Distinct functions of POT1 at telomeres

The mammalian protein POT1 binds to telomeric single-stranded DNA (ssDNA), protecting chromosome ends from being detected as sites of DNA damage. POT1 is composed of an N-terminal ssDNA-binding domain and a C-terminal protein interaction domain. With regard to the latter, POT1 heterodimerizes with the protein TPP1 to foster binding to telomeric ssDNA in vitro and binds the telomeric double-stranded-DNA-binding protein TRF2. We sought to determine which of these functions-ssDNA, TPP1, or TRF2 binding-was required to protect chromosome ends from being detected as DNA damage. Using separation-of-function POT1 mutants deficient in one of these three activities, we found that binding to TRF2 is dispensable for protecting telomeres but fosters robust loading of POT1 onto telomeric chromatin. Furthermore, we found that the telomeric ssDNA-binding activity and binding to TPP1 are required in cis for POT1 to protect telomeres. Mechanistically, binding of POT1 to telomeric ssDNA and association with TPP1 inhibit the localization of RPA, which can function as a DNA damage sensor, to telomeres.     

Secreted HHIP1 interacts with heparan sulfate and regulates Hedgehog ligand localization and function

Vertebrate Hedgehog (HH) signaling is controlled by several ligand-binding antagonists including Patched-1 (PTCH1), PTCH2, and HH-interacting protein 1 (HHIP1), whose collective action is essential for proper HH pathway activity. However, the molecular mechanisms used by these inhibitors remain poorly understood. In this paper, we investigated the mechanisms underlying HHIP1 antagonism of HH signaling. Strikingly, we found evidence that HHIP1 non–cell-autonomously inhibits HH-dependent neural progenitor patterning and proliferation. Furthermore, this non–cell-autonomous antagonism of HH signaling results from the secretion of HHIP1 that is modulated by cell type–specific interactions with heparan sulfate (HS). These interactions are mediated by an HS-binding motif in the cysteine-rich domain of HHIP1 that is required for its localization to the neuroepithelial basement membrane (BM) to effectively antagonize HH pathway function. Our data also suggest that endogenous, secreted HHIP1 localization to HS-containing BMs regulates HH ligand distribution. Overall, the secreted activity of HHIP1 represents a novel mechanism to regulate HH ligand localization and function during embryogenesis.     

POT1b protects telomeres from end-to-end chromosomal fusions and aberrant homologous recombination

POT1 (protection of telomere 1) is a highly conserved single-stranded telomeric binding protein that is essential for telomere end protection. Here, we report the cloning and characterization of a second member of the mouse POT family. POT1b binds telomeric DNA via conserved DNA binding oligonucleotide/oligosaccharide (OB) folds. Compared to POT1a, POT1b OB-folds possess less sequence specificity for telomeres. In contrast to POT1a, truncated POT1b possessing only the OB-folds can efficiently localize to telomeres in vivo. Overexpression of a mutant Pot1b allele that cannot bind telomeric DNA initiated a DNA damage response at telomeres that led to p53-dependent senescence. Furthermore, a reduction of the 3' G-rich overhang, increased chromosomal fusions and elevated homologous recombination (HR) were observed at telomeres. shRNA mediated depletion of endogenous Pot1b in Pot1a deficient cells resulted in increased chromosomal aberrations. Our results indicate that POT1b plays important protective functions at telomeres and that proper maintenance of chromosomal stability requires both POT proteins.     

The EGF receptor interacts with the type 1 IGF receptor and regulates its stability

Both the epidermal growth factor receptor (EGFR) and type 1 insulin-like growth factor receptor (IGF1R) require homo- and hetero-dimerisation with their own family members to acquire full function. We recently showed that IGF1R gene silencing led to EGFR hyper-phosphorylation in human breast cancer cells, and hypothesised that this crosstalk might be associated with direct IGF1R:EGFR interaction. Indeed we could detect reciprocal co-precipitation between the IGF1R and EGFR when overexpressed in SKUT-1 cells, and between endogenous IGF1R and EGFR in MDA-MB-468 breast carcinoma cells, two squamous cancer cell lines, and clinical samples of breast cancer. Interaction was abolished by knockdown of either receptor, and we noted that EGFR knockdown also suppressed IGF1R protein levels. Further investigation revealed that EGFR depletion induced enhancement of IGF1R ubiquitylation and degradation. These results indicate novel evidence of crosstalk between two key cancer treatment targets, capable of modifying the stability of IGF1R protein.     

