Maria Alvarado-Kristensson

p38-MAPK Signals Survival by Phosphorylation of Caspase-8 and Caspase-3 in Human Neutrophils.

Neutrophil apoptosis occurs both in the bloodstream and in the tissue and is considered essential for the resolution of an inflammatory process. Here, we show that p38–mitogen-activated protein kinase (MAPK) associates to caspase-8 and caspase-3 during neutrophil apoptosis and that p38-MAPK activity, previously shown to be a survival signal in these primary cells, correlates with the levels of caspase-8 and caspase-3 phosphorylation. In in vitro experiments, immunoprecipitated active p38-MAPK phosphorylated and inhibited the activity of the active p20 subunits of caspase-8 and caspase-3. Phosphopeptide mapping revealed that these phosphorylations occurred on serine-364 and serine-150, respectively. Introduction of mutated (S150A), but not wild-type, TAT-tagged caspase-3 into primary neutrophils made the Fas-induced apoptotic response insensitive to p38-MAPK inhibition. Consequently, p38-MAPK can directly phosphorylate and inhibit the activities of caspase-8 and caspase-3 and thereby hinder neutrophil apoptosis, and, in so doing, regulate the inflammatory response.

p38 Mitogen-activated protein kinase and phosphatidylinositol 3-kinase activities have opposite effects on human neutrophil apoptosis.

Neutrophil apoptosis is essential for resolution of inflammatory reactions. Here, we studied the role of two apoptosis/survival‐associated protein kinases in this process. We discovered a previously undetected early and transient inhibition of the activity of p38 mitogen‐activated protein kinase (p38 MAPK) during both spontaneous and Fas‐induced apoptosis. Pharmacological inhibition of this enzyme augmented the activation of caspases and the apoptotic response, which suggests that the p38 MAPK signals survival in neutrophils. Our finding that caspase‐3 activity was initiated during the transient inhibition of p38 MAPK suggests that apoptosis is initiated during this inhibition. Furthermore, such transient inhibition was counteracted by granulocyte‐macrophage colony‐stimulating factor, which elicits survival. We also found that neither this inhibition of p38 MAPK nor the spontaneous apoptotic response depended on Fas. Instead, the early inhibition of p38 MAPK concurred with a Fas‐induced activation of phosphatidylinositol 3‐kinase, inhibition of which reduced apoptosis. Thus, the Fas‐induced augmentation of spontaneous apoptosis can be explained by its activation of phosphatidylinositol 3‐kinase. We conclude that p38 MAPK activity represents a survival signal that is inactivated transiently during both spontaneous and Fas‐induced apoptosis, whereas Fas‐induced phosphatidylinositol 3‐kinase activity is a proapoptotic signal in isolated human neutrophils.

SADB phosphorylation of gamma-tubulin regulates centrosome duplication.

Symmetrical cell division requires duplication of DNA and protein content to generate two daughter cells. Centrosomes also duplicate during cell division, but the mechanism controlling this process is incompletely understood. We describe an alternative splice form of SadB encoding a short SADB Ser/Thr kinase whose activity fluctuates during the cell cycle, localizes to centrosomes, and controls centrosome duplication. Reduction of endogenous SADB levels diminished centrosome numbers, whereas enhanced SADB expression induced centrosome amplification. SADB exerted this action through phosphorylation of γ-tubulin on Ser 131, as expression of a phosphomimetic Ser 131-to-Asp γ-tubulin mutant alone increased centrosome numbers, whereas non-phosphorylatable Ala 131-γ-tubulin impaired centrosome duplication. We propose that SADB kinase activity controls centrosome homeostasis by regulating phosphorylation of γ-tubulin.

Expression of the RNA-binding protein RBM3 is associated with a favourable prognosis and cisplatin sensitivity in epithelial ovarian cancer

