Hanne Hollås

Annexin A2 binds to the localization signal in the 3′ untranslated region of c‐myc mRNA

Messenger RNA trafficking, which provides a mechanism for local protein synthesis, is dependent on cis‐acting sequences in the 3′ untranslated regions (3′UTRs) of the mRNAs concerned acting together with trans‐acting proteins. The C‐MYC transcription factor is a proto‐oncogene product involved in cell proliferation, differentiation and apoptosis. Localization of c‐myc mRNA to the perinuclear cytoplasm and its association with the cytoskeleton is determined by a signal in the 3′UTR. Here we show the specific binding of a trans‐acting factor to the perinuclear localization element in the 3′UTR of c‐myc mRNA and identify this protein as annexin A2. Gel retardation and UV cross‐linking experiments showed that proteins in fibroblast extracts formed complexes with the region of c‐myc 3′UTR implicated in localization; a protein of ≈ 36 kDa exhibited specific, Ca2+‐dependent binding. Binding was reduced by introduction of a mutation that abrogates localization. Using RNA‐affinity columns followed by gel electrophoresis and mass spectrometry this protein was identified as annexin A2. The RNA–protein complex formed by cell extracts was further retarded by anti‐(annexin A2). Purified annexin A2 bound to the same region of the c‐myc 3′UTR but binding was reduced by introduction of a mutation, as with cell extracts. It is proposed that binding of annexin A2 to the localization signal in the c‐myc mRNA leads to association with the cytoskeleton and perinuclear localization. The data indicate a novel functional role for the RNA‐binding properties of annexin A2 in perinuclear localization of mRNA and the association with the cytoskeleton.

Ubiquitinated annexin A2 is enriched in the cytoskeleton fraction

Annexin A2 is a multifunctional protein and its cellular functions are regulated by post‐translational modifications and ligand binding. When purified from porcine intestinal mucosa and transformed mouse Krebs II cells, SDS–PAGE revealed high‐molecular‐mass forms in addition to the 36 kDa protomer. These forms were identified as poly‐/multi‐ubiquitin conjugates of annexin A2, and ubiquitination represents a novel post‐translational modification of this protein. Subcellular fractionation of mouse Krebs II cells revealed an enrichment of annexin A2‐ubiquitin conjugates in the Triton X‐100 resistant cytoskeleton fraction, suggesting that ubiquitinated annexin A2 may have a role associated with its function as an actin‐binding protein.

Domains I and IV of Annexin A2 Affect the Formation and Integrity of In Vitro Capillary-Like Networks

Annexin A2 (AnxA2) is a widely expressed multifunctional protein found in different cellular compartments. In spite of lacking a hydrophobic signal peptide, AnxA2 is found at the cell surface of endothelial cells, indicative of a role in angiogenesis. Increased extracellular levels of AnxA2 in tumours correlate with neoangiogenesis, metastasis and poor prognosis. We hypothesised that extracellular AnxA2 may contribute to angiogenesis by affecting endothelial cell-cell interactions and motility. To address this question, we studied the effect of heterotetrameric and monomeric forms of AnxA2, as well as its two soluble domains on the formation and maintenance of capillary-like structures by using an in vitro co-culture system consisting of endothelial and smooth muscle cells. In particular, addition of purified domains I and IV of AnxA2 potently inhibited the vascular endothelial growth factor (VEGF)-dependent formation of the capillary-like networks in a dose-dependent manner. In addition, these AnxA2 domains disrupted endothelial cell-cell contacts in preformed capillary-like networks, resulting in the internalisation of vascular endothelial (VE)-cadherin and the formation of VE-cadherin-containing filopodia-like structures between the endothelial cells, suggesting increased cell motility. Addition of monoclonal AnxA2 antibodies, in particular against Tyr23 phosphorylated AnxA2, also strongly inhibited network formation in the co-culture system. These results suggest that extracellular AnxA2, most likely in its Tyr phosphorylated form, plays a pivotal role in angiogenesis. The exogenously added AnxA2 domains most likely mediate their effects by competing with endogenous AnxA2 for extracellular factors necessary for the initiation and maintenance of angiogenesis, such as those involved in the formation/integrity of cell-cell contacts.

