Angiopoietin 2 regulates the transformation and integrity of lymphatic endothelial cell junctions.

Primitive lymphatic vessels are remodeled into functionally specialized initial and collecting lymphatics during development. Lymphatic endothelial cell (LEC) junctions in initial lymphatics transform from a zipper-like to a button-like pattern during collecting vessel development, but what regulates this process is largely unknown. Angiopoietin 2 (Ang2) deficiency leads to abnormal lymphatic vessels. Here we found that an ANG2-blocking antibody inhibited embryonic lymphangiogenesis, whereas endothelium-specific ANG2 overexpression induced lymphatic hyperplasia. ANG2 inhibition blocked VE-cadherin phosphorylation at tyrosine residue 685 and the concomitant formation of button-like junctions in initial lymphatics. The defective junctions were associated with impaired lymph uptake. In collecting lymphatics, adherens junctions were disrupted, and the vessels leaked upon ANG2 blockade or gene deletion. ANG2 inhibition also suppressed the onset of lymphatic valve formation and subsequent valve maturation. These data identify ANG2 as the first essential regulator of the functionally important interendothelial cell-cell junctions that form during lymphatic development.

Internalization and homologous desensitization of the GLP-1 receptor depend on phosphorylation of the receptor carboxyl tail at the same three sites.

Homologous desensitization and internalization of the GLP-1 receptor correlate with phosphorylation of the receptor in a 33-amino acid segment of the cytoplasmic tail. Here, we identify the sites of phosphorylation as being three serine doublets located at positions 441/442, 444/445, and 451/452. The role of phosphorylation on homologous desensitization was assessed after stable expression in fibroblasts of the wild type or of mutant receptors in which phosphorylation sites were changed in various combinations to alanines. We showed that desensitization, as measured by a decrease in the maximal production of cAMP after a first exposure of the cells to GLP-1, was strictly dependent on phosphorylation. Furthermore, the number of phosphorylation sites correlated with the extent of desensitization with no, intermediate, or maximal desensitization observed in the presence of one, two, or three phosphorylation sites, respectively. Internalization of the receptor-ligand complex was assessed by measuring the rate of internalization of bound [125I]GLP-1 or the redistribution of the receptor to an endosomal compartment after agonist binding. Our data demonstrate that internalization was prevented in the absence of receptor phosphorylation and that intermediate rates of endocytosis were obtained with receptors containing one or two phosphorylation sites. Thus, homologous desensitization and internalization require phosphorylation of the receptor at the same three sites. However, the differential quantitative impairment of these two processes in the single and double mutants suggests different molecular mechanisms controlling desensitization and internalization.

Differential phosphorylation of MAP1b during postnatal development of the cat brain.

Microtubule-associated protein 1b, previously also referred to as microtubule-associated protein 5 or microtubule-associated protein 1x, is a major component of the juvenile cytoskeleton, and is essential during the early differentiation of neurons. It is required for axonal growth and its function is influenced by phosphorylation. The distribution of microtubule-associated protein 1b in kitten cerebellum and cortex during postnatal development was studied with two monoclonal antibodies. Hybridoma clone AA6 detected a non-phosphorylated site, while clone 125 detected a site phosphorylated by casein-kinase II. On blots, both monoclonal antibodies stained the same two proteins of similar molecular weights, also referred to as microtubule-associated protein 5a and 5b. Antibody 125 detected a phosphorylated epitope on both microtubule-associated protein 1b forms; dephosphorylation by alkaline phosphatase abolished the immunological detection. During development of cat cortex and cerebellum, AA6 stained the perikarya and dendrites of neurons during their early differentiation, and especially labelled newly generated axons. The staining decreased during development, and axonal staining was reduced in adult tissue. In contrast to previous reports which demonstrated that antibodies against phosphorylated microtubule-associated protein 1b label exclusively axons, antibody 125 also localized microtubule-associated protein 1b in cell bodies and dendrites, even in adulthood. Some nuclear staining was observed, indicating that a phosphorylated form of microtubule-associated protein 1b may participate in nuclear function. These results demonstrate that microtubule-associated protein 1b is subject to CK2-type phosphorylation throughout neuronal maturation and suggest that phosphorylation of microtubule-associated protein 1b may participate in juvenile and mature-type microtubule functions throughout development.

