Glioblastoma (GBM) is one of the most aggressive human brain tumors, with a median survival of 15-18 months. There is a desperate need to find novel therapeutic targets. Various receptor protein ...kinases have been identified as potential targets; however, response rates in clinical studies have been somewhat disappointing. Targeting the spleen tyrosine kinase (SYK), which acts downstream of a range of oncogenic receptors, may therefore show more promising results.
Kinase expression of brain tumor samples including GBM and low-grade tumors were compared with normal brain and normal human astrocytes by microarray analysis. Furthermore, SYK, LYN, SLP76, and PLCG2 protein expressions were analyzed by immunohistochemistry, western blot, and immunofluorescence of additional GBM patient samples, murine glioma samples, and cell lines. SYK was then blocked chemically and genetically in vitro and in vivo in 2 different mouse models. Multiphoton intravital imaging and multicolor flow cytometry were performed in a syngeneic immunocompetent C57BL/6J mouse GL261 glioma model to study the effect of these inhibitors on the tumor microenvironment.
SYK, LYN, SLP76, and PLCG2 were found expressed in human and murine glioma samples and cell lines. SYK inhibition blocked proliferation, migration, and colony formation. Flow cytometric and multiphoton imaging imply that targeting SYK in vivo attenuated GBM tumor growth and invasiveness and reduced B and CD11b+ cell mobility and infiltration.
Our data suggest that gliomas express a SYK signaling network important in glioma progression, inhibition of which results in reduced invasion with slower tumor progression.
The tumor suppressor p53 is commonly inhibited under conditions in which the phosphatidylinositide 3â²-OH kinase/protein kinase
B (PKB)Akt pathway is activated. Intracellular levels of p53 are ...controlled by the E3 ubiquitin ligase Mdm2. Here we show
that PKB inhibits Mdm2 self-ubiquitination via phosphorylation of Mdm2 on Ser 166 and Ser 188 . Stimulation of human embryonic kidney 293 cells with insulin-like growth factor-1 increased Mdm2 phosphorylation on Ser 166 and Ser 188 in a phosphatidylinositide 3â²-OH kinase-dependent manner, and the treatment of both human embryonic kidney 293 and COS-1
cells with phosphatidylinositide 3â²-OH kinase inhibitor LY-294002 led to proteasome-mediated Mdm2 degradation. Introduction
of a constitutively active form of PKB together with Mdm2 into cells induced phosphorylation of Mdm2 at Ser 166 and Ser 188 and stabilized Mdm2 protein. Moreover, mouse embryonic fibroblasts lacking PKBα displayed reduced Mdm2 protein levels with
a concomitant increase of p53 and p21 Cip1 , resulting in strongly elevated apoptosis after UV irradiation. In addition, activation of PKB correlated with Mdm2 phosphorylation
and stability in a variety of human tumor cells. These findings suggest that PKB plays a critical role in controlling of the
Mdm2·p53 signaling pathway by regulating Mdm2 stability.
Protein kinase B/Akt plays crucial roles in promoting cell survival and mediating insulin responses. The enzyme is stimulated by phosphorylation at two regulatory sites: Thr 309 of the activation ...segment and Ser 474 of the hydrophobic motif, a conserved feature of many AGC kinases. Analysis of the crystal structures of the unphosphorylated and Thr 309 phosphorylated states of the PKB kinase domain provides a molecular explanation for regulation by Ser 474 phosphorylation. Activation by Ser 474 phosphorylation occurs via a disorder to order transition of the αC helix with concomitant restructuring of the activation segment and reconfiguration of the kinase bilobal structure. These conformational changes are mediated by a phosphorylation-promoted interaction of the hydrophobic motif with a channel on the N-terminal lobe induced by the ordered αC helix and are mimicked by peptides corresponding to the hydrophobic motif of PKB and potently by the hydrophobic motif of PRK2.