Transient Up‐Regulation of Liver Mitochondrial Thymidine Kinase Activity in Proliferating Mitochondria

Administration of the fatty acid analogue tetradecylthioacetic acid (TTA) to rodents up‐regulates peroxisomal and mitochondrial lipid‐metabolizing enzymes and induces a proliferation of these organelles in hepatocytes. We show here that male NMRI mice fed a diet containing 0.3% (w/w) TTA revealed a transient two‐fold increase in the incorporation of [3

Reactive oxygen species exert opposite effects on Tyr23 phosphorylation of the nuclear and cortical pools of Annexin A2

ABSTRACT Annexin A2 (AnxA2) is a multi-functional and -compartmental protein whose subcellular localisation and functions are tightly regulated by its post-translational modifications. AnxA2 and its Tyr23-phosphorylated form (pTyr23AnxA2) are involved in malignant cell transformation, metastasis and angiogenesis. Here, we show that H2O2 exerts rapid, simultaneous and opposite effects on the Tyr23 phosphorylation status of AnxA2 in two distinct compartments of rat pheochromocytoma (PC12) cells. Reactive oxygen species induce dephosphorylation of pTyr23AnxA2 located in the PML bodies of the nucleus, whereas AnxA2 associated with F-actin at the cell cortex is Tyr23 phosphorylated. The H2O2-induced responses in both compartments are transient and the pTyr23AnxA2 accumulating at the cell cortex is subsequently incorporated into vesicles and then released to the extracellular space. Blocking nuclear export by leptomycin B does not affect the nuclear pool of pTyr23AnxA2, but increases the amount of total AnxA2 in this compartment, indicating that the protein might have several functions in the nucleus. These results suggest that Tyr23 phosphorylation can regulate the function of AnxA2 at distinct subcellular sites. Summary: Reactive oxygen species cause two opposite and transient Tyr23-based modifications of annexin A2; its dephosphorylation in the nucleus and phosphorylation at the cell cortex, resulting in release of the protein in exosomes.

The native structure of annexin A2 peptides in hydrophilic environment determines their anti-angiogenic effects

The progression of aggressive cancer occurs via angiogenesis and metastasis makes these processes important targets for the development of anti-cancer agents. However, recent studies have raised the concern that selective inhibition of angiogenesis results in a switch towards increased tumour growth and metastasis. Since Annexin A2 (AnxA2) is involved in both angiogenesis and metastasis, it may serve as an ideal target for the simultaneous inhibition of both processes. Based on the discovery that domains I (D(I)) and IV (D(IV)) of AnxA2 are potent inhibitors of angiogenesis, we designed seven peptides derived from these domains based on AnxA2 crystal structures. The peptides were expressed as fusion peptides to increase their folding and solubility. Light scattering, far-UV circular dichroism and thermal transition analyses were employed to investigate their aggregation tendencies, α-helical propensity and stability, respectively. 2,2,2-trifluoroethanol (50%) increased the α-helical propensities of all peptides, indicating that they may favour a hydrophobic environment, but did not enhance their thermal stability. D(I)-P2 appears to be the most stable and folded peptide in a hydrophilic environment. The secondary structure of D(I)-P2 was confirmed by nuclear magnetic resonance spectra. The effect of the seven AnxA2 peptides on the formation and integrity of capillary-like networks was studied in a co-culture system mimicking many of the angiogenesis-related processes. Notably, D(I)-P2 inhibited significantly network formation in this system, indicating that the folded D(I)-P2 peptide interferes with vascular endothelial growth factor-dependent pro-angiogenic processes. Thus, this peptide has the potential of being developed further as an anti-angiogenic drug.

Post‐translational modifications of Annexin A2 are linked to its association with perinuclear nonpolysomal mRNP complexes

Various post‐translational modifications (PTMs) regulate the localisation and function of the multifunctional protein Annexin A2 (AnxA2). In addition to its various tasks as a cytoskeletal‐ and membrane‐associated protein, AnxA2 can function as a trans‐acting protein binding to cis‐acting sequences of specific mRNAs. In the present study, we have examined the role of Ser25 phosphorylation in subcellular localisation of AnxA2 and its interaction with mRNP complexes. Subcellular fractionation and confocal microscopy of rat neuroendocrine PC12 cells showed that Ser25‐phosphorylated AnxA2 (pSer25AnxA2) is absent from the nucleus and mainly localised to the perinuclear region, evidently associating with both membranes and cytoskeletal elements. Perinuclear targeting of AnxA2 was abolished by inhibition of protein kinase C activity, which resulted in cortical enrichment of the protein. Although oligo(dT)‐affinity purification of mRNAs revealed that pSer25AnxA2 associates with nonpolysomal, translationally inactive mRNP complexes, it displayed only partial overlap with a marker of P‐bodies. The phosphorylated protein is present as high‐molecular‐mass forms, indicating that it contains additional covalent PTMs, apparently triggered by its Ser25 phosphorylation. The subcellular distributions of these forms clearly differ from the main form of AnxA2 and are also distinct from that of Tyr23‐phosphorylated AnxA2. Immunoprecipitation verified that these high‐molecular‐mass forms are due to ubiquitination and/or sumoylation. Moreover, these results indicate that Ser25 phosphorylation and ubiquitin/SUMO1 conjugation of AnxA2 promote its association with nonpolysomal mRNAs, providing evidence of a possible mechanism to sequester a subpopulation of mRNAs in a translationally inactive and transport competent form at a distinct subcellular localisation.