implicated in the positive and negative control of proliferation, the underlying cell cycle mechanisms regulated by individual PKC isozymes are only partially understood. In this report, we show that PKCdelta mediates phorbol ester-induced G1 arrest in lung adenocarcinoma cells and establish an essential role for this novel PKC in controlling the expression of the cell cycle inhibitor p21. Activation of PKC with phorbol 12-myristate 13-acetate (PMA) in early G1 phase impaired progression of lung adenocarcinoma cells into S phase, an effect that was completely abolished by specific depletion of PKCdelta, but not PKCalpha. Although the PKC effect was unrelated to the inhibition of cyclin D1 expression, PKC activation significantly up-regulated p21 and down-regulated Rb hyperphosphorylation and cyclin A expression. Elevations in p21 mRNA and protein by PMA were mediated by PKCdelta but not PKCalpha. Studies using luciferase reporters also revealed an essential role for PKCdelta in the PMA-induced inhibition of Rb-dependent cyclin A promoter activity. Finally, we showed that the cell cycle inhibitory effect of PKCdelta is greatly attenuated by RNA interference-mediated knock-down of p21. Our results identify a novel link between PKCdelta and G1 arrest via p21 up-regulation and highlight the complexities in the downstream effectors of PKC isozymes in the context of cell cycle progression and proliferation.

The P34G mutation reduces the transforming activity of K-Ras and N-Ras in NIH 3T3 cells but not of H-Ras.

Ras proteins (H-, N-, and K-Ras) operate as molecular switches in signal transduction cascades controlling cell proliferation, differentiation, or apoptosis. The interaction of Ras with its effectors is mediated by the effector-binding loop, but different data about Ras location to plasma membrane subdomains and new roles for some docking/scaffold proteins point to signaling specificities of the different Ras proteins. To investigate the molecular mechanisms for these specificities, we compared an effector loop mutation (P34G) of three Ras isoforms (H-, N-, and K-Ras4B) for their biological and biochemical properties. Although this mutation diminished the capacity of Ras proteins to activate the Raf/ERK and the phosphatidylinositol 3-kinase/AKT pathways, the H-Ras V12G34 mutant retained the ability to cause morphological transformation of NIH 3T3 fibroblasts, whereas both the N-Ras V12G34 and the K-Ras4B V12G34 mutants were defective in this biological activity. On the other hand, although both the N-Ras V12G34 and the K-Ras4B V12G34 mutants failed to promote activation of the Ral-GDS/Ral A/PLD and the Ras/Rac pathways, the H-Ras V12G34 mutant retained the ability to activate these signaling pathways. Interestingly, the P34G mutation reduced specifically the N-Ras and K-Ras4B in vitro binding affinity to Ral-GDS, but not in the case of H-Ras. Thus, independently of Ras location to membrane subdomains, there are marked differences among Ras proteins in the sensitivity to an identical mutation (P34G) affecting the highly conserved effector-binding loop.

The cyclopentenone 15-deoxy-delta 12,14-prostaglandin J2 binds to and activates H-Ras.

The cyclopentenone 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) induces cell proliferation and mitogen-activated protein kinase activation. Here, we describe that these effects are mediated by 15d-PGJ(2)-elicited H-Ras activation. We demonstrate that this pathway is specific for H-Ras through the formation of a covalent adduct of 15d-PGJ(2) with Cys-184 of H-Ras, but not with N-Ras or K-Ras. Mutation of C184 inhibited H-Ras modification and activation by 15d-PGJ(2), whereas serum-elicited stimulation was not affected. These results describe a mechanism for the activation of the Ras signaling pathway, which results from the chemical modification of H-Ras by formation of a covalent adduct with cyclopentenone prostaglandins.

H-Ras-specific activation of NF-kappaB protects NIH 3T3 cells against stimulus-dependent apoptosis.