Peptide modification or blocking of CD8, resulting in weak TCR signaling, can activate CTL for Fas- but not perforin-dependent cytotoxicity or cytokine production.

This study describes a form of partial agonism for a CD8+ CTL clone, S15, in which perforin-dependent killing and IFN-gamma production were lost but Fas (APO1 or CD95)-dependent cytotoxicity preserved. Cloned S15 CTL are H-2Kd restricted and specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). The presence of a photoactivatable group in the epitope permitted assessment of TCR-ligand binding by TCR photoaffinity labeling. Selective activation of Fas-dependent killing was observed for a peptide-derivative variant containing a modified photoreactive group. A similar functional response was obtained after binding of the wild-type peptide derivative upon blocking of CD8 participation in TCR-ligand binding. The epitope modification or blocking of CD8 resulted in an > or = 8-fold decrease in TCR-ligand binding. In both cases, phosphorylation of zeta-chain and ZAP-70, as well as calcium mobilization were reduced close to background levels, indicating that activation of Fas-dependent cytotoxicity required weaker TCR signaling than activation of perforin-dependent killing or IFN-gamma production. Consistent with this, we observed that depletion of the protein tyrosine kinase p56(lck) by preincubation of S15 CTL with herbimycin A severely impaired perforin- but not Fas-dependent cytotoxicity. Together with the observation that S15 CTL constitutively express Fas ligand, these results indicate that TCR signaling too weak to elicit perforin-dependent cytotoxicity or cytokine production can induce Fas-dependent cytotoxicity, possibly by translocation of preformed Fas ligand to the cell surface.

Long-term swimming exercise does not modulate the Akt-dependent endothelial nitric oxide synthase phosphorylation in healthy mice.

Molecular mechanisms by which exercise exerts cardiovascular benefits are poorly understood. Exercise-induced increase of endothelial NO synthase (eNOS) phosphorylation through the protein kinase Akt has been shown to be a key mechanism underlying the beneficial effect of exercise in coronary artery disease patients. We examined whether this protective pathway might also be activated in long-term-exercised healthy mice. C57BL/6 wild-type mice swam for 24 weeks. A group of sedentary animals were used as controls. Aortic levels of total protein kinase Akt (protein kinase B), phosphorylated Akt at ser473 (p-Akt), total eNOS, phosphorylated eNOS at Ser1177 (p-eNOS), and PECAM-1 (platelet endothelial cell adhesion molecule-1) were assessed by Western blotting. Protein expressions of Akt, p-Akt, eNOS, p-eNOS, and PECAM-1 were not modulated by 24 weeks of exercise. The Akt-dependent eNOS phosphorylation did not seem to be a primary molecular adaptation in response to long-term exercise in healthy mice.

Equine herpesvirus protein E10 induces membrane recruitment and phosphorylation of its cellular homologue, bcl-10.

v-E10, a caspase recruitment domain (CARD)-containing gene product of equine herpesvirus 2, is the viral homologue of the bcl-10 protein whose gene was found to be translocated in mucosa-associated lymphoid tissue (MALT) lymphomas. v-E10 efficiently activates the c-jun NH(2)-terminal kinase (JNK), p38 stress kinase, and the nuclear factor (NF)-kappaB transcriptional pathway and interacts with its cellular homologue, bcl-10, via a CARD-mediated interaction. Here we demonstrate that v-E10 contains a COOH-terminal geranylgeranylation consensus site which is responsible for its plasma membrane localization. Expression of v-E10 induces hyperphosphorylation and redistribution of bcl-10 from the cytoplasm to the plasma membrane, a process which is dependent on the intactness of the v-E10 CARD motif. Both membrane localization and a functional CARD motif are important for v-E10-mediated NF-kappaB induction, but not for JNK activation, which instead requires a functional v-E10 binding site for tumor necrosis factor receptor-associated factor (TRAF)6. Moreover, v-E10-induced NF-kappaB activation is inhibited by a dominant negative version of the bcl-10 binding protein TRAF1, suggesting that v-E10-induced membrane recruitment of cellular bcl-10 induces constitutive TRAF-mediated NF-kappaB activation.