Second heart field (SHF) progenitors exhibit continued proliferation and delayed differentiation, which are modulated by FGF4/8/10, BMP and canonical Wnt/β-catenin signaling. PTEN-Akt signaling ...regulates the stem cell/progenitor cell homeostasis in several systems, such as hematopoietic stem cells, intestinal stem cells and neural progenitor cells. To address whether PTEN-Akt signaling is involved in regulating cardiac progenitors, we deleted Pten in SHF progenitors. Deletion of Pten caused SHF expansion and increased the size of the SHF derivatives, the right ventricle and the outflow tract. Cell proliferation of cardiac progenitors was enhanced, whereas cardiac differentiation was unaffected by Pten deletion. Removal of Akt1 rescued the phenotype and early lethality of Pten deletion mice, suggesting that Akt1 was the key downstream target that was negatively regulated by PTEN in cardiac progenitors. Furthermore, we found that inhibition of FOXO by Akt1 suppressed the expression of the gene encoding the BMP ligand (BMP7), leading to dampened BMP signaling in the hearts of Pten deletion mice. Cardiac activation of Akt also increased the Ser552 phosphorylation of β-catenin, thus enhancing its activity. Reducing β-catenin levels could partially rescue heart defects of Pten deletion mice. We conclude that Akt signaling regulates the cell proliferation of SHF progenitors through coordination of BMP signaling and β-catenin activity.
Studies of mammalian tissue culture cells indicate that the conserved and distinct NDR isoforms, NDR1 and NDR2, play essential cell biological roles. However, mice lacking either Ndr1 or Ndr2 alone ...develop normally. Here, we studied the physiological consequences of inactivating both NDR1 and NDR2 in mice, showing that the lack of both Ndr1/Ndr2 (called Ndr1/2-double null mutants) causes embryonic lethality. In support of compensatory roles for NDR1 and NDR2, total protein and activating phosphorylation levels of the remaining NDR isoform were elevated in mice lacking either Ndr1 or Ndr2. Mice retaining one single wild-type Ndr allele were viable and fertile. Ndr1/2-double null embryos displayed multiple phenotypes causing a developmental delay from embryonic day E8.5 onwards. While NDR kinases are not required for notochord formation, the somites of Ndr1/2-double null embryos were smaller, irregularly shaped and unevenly spaced along the anterior-posterior axis. Genes implicated in somitogenesis were down-regulated and the normally symmetric expression of Lunatic fringe, a component of the Notch pathway, showed a left-right bias in the last forming somite in 50% of all Ndr1/2-double null embryos. In addition, Ndr1/2-double null embryos developed a heart defect that manifests itself as pericardial edemas, obstructed heart tubes and arrest of cardiac looping. The resulting cardiac insufficiency is the likely cause of the lethality of Ndr1/2-double null embryos around E10. Taken together, we show that NDR kinases compensate for each other in vivo in mouse embryos, explaining why mice deficient for either Ndr1 or Ndr2 are viable. Ndr1/2-double null embryos show defects in somitogenesis and cardiac looping, which reveals their essential functions and shows that the NDR kinases are critically required during the early phase of organogenesis.
Glycogen synthase kinase-3 (GSK3) is implicated in the regulation of several physiological processes, including the control of glycogen and protein synthesis by insulin, modulation of the ...transcription factors AP-1 and CREB, the specification of cell fate in Drosophila and dorsoventral patterning in Xenopus embryos. GSK3 is inhibited by serine phosphorylation in response to insulin or growth factors and in vitro by either MAP kinase-activated protein (MAPKAP) kinase-1 (also known as p90rsk) or p70 ribosomal S6 kinase (p70S6k). Here we show, however, that agents which prevent the activation of both MAPKAP kinase-1 and p70S6k by insulin in vivo do not block the phosphorylation and inhibition of GSK3. Another insulin-stimulated protein kinase inactivates GSK3 under these conditions, and we demonstrate that it is the product of the proto-oncogene protein kinase B (PKB, also known as Akt/RAC). Like the inhibition of GSK3 (refs 10, 14), the activation of PKB is prevented by inhibitors of phosphatidylinositol (PI) 3-kinase.
The transcription factor Twist plays vital roles during embryonic development through regulating/controlling cell migration. However, postnatally, in normal physiological settings, Twist is either ...not expressed or inactivated. Increasing evidence shows a strong correlation between Twist reactivation and both cancer progression and malignancy, where the transcriptional activities of Twist support cancer cells to disseminate from primary tumours and subsequently establish a secondary tumour growth in distant organs. However, it is largely unclear how this signalling programme is reactivated or what signalling pathways regulate its activity. The present review discusses recent advances in Twist regulation and activity, with a focus on phosphorylation-dependent Twist activity, potential upstream kinases and the contribution of these factors in transducing biological signals from upstream signalling complexes. The recent advances in these areas have shed new light on how phosphorylation-dependent regulation of the Twist proteins promotes or suppresses Twist activity, leading to differential regulation of Twist transcriptional targets and thereby influencing cell fate.
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