Pin1 interacts with c-Myb in a phosphorylation-dependent manner and regulates its transactivation activity

Activity and stability of the proto-oncogene c-Myb are regulated by post-translational modifications, though the molecular mechanisms underlying such control are only partially understood. Here we describe the functional interaction of c-Myb with Pin1, an isomerase that binds to phosphorylated Ser/Thr-Pro motifs. We found that co-expression of c-Myb and Pin1 led to a net increase of c-Myb transactivation activity, both on reporter constructs as well as on an endogenous target gene. DNA-binding studies revealed that Pin1 did not increase the association of c-Myb with its response element in DNA. The increase of c-Myb transactivation activity was strictly dependent on the presence of an active catalytic center in Pin1. We provide evidence that c-Myb and Pin1 physically interacted, both upon ectopic expression of the proteins in HEK-293 cells as well as in the more physiological setting of HL60 cells, where c-Myb and Pin1 are resident proteins. By point mutating each individual Ser/Thr-Pro motif in c-Myb as well as by using deletion mutants we show that S528 in the EVES-motif was the docking site for Pin1. Mass spectrometry confirmed that S528 is phosphorylated in vivo. Finally, functional studies showed that mutation of S528 to alanine almost abolished the increase of transactivation activity by Pin1. This study reveals a new paradigm by which phosphorylation controls c-Myb function.     

ZNRF1 interacts with tubulin and regulates cell morphogenesis

The ubiquitin-proteasome system has been implicated in neuronal degeneration and regeneration. We demonstrated that overexpression of ZNRF1, which has been identified as a crucial molecule in nerve regeneration, causes morphological changes such as neurite-like elongation. Molecular dissections showed that both the RING finger domain and zinc finger domain are required for morphological changes. Furthermore, we identified β-tubulin type 2 (Tubb2) as a ZNRF1-binding protein by yeast two-hybrid screening. In vivo binding assay showed that ZNRF1 interacts with Tubb2 and immunofluorescent staining suggests that ZNRF1 is colocalized with Tubb2. These results suggest that ZNRF1 mediates regulation of neuritogenesis via interaction with tubulin.     

HSP60 interacts with YB-1 and affects its polysome association and subcellular localization

YB-1 is a DNA/RNA-binding protein which, in the cytoplasm, associates with polysomes and regulates translation. However, YB-1 has a novel nuclear localization signal, and its nuclear accumulation is correlated with cancer induction. Here we designated the amino-acid sequence as YB-NLS and demonstrated that YB-NLS is necessary for the nuclear translocation of overexpressed YB-1 in NG108-15 cells. In addition, we found that a heat shock protein, HSP60, binds to YB-NLS in the cytoplasm. Interestingly, when HSP60 expression was repressed, an increase of polysome-associated YB-1 was observed in heavy-sedimenting fractions on a sucrose gradient. Overexpression of HSP60 resulted in a decrease of YB-1 in the heavy-sedimenting fractions and suppression of YB-NLS activity. Furthermore, the NLS-deleted YB-1 was apparently associated with the heavy-sedimenting polysomes. These results suggest that HSP60 interacts with YB-1 at the YB-NLS region and acts as a regulator of polysome association and the subcellular distribution of YB-1.     

Axin directly interacts with plakoglobin and regulates its stability.

Plakoglobin is homologous to beta-catenin. Axin, a Wnt signal negative regulator, enhances glycogen synthase kinase (GSK)-3beta-dependent phosphorylation of beta-catenin and stimulates the degradation of beta-catenin. Therefore, we examined the effect of Axin on plakoglobin stability. Axin formed a complex with plakoglobin in COS cells and SW480 cells. Axin directly bound to plakoglobin, and this binding was inhibited by beta-catenin. Axin promoted GSK-3beta-dependent phosphorylation of plakoglobin. Furthermore, overexpression of Axin down-regulated the level of plakoglobin in SW480 cells. These results suggest that Axin regulates the stability of plakoglobin by enhancing its phosphorylation by GSK-3beta and that Axin may act on beta-catenin and plakoglobin in similar manners.     

Migfilin interacts with vasodilator-stimulated phosphoprotein (VASP) and regulates VASP localization to cell-matrix adhesions and migration

Cell migration is a complex process that is coordinately regulated by cell-matrix adhesion and actin cytoskeleton. We report here that migfilin, a recently identified component of cell-matrix adhesions, is a biphasic regulator of cell migration. Loss of migfilin impairs cell migration. Surprisingly, overexpression of migfilin also reduces cell migration. Molecularly, we have identified vasodilator-stimulated phosphoprotein (VASP) as a new migfilin-binding protein. The interaction is mediated by the VASP EVH1 domain and a single L104PPPPP site located within the migfilin proline-rich domain. Migfilin and VASP form a complex in both suspended and adhered cells, and in the latter, they co-localize in cell-matrix adhesions. Functionally, migfilin facilitates VASP localization to cell-matrix adhesions. Using two different approaches (VASP-binding defective migfilin mutants and small interfering RNA-mediated VASP knockdown), we show that the interaction with VASP is crucially involved in migfilin-mediated regulation of cell migration. Our results identify migfilin as an important regulator of cell migration and provide new information on the mechanism by which migfilin regulates this process.     