Ras signaling involves the activation of several downstream pathways that exhibit isoform specificity. In this study, the basal and tumor necrosis factor alpha (TNFalpha)-induced activation of NF-kappaB has been examined in cells overexpressing H-Ras, K-Ras or N-Ras. Cells expressing H-Ras exhibited a basal kappaB activity that correlated with sustained IkappaB kinase activation and lower steady-state levels of IkappaBalpha in the cytosol. Upon activation with TNFalpha, the cells expressing the distinct Ras isoforms behaved similarly in terms of binding of nuclear proteins to a kappaB sequence and induction of a kappaB-dependent reporter gene. The basal activation of NF-kappaB in cells expressing H-Ras impaired staurosporine-induced apoptosis in these cells, through a mechanism that was NF-kappaB-dependent and inhibitable in the presence of z-VAD. Moreover, titration of caspase activation in response to staurosporine showed a significant resistance in cells expressing H-Ras when compared with the void vector or the N-Ras counterparts. These results indicate that the distinct Ras proteins have specific effects on the NF-kappaB pathway and that this action contributes to protect cells against apoptosis.

HSos1 contains a new amino-terminal regulatory motif with specific binding affinity for its pleckstrin homology domain.

The protein hSos1 is a Ras guanine nucleotide exchange factor. In the present study, we investigated the function of the amino-terminal region of the hSos1 protein, corresponding to the first 600 residues, which includes the Dbl and pleckstrin homology (DH and PH) domains. We demonstrated, using a series of truncated mutants, that this region is absolutely necessary for hSos1 activity. Our results suggest that the first 200 residues (upstream of DH domain), which we called the HF motif on the basis of their homology with histone H2A, may exert negative control over the functional activity of the whole hSos1 protein. In vitro binding analysis showed that the HF motif is able to interact specifically with the PH domain of hSos1. The amino-terminal region of hSos1 may be associated in vivo with an expressed HF motif. These findings document the existence of the HF motif located upstream of the DH domain in the hSos1 protein. This motif may be responsible for the negative control of hSos1, probably by intramolecular binding with the PH domain.

Importância da ventilaçäo pulmonar no transporte de O2 e equilíbrio ácido-base após desclampeamento intermitente de aorta na revascularizaçäo cirúrgica do miocárdio / Significance of pulmonary ventilation in the O2 transport and acid-base equilibrium after intermittent aortic desclamping in myocardial revascularization

Na revascularizaçao cirúrgica do miocárdio, empregando-se a técnica de pinçamento intermitente de aorta, após o desclampeamento e a recuperaçao dos batimentos cardíacos, surgiu a polêmica da necessidade da ventilaçao pulmonar na prevençao de hipoxemia. O objetivo deste trabalho foi analisar a importância da ventilaçao pulmonar no transporte de oxigênio e equilíbrio ácido-base do sangue que irá perfundir o miocárdio após desclampeamento de aorta e recuperaçao dos batimentos cardíacos. Foram estudados dez pacientes submetidos a revascularizaçao cirúrgica do miocárdio, empregando-se a técnica de pinçamento intermitente de aorta com hipotermia moderada (ñ 32 graus Celsius). Em cinco pacientes (Grupo I), após o 1( desclampeamento de aorta, a ventilaçao pulmonar foi realizada simultaneamente à recuperaçao dos batimentos cardíacos. Nos outros cinco pacientes (Grupo II) nao se realizou a ventilaçao pulmonar. Foram analisados (Saturaçao de 0(2') PO(2') PCO(2) e pH) do sangue do átrio direito (AD), tronco pulmonar (AP), átrio esquerdo (AE), aorta (Ao), artéria radial (art. radial) e circuito da circulaçao extracorpórea (CEC) (arterial [art. CEC] e venoso [ven. CEC]), comparando-se os dois grupos. Nao houve diferença estatisticamente nos valores de saturaçao de 0(2) ambos os grupos. No Grupo I, os valores do PO(2) aumentaram significativamente. Houve aumento significante da PO(2) no Grupo II, contribuindo para reduçao significante de pH neste grupo. Os autores concluem que, embora nao tenha ocorrido hipoxemia, a acidose respiratória observada no Grupo II permite recomendar a ventilaçao pulmonar ao utilizar-se do pinçamento intermitente de aorta como forma de proteçao miócardica, pois sabe-se dos efeitos deletérios do aumento da concentraçao de íons hidrogênio ([H+]) na funçao contrátil do miocárdio.