PIDD death-domain phosphorylation by ATM controls prodeath versus prosurvival PIDDosome signaling.

Biochemical evidence implicates the death-domain (DD) protein PIDD as a molecular switch capable of signaling cell survival or death in response to genotoxic stress. PIDD activity is determined by binding-partner selection at its DD: whereas recruitment of RIP1 triggers prosurvival NF-κB signaling, recruitment of RAIDD activates proapoptotic caspase-2 via PIDDosome formation. However, it remains unclear how interactor selection, and thus fate decision, is regulated at the PIDD platform. We show that the PIDDosome functions in the "Chk1-suppressed" apoptotic response to DNA damage, a conserved ATM/ATR-caspase-2 pathway antagonized by Chk1. In this pathway, ATM phosphorylates PIDD on Thr788 within the DD. This phosphorylation is necessary and sufficient for RAIDD binding and caspase-2 activation. Conversely, nonphosphorylatable PIDD fails to bind RAIDD or activate caspase-2, and engages prosurvival RIP1 instead. Thus, ATM phosphorylation of the PIDD DD enables a binary switch through which cells elect to survive or die upon DNA injury.

Low-dose angiostatic tyrosine kinase inhibitors improve photodynamic therapy for cancer: lack of vascular normalization.

Photodynamic therapy (PDT) is an effective clinical treatment for a number of different cancers. PDT can induce hypoxia and inflammation, pro-angiogenic side effects, which may counteract its angio-occlusive mechanism. The combination of PDT with anti-angiogenic drugs offers a possibility for improved anti-tumour outcome. We used two tumour models to test the effects of the clinically approved angiostatic tyrosine kinase inhibitors sunitinib, sorafenib and axitinib in combination with PDT, and compared these results with the effects of bevacizumab, the anti-VEGF antibody, for the improvement of PDT. Best results were obtained from the combination of PDT and low-dose axitinib or sorafenib. Molecular analysis by PCR revealed that PDT in combination with axitinib suppressed VEGFR-2 expression in tumour vasculature. Treatment with bevacizumab, although effective as monotherapy, did not improve PDT outcome. In order to test for tumour vessel normalization effects, axitinib was also applied prior to PDT. The absence of improved PDT outcome in these experiments, as well as the lack of increased oxygenation in axitinib-treated tumours, suggests that vascular normalization did not occur. The current data imply that there is a future for certain anti-angiogenic agents to further improve the efficacy of photodynamic anti-cancer therapy.

Imp2 controls oxidative phosphorylation and is crucial for preserving glioblastoma cancer stem cells.

Growth of numerous cancer types is believed to be driven by a subpopulation of poorly differentiated cells, often referred to as cancer stem cells (CSCs), that have the capacity for self-renewal, tumor initiation, and generation of nontumorigenic progeny. Despite their potentially key role in tumor establishment and maintenance, the energy requirements of these cells and the mechanisms that regulate their energy production are unknown. Here, we show that the oncofetal insulin-like growth factor 2 mRNA-binding protein 2 (IMP2, IGF2BP2) regulates oxidative phosphorylation (OXPHOS) in primary glioblastoma (GBM) sphere cultures (gliomaspheres), an established in vitro model for CSC expansion. We demonstrate that IMP2 binds several mRNAs that encode mitochondrial respiratory chain complex subunits and that it interacts with complex I (NADH:ubiquinone oxidoreductase) proteins. Depletion of IMP2 in gliomaspheres decreases their oxygen consumption rate and both complex I and complex IV activity that results in impaired clonogenicity in vitro and tumorigenicity in vivo. Importantly, inhibition of OXPHOS but not of glycolysis abolishes GBM cell clonogenicity. Our observations suggest that gliomaspheres depend on OXPHOS for their energy production and survival and that IMP2 expression provides a key mechanism to ensure OXPHOS maintenance by delivering respiratory chain subunit-encoding mRNAs to mitochondria and contributing to complex I and complex IV assembly.