The anti-apoptotic protein HAX-1 interacts with SERCA2 and regulates its protein levels to promote cell survival

Cardiac contractility is regulated through the activity of various key Ca²⁺-handling proteins. The sarco(endo)plasmic reticulum (SR) Ca²⁺ transport ATPase (SERCA2a) and its inhibitor phospholamban (PLN) control the uptake of Ca²⁺ by SR membranes during relaxation. Recently, the antiapoptotic HS-1–associated protein X-1 (HAX-1) was identified as a binding partner of PLN, and this interaction was postulated to regulate cell apoptosis. In the current study, we determined that HAX-1 can also bind to SERCA2. Deletion mapping analysis demonstrated that amino acid residues 575–594 of SERCA2's nucleotide binding domain are required for its interaction with the C-terminal domain of HAX-1, containing amino acids 203-245. In transiently cotransfected human embryonic kidney 293 cells, recombinant SERCA2 was specifically targeted to the ER, whereas HAX-1 selectively concentrated at mitochondria. On triple transfections with PLN, however, HAX-1 massively translocated to the ER membranes, where it codistributed with PLN and SERCA2. Overexpression of SERCA2 abrogated the protective effects of HAX-1 on cell survival, after hypoxia/reoxygenation or thapsigargin treatment. Importantly, HAX-1 overexpression was associated with down-regulation of SERCA2 expression levels, resulting in significant reduction of apparent ER Ca²⁺ levels. These findings suggest that HAX-1 may promote cell survival through modulation of SERCA2 protein levels and thus ER Ca²⁺ stores.     

14-3-3 protein interacts with Huntingtin-associated protein 1 and regulates its trafficking

HAP1 (Huntingtin-associated protein 1) consists of two alternately spliced isoforms (HAP1A and HAP1B, which have unique C-terminal sequences) and participates in intracellular trafficking. The C terminus of HAP1A is phosphorylated, and this phosphorylation was found to decrease the association of HAP1A with kinesin light chain, a protein involved in anterograde transport in cells. It remains unclear how this phosphorylation functions to regulate the association of HAP1 with trafficking proteins. Using the yeast two-hybrid system, we found that HAP1 also interacts with 14-3-3 proteins, which are involved in the assembly of protein complexes and the regulation of protein trafficking. The interaction of HAP1 with 14-3-3 is confirmed by their immunoprecipitation and colocalization in mouse brain. Moreover, this interaction is specific to HAP1A and is increased by the phosphorylation of the C terminus of HAP1A. We also found that expression of 14-3-3 decreases the association of HAP1A with kinesin light chain. As a result, there is less HAP1A distributed in neurite tips of PC12 cells that overexpress 14-3-3. Also, overexpression of 14-3-3 reduces the effect of HAP1A in promoting neurite outgrowth of PC12 cells. We propose that the phosphorylation-dependent interaction of HAP1A with 14-3-3 regulates HAP1 function by influencing its association with kinesin light chain and trafficking in neuronal processes.     

Adaptor protein Nck1 interacts with p120 Ras GTPase-activating protein and regulates its activity

Adaptor protein Nck1 binds a number of intracellular proteins and influences various signaling pathways. Here we show that Nck1 directly binds and activates the GTPase-activating protein of Ras (RasGAP), which is responsible for the down-regulation of Ras. The first and the third SH3 domains of Nck1 and the NH₂-terminal proline-rich sequence of RasGAP contribute most to the complex formation causing direct molecular interaction between the two proteins. Cell adhesion to the substrate is obligatory for the Nck1 and RasGAP association, as cell detachment makes RasGAP incapable of associating with Nck1. This leads to the complex dissipation, decrease of RasGAP activity and the increase of H-Ras-GTP level in the detached cells. Our findings reveal unexpected feature of adaptor protein Nck1 as the regulator of RasGAP activity.     

KIAA0101 interacts with BRCA1 and regulates centrosome number

To find genes and proteins that collaborate with BRCA1 or BRCA2 in the pathogenesis of breast cancer, we used an informatics approach and found a candidate BRCA interactor, KIAA0101, to function like BRCA1 in exerting a powerful control over centrosome number. The effect of KIAA0101 on centrosomes is likely direct, as its depletion does not affect the cell cycle, KIAA0101 localizes to regions coincident with the centrosomes, and KIAA0101 binds to BRCA1. We analyzed whether KIAA0101 protein is overexpressed in breast cancer tumor samples in tissue microarrays, and we found that overexpression of KIAA0101 correlated with positive Ki67 staining, a biomarker associated with increased disease severity. Furthermore, overexpression of the KIAA0101 gene in breast tumors was found to be associated with significantly decreased survival time. This study identifies KIAA0101 as a protein important for breast tumorigenesis, and as this factor has been reported as a UV repair factor, it may link the UV damage response to centrosome control.