BackgroundWe recently demonstrated that increased expression of the RNA-binding protein RBM3 is associated with a favourable prognosis in breast cancer. The aim of this study was to examine the prognostic value of RBM3 mRNA and protein expression in epithelial ovarian cancer (EOC) and the cisplatin response upon RBM3 depletion in a cisplatin-sensitive ovarian cancer cell line.MethodsRBM3 mRNA expression was analysed in tumors from a cohort of 267 EOC cases (Cohort I) and RBM3 protein expression was analysed using immunohistochemistry (IHC) in an independent cohort of 154 prospectively collected EOC cases (Cohort II). Kaplan Meier analysis and Cox proportional hazards modelling were applied to assess the relationship between RBM3 and recurrence free survival (RFS) and overall survival (OS). Immunoblotting and IHC were used to examine the expression of RBM3 in a cisplatin-resistant ovarian cancer cell line A2780-Cp70 and its cisplatin-responsive parental cell line A2780. The impact of RBM3 on cisplatin response in EOC was assessed using siRNA-mediated silencing of RBM3 in A2780 cells followed by cell viability assay and cell cycle analysis.ResultsIncreased RBM3 mRNA expression was associated with a prolonged RFS (HR = 0.64, 95% CI = 0.47-0.86, p = 0.003) and OS (HR = 0.64, 95% CI = 0.44-0.95, p = 0.024) in Cohort I. Multivariate analysis confirmed that RBM3 mRNA expression was an independent predictor of a prolonged RFS, (HR = 0.61, 95% CI = 0.44-0.84, p = 0.003) and OS (HR = 0.62, 95% CI = 0.41-0.95; p = 0.028) in Cohort I. In Cohort II, RBM3 protein expression was associated with a prolonged OS (HR = 0.53, 95% CI = 0.35-0.79, p = 0.002) confirmed by multivariate analysis (HR = 0.61, 95% CI = 0.40-0.92, p = 0.017). RBM3 mRNA and protein expression levels were significantly higher in the cisplatin sensitive A2780 cell line compared to the cisplatin resistant A2780-Cp70 derivative. siRNA-mediated silencing of RBM3 expression in the A2780 cells resulted in a decreased sensitivity to cisplatin as demonstrated by increased cell viability and reduced proportion of cells arrested in the G2/M-phase.ConclusionsThese data demonstrate that RBM3 expression is associated with cisplatin sensitivity in vitro and with a good prognosis in EOC. Taken together these findings suggest that RBM3 may be a useful prognostic and treatment predictive marker in EOC.

PTEN Regulation, a Novel Function for the p85 Subunit of Phosphoinositide 3-Kinase

Timely regulation of phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] and phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] abundance in cells is essential for the control of cellular homeostasis. The concentrations of these lipids are low in quiescent cells but rapidly and transiently increase following growth factor receptor (GFR) stimulation, which triggers cellular metabolic changes, proliferation, survival, and motility. Class IA phosphatidylinositol 3-kinase (PI3K), which is composed of a p85 (regulatory) and p110 (catalytic) subunits, is the enzyme generating PI(3,4)P2 and PI(3,4,5)P3 following GFR stimulation. Although the steps in GFR-induced activation of PI3K , are relatively well known, the mechanisms for subsequent 3-polyphospho-PI down-regulation are less understood. Examination of frequent genetic alterations in human cancer showed that PTEN (phosphatase with tensin homology on chromosome 10) is the major enzyme that decreases PI(3,4)P2 and PI(3,4,5)P3 cell content. Nonetheless, interpretation of the complexity of PTEN regulation remains a matter of debate. The recent description of diminished PTEN activity in liver-conditional knockout mice lacking the p85α PI3K regulatory subunit reveals a previously unknown p85α-dependent negative-feedback pathway that controls PI(3,4)P2 and PI(3,4,5)P3 half-life by regulating PTEN.

Nuclear localization of γ-tubulin affects E2F transcriptional activity and S-phase progression

We show that the centrosome‐ and microtubule‐regulating protein γ‐tubulin interacts with E2 promoter binding factors (E2Fs) to modulate E2F transcriptional activity and thereby control cell cycle progression. γ‐Tubulin contains a C‐terminal signal that results in its translocation to the nucleus during late G1 to early S phase. γ‐Tubulin mutants showed that the C terminus interacts with the transcription factor E2F1 and that the E2F1–γ‐tubulin complex is formed during the G1/S transition, when E2F1 is transcriptionally active. Furthermore, E2F transcriptional activity is altered by reduced expression of γ‐tubulin or by complex formation between γ‐tubulin and E2F1, E2F2, or E2F3, but not E2F6. In addition, the γ‐tubulin C terminus encodes a DNA‐binding domain that interacts with E2F‐regulated promoters, resulting in γ‐tubulin‐mediated transient activation of E2Fs. Thus, we report a novel mechanism regulating the activity of E2Fs, which can help explain how these proteins affect cell cycle progression in mammalian cells.—Höög, G., Zarrizi, R., von Stedingk, K., Jonsson, K., Alvarado‐Kristensson, M. Nuclear localization of γ‐tubulin affects E2F transcriptional activity and S‐phase progression. FASEB J. 25, 3815–3827 (2011). www.fasebj.org

Tumors with non-functional RB1 are killed by reduced gamma-tubulin levels.