Caveolin-1 interacts with the chaperone complex TCP-1 and modulates its protein folding activity.

We report that caveolin-1, one of the major structural protein of caveolae, interacts with TCP-1, a hetero-oligomeric chaperone complex present in all eukaryotic cells that contributes mainly to the folding of actin and tubulin. The caveolin-TCP-1 interaction entails the first 32 amino acids of the N-terminal segment of caveolin. Our data show that caveolin-1 expression is needed for the induction of TCP-1 actin folding function in response to insulin stimulation. Caveolin-1 phosphorylation at tyrosine residue 14 induces the dissociation of caveolin-1 from TCP-1 and activates actin folding. We show that the mechanism by which caveolin-1 modulates TCP-1 activity is indirect and involves the cytoskeleton linker filamin. Filamin is known to bind caveolin-1 and to function as a negative regulator of insulin-mediated signaling. Our data support the notion that the caveolin-filamin interaction contributes to restore insulin-mediated phosphorylation of caveolin, thus allowing the release of active TCP-1.

Difference in distribution of microtubule-associated proteins 5a and 5b during the development of cerebral cortex and corpus callosum in cats: dependence on phosphorylation.

MAP5, a microtubule-associated protein characteristic of differentiating neurons, was studied in the developing visual cortex and corpus callosum of the cat. In juvenile cortical tissue, during the first month after birth, MAP5 is present as a protein doublet of molecular weights of 320 and 300 kDa, defined as MAP5a and MAP5b, respectively. MAP5a is the phosphorylated form. MAP5a decreases two weeks after birth and is no longer detectable at the beginning of the second postnatal month; MAP5b also decreases after the second postnatal week but more slowly and it is still present in the adult. In the corpus callosum only MAP5a is present between birth and the end of the first postnatal month. Afterwards only MAP5b is present but decreases in concentration more than 3-fold towards adulthood. Our immunocytochemical studies show MAP5 in somata, dendrites and axonal processes of cortical neurons. In adult tissue it is very prominent in pyramidal cells of layer V. In the corpus callosum MAP5 is present in axons at all ages. There is strong evidence that MAP5a is located in axons while MAP5b seems restricted to somata and dendrites until P28, but is found in callosal axons from P39 onwards. Biochemical experiments indicate that the state of phosphorylation of MAP5 influences its association with structural components. After high speed centrifugation of early postnatal brain tissue, MAP5a remains with pellet fractions while most MAP5b is soluble. In conclusion, phosphorylation of MAP5 may regulate (1) its intracellular distribution within axons and dendrites, and (2) its ability to interact with other subcellular components.

Zoledronate sensitizes endothelial cells to tumor necrosis factor-induced programmed cell death: evidence for the suppression of sustained activation of focal adhesion kinase and protein kinase B/Akt.

Bisphosphonates are potent inhibitors of osteoclast function widely used to treat conditions of excessive bone resorption, including tumor bone metastases. Recent evidence indicates that bisphosphonates have direct cytotoxic activity on tumor cells and suppress angiogenesis, but the associated molecular events have not been fully characterized. In this study we investigated the effects of zoledronate, a nitrogen-containing bisphosphonate, and clodronate, a non-nitrogen-containing bisphosphonate, on human umbilical vein endothelial cell (HUVEC) adhesion, migration, and survival, three events essential for angiogenesis. Zoledronate inhibited HUVEC adhesion mediated by integrin alphaVbeta3, but not alpha5beta1, blocked migration and disrupted established focal adhesions and actin stress fibers without modifying cell surface integrin expression level or affinity. Zoledronate treatment slightly decreased HUVEC viability and strongly enhanced tumor necrosis factor (TNF)-induced cell death. HUVEC treated with zoledronate and TNF died without evidence of enhanced annexin-V binding, chromatin condensation, or nuclear fragmentation and caspase dependence. Zoledronate inhibited sustained phosphorylation of focal adhesion kinase (FAK) and in combination with TNF, with and without interferon (IFN) gamma, of protein kinase B (PKB/Akt). Constitutive active PKB/Akt protected HUVEC from death induced by zoledronate and TNF/IFNgamma. Phosphorylation of c-Src and activation of NF-kappaB were not affected by zoledronate. Clodronate had no effect on HUVEC adhesion, migration, and survival nor did it enhanced TNF cytotoxicity. Taken together these data demonstrate that zoledronate sensitizes endothelial cells to TNF-induced, caspase-independent programmed cell death and point to the FAK-PKB/Akt pathway as a novel zoledronate target. These results have potential implications to the clinical use of zoledronate as an anti-angiogenic or anti-cancer agent.