Background: γ-Tubulin moderates the expression of E2F-regulated promoters by direct binding to DNA. Results: RB1 and γ-tubulin proteins moderate each others expression by binding to their respective gene promoters. Conclusion: Reduction of γ-tubulin protein levels in tumors with nonfunctional RB1 leads to induction of apoptosis. Significance: The RB1/γ-tubulin signal network can be considered as a new target for cancer treatment. In various tumors inactivation of growth control is achieved by interfering with the RB1 signaling pathway. Here, we describe that RB1 and γ-tubulin proteins moderate each others expression by binding to their respective gene promoters. Simultaneous reduction of RB1 and γ-tubulin protein levels results in an E2F1-dependent up-regulation of apoptotic genes such as caspase 3. We report that in various tumors types, there is an inverse correlation between the expression levels of γ-tubulin and RB1 and that in tumor cell lines with a nonfunctioning RB1, reduction of γ-tubulin protein levels leads to induction of apoptosis. Thus, the RB1/γ-tubulin signal network can be considered as a new target for cancer treatment.

The Nuclear Localization of γ-Tubulin Is Regulated by SadB-mediated Phosphorylation

Background: γ-Tubulin moderates gene expression by accumulating in the nucleus during early cell division. Results: SadB-mediated phosphorylation of Ser385-γ-tubulin exposes the nuclear localization signal of γ-tubulin. Conclusion: SadB kinases regulate the cellular location of γ-tubulin and in this way control cell growth. Significance: All knowledge on tubulins can aid the design of more efficient chemotherapeutic agents. γ-Tubulin is an important cell division regulator that arranges microtubule assembly and mitotic spindle formation. Cytosolic γ-tubulin nucleates α- and β-tubulin in a growing microtubule by forming the ring-shaped protein complex γTuRC. Nuclear γ-tubulin also regulates S-phase progression by moderating the activities of E2 promoter-binding factors. The mechanism that regulates localization of γ-tubulin is currently unknown. Here, we demonstrate that the human Ser/Thr kinase SadB short localizes to chromatin and centrosomes. We found that SadB-mediated phosphorylation of γ-tubulin on Ser385 formed chromatin-associated γ-tubulin complexes that moderate gene expression. In this way, the C-terminal region of γ-tubulin regulates S-phase progression. In addition, chromatin levels of γ-tubulin were decreased by the reduction of SadB levels or expression of a non-phosphorylatable Ala385-γ-tubulin but were enhanced by expression of SadB, wild-type γ-tubulin, or a phosphomimetic Asp385-γ-tubulin mutant. Our results demonstrate that SadB kinases regulate the cellular localization of γ-tubulin and thereby control S-phase progression.

Sphingosine 1-phosphate antagonizes human neutrophil apoptosis via p38 mitogen-activated protein kinase.

Abstract. Sphingosine 1-phosphate (SPP) is associated with the regulation of apoptosis, although its role in neutrophil apoptosis remains poorly investigated. Here, we show that exogenous SPP antagonizes spontaneous and anti-Fas-induced apoptosis in neutrophils. Pre-treatment with pertussis toxin clearly reduced the apoptosis-inhibiting capacity of SPP. Consequently, we investigated the involvement of potential modulators of apoptosis that are activated downstream of Gi/G0-coupled receptors. Neither Akt activity nor change in basal activity of c-Jun N-terminal kinases was detected during apoptosis or after adding SPP. In contrast, there was a transient decrease in phosphorylation of both extracellular-regulated kinase (ERK) and p38mitogen-activated protein kinase (MAPK) during both spontaneous and anti-Fas-induced apoptosis. Although exogenous SPP reversed these reductions in kinase activity, experiments with inhibitors of ERK (PD98059) and p38 MAPK (SB203580) revealed that only SB203580 counteracted the effect of SPP. Thus, SPP counteracts neutrophil apoptosis via a Gi/G0protein survival-signalling pathway that includes modulation of p38 MAPK activity.

Gamma-tubulin coordinates nuclear envelope assembly around chromatin

The cytosolic role of γ-tubulin as a microtubule organizer has been studied thoroughly, but its nuclear function is poorly understood. Here, we show that γ-tubulin is located throughout the chromatin of demembranated Xenopus laevis sperm and, as the nucleus is formed, γ-tubulin recruits lamin B3 and nuclear membranes. Immunodepletion of γ-tubulin impairs X. laevis assembly of both the lamina and the nuclear membrane. During nuclear formation in mammalian cell lines, γ-tubulin establishes a cellular protein boundary around chromatin that coordinates nuclear assembly of the daughter nuclei. Furthermore, expression of a γ-tubulin mutant that lacks the DNA-binding domain forms chromatin-empty nuclear like structures and demonstrate that a constant interplay between the chromatin-associated and the cytosolic pools of γ-tubulin is required and, when the balance between pools is impaired, aberrant nuclei are formed. We therefore propose that the nuclear protein meshwork formed by γ-tubulin around chromatin coordinates nuclear formation in eukaryotic cells.