Differential ubiquitylation of the mineralocorticoid receptor is regulated by phosphorylation.

Aldosterone stimulation of the mineralocorticoid receptor (MR) is involved in numerous physiological responses, including Na+ homeostasis, blood pressure control, and heart failure. Aldosterone binding to MR promotes different post-translational modifications that regulate MR nuclear translocation, gene expression, and finally receptor degradation. Here, we show that aldosterone stimulates rapid phosphorylation of MR via ERK1/2 in a dose-dependent manner (from 0.1 to 10 nM) in renal epithelial cells. This phosphorylation induces an increase of MR apparent molecular weight, with a maximal upward shift of 30 kDa. Strikingly, these modifications are critical for the regulation of the MR ubiquitylation state. Indeed, we find that MR is monoubiquitylated in its basal state, and this status is sustained by the tumor suppressor gene 101 (Tsg101). Phosphorylation leads to disruption of MR/Tsg101 association and monoubiquitin removal. These events prompt polyubiquitin-dependent destabilization of MR and degradation. Preventing MR phosphorylation by ERK1/2 inhibition or mutation of target serines affects the sequential mechanisms of MR ubiquitylation and inhibits the aldosterone-mediated degradation. Our data provide a novel model of negative feedback of aldosterone signaling, involving sequential phosphorylation, monoubiquitin removal and subsequent polyubiquitylation/degradation of MR.

Carma1, a CARD-containing binding partner of Bcl10, induces Bcl10 phosphorylation and NF-kappaB activation.

Bcl10, a caspase recruitment domain (CARD)-containing protein identified from a breakpoint in mucosa-associated lymphoid tissue (MALT) B lymphomas, is essential for antigen-receptor-mediated nuclear factor kappaB (NF-kappaB) activation in lymphocytes. We have identified a novel CARD-containing protein and interaction partner of Bcl10, named Carma1. Carma1 is predominantly expressed in lymphocytes and represents a new member of the membrane-associated guanylate kinase family. Carma1 binds Bcl10 via its CARD motif and induces translocation of Bcl10 from the cytoplasm into perinuclear structures. Moreover, expression of Carma1 induces phosphorylation of Bcl10 and activation of the transcription factor NF-kappaB. We propose that Carma1 is a crucial component of a novel Bcl10-dependent signaling pathway in T-cells that leads to the activation of NF-kappaB.

CCAAT/enhancer-binding protein family members recruit the coactivator CREB-binding protein and trigger its phosphorylation.

CCAAT/enhancer-binding protein (C/EBP) family members are transcription factors involved in important physiological processes, such as cellular proliferation and differentiation, regulation of energy homeostasis, inflammation, and hematopoiesis. Transcriptional activation by C/EBPalpha and C/EBPbeta involves the coactivators CREB-binding protein (CBP) and p300, which promote transcription by acetylating histones and recruiting basal transcription factors. In this study, we show that C/EBPdelta is also using CBP as a coactivator. Based on sequence homology with C/EBPalpha and -beta, we identify in C/EBPdelta two conserved amino acid segments that are necessary for the physical interaction with CBP. Using reporter gene assays, we demonstrate that mutation of these residues prevents CBP recruitment and diminishes the transactivating potential of C/EBPdelta. In addition, our results indicate that C/EBP family members not only recruit CBP but specifically induce its phosphorylation. We provide evidence that CBP phosphorylation depends on its interaction with C/EBPdelta and define point mutations within one of the two conserved amino acid segments of C/EBPdelta that abolish CBP phosphorylation as well as transcriptional activation, suggesting that this new mechanism could be important for C/EBP-mediated transcription.