Protein kinase A-mediated phosphorylation of the NF2 tumor suppressor protein merlin at serine 10 affects the actin cytoskeleton.

Mutations in the neurofibromatosis 2 tumor suppressor gene (NF2) encoding merlin (moesin-ezrin-radixin like-protein) induce tumors of the nervous system. Merlin localizes to the cell membrane where it links the actin cytoskeleton to membrane proteins. Cell proliferation is regulated by merlin in many cell types, but merlin's tumor suppressor function still remains unclear. Phosphorylation has been suggested to regulate merlin's activity. The C-terminal serine 518 is phosphorylated both by p21-activated kinases (PAKs) and protein kinase A (PKA). In this work, we identify a novel PKA phosphorylation site, serine 10, in the N terminus of merlin. We show that a non-phosphorylatable form of serine 10 (S10A) affects cellular morphology. Regulation of this site also influences actin cytoskeleton organization and dynamics in vivo, as merlin S10A reduces the amount of cellular F-actin and merlin S10D stabilizes F-actin filaments. By using a wound-healing assay and live cell imaging, we demonstrate that dephosphorylation of serine 10 leads to defects in migration, possibly through altered ability of the cells to form lamellipodia. This study suggests a role for merlin in mediating PKA-induced changes of the actin cytoskeleton.

A functional association between merlin and HEI10, a cell cycle regulator.

Merlin and ezrin are homologous proteins with opposite effects on neoplastic growth. Merlin is a tumor suppressor inactivated in the neurofibromatosis 2 disease, whereas upregulated ezrin expression is associated with increased malignancy. Merlin's tumor suppressor mechanism is not known, although participation in cell cycle regulation has been suggested. To characterize merlin's biological activities, we screened for molecules that would interact with merlin but not ezrin. We identified the cyclin B-binding protein and cell cycle regulator HEI10 as a novel merlin-binding partner. The interaction is mediated by the alpha-helical domain in merlin and the coiled-coil domain in HEI10 and requires conformational opening of merlin. The two proteins show partial subcellular colocalization, which depends on cell cycle stage and cell adhesion. Comparison of Schwann cells and schwannoma cultures demonstrated that the distribution of HEI10 depends on merlin expression. In transfected cells, a constitutively open merlin construct affected HEI10 protein integrity. These results link merlin to the cell cycle control machinery and may help to understand its tumor suppressor function.

Characterization of human palladin, a microfilament-associated protein.

Actin-containing microfilaments control cell shape, adhesion, and contraction. In striated muscle, alpha-actinin and other Z-disk proteins coordinate the organization and functions of actin filaments. In smooth muscle and nonmuscle cells, periodic structures termed dense bodies and dense regions, respectively, are thought to serve functions analogous to Z-discs. We describe here identification and characterization of human palladin, a protein expressed mainly in smooth muscle and nonmuscle and distributed along microfilaments in a periodic manner consistent with dense regions/bodies. Palladin contains three Ig-domains most homologous to the sarcomeric Z-disk protein myotilin. The N terminus includes an FPPPP motif recognized by the Ena-Vasp homology domain 1 domain in Ena/vasodilatator-stimulated phosphoprotein (VASP)/Wiscott-Aldrich syndrome protein (WASP) protein family. Cytoskeletal proteins with FPPPP motif target Ena/VASP/WASP proteins to sites of actin modulation. We identified palladin in a yeast two-hybrid search as an ezrin-associated protein. An interaction between palladin and ezrin was further verified by affinity precipitation and blot overlay assays. The interaction was mediated by the alpha-helical domain of ezrin and by Ig-domains 2-3 of palladin. Ezrin is typically a component of the cortical cytoskeleton, but in smooth muscle cells it is localized along microfilaments. These cells express palladin abundantly and thus palladin may be involved in the microfilament localization of ezrin. Palladin expression was up-regulated in differentiating dendritic cells (DCs), coinciding with major cytoskeletal and morphological alterations. In immature DCs, palladin localized in actin-containing podosomes and in mature DCs along actin filaments. The regulated expression and localization suggest a role for palladin in the assembly of DC cytoskeleton.

A comparison between on-site immunoassay drug-testing devices and laboratory results.

The aim with this study was to evaluate the accuracy of several on-site testing devices on the market. A part of this study is included in the European Union's (EU's) roadside testing assessment project (ROSITA). An other request for this kind of study came from the Finnish prison department in the Ministry of Justice. The evaluation was performed on both urine assays and oral fluid assays. The on-site test results were compared with laboratory results (gas chromatography-mass spectrometry (GC/MS)). The samples were tested on amphetamines (AMP), cannabinoids (THC), opiates (OPI) and cocaine metabolites (COC). Some of the tests also included a metamphetamine (MET) and a benzodiazepine (BZO) test. Both positive and negative samples were tested. A total of 800 persons and eight on-site devices for urine and two for oral fluid testing were included in this study. Good results were obtained for the urine on-site devices, with accuracies of 93-99% for amphetamines, 97-99% for cannabinoids, 94-98% for opiates and 90-98% for benzodiazepines. However, differences in the ease of performance and interpretation of test result were observed. It was possible to detect amphetamines and opiates in oral fluid by the used on-site devices, but the benzodiazepines and cannabinoids did not fulfil the needs of sensitivity.

Comparison of eight commercial on-site screening devices for drugs-of-abuse testing.

Eight commercially available on-site drugs-of-abuse testing devices for detecting cannabinoids (THC-COOH), opiates (OPI), cocaine (COC), amphetamines (AMP), metamphetamines (MET) and benzodiazepines (BZO) were evaluated. The used urine specimens suspected of being drug positive were all confirmed by gas chromatographic/mass spectrometry (GC/MS). For AMP and MET, sensitivities varied between 83 and 95% and specificities between 98 and 100%. Correspondingly, sensitivities between 88 and 98% and specificities between 95 and 100% were observed for THC-COOH. For BZO, sensitivities varied between 91 and 97% and specificities between 97 and 100%. Only a few confirmed positive samples were available for OPI and COC, the sensitivities being between 83 and 100% and 100%, respectively. On-site devices did not always find extremely high drug concentrations. False-negative results were found with AMP in particular. Pholcodine, commonly used as medicine, was observed to give false-positive results with most of the devices and was not, however, included in given cross-reactivity tables. It was found that the devices differed markedly with respect to interpretation of test results and to ease of test performance, leading to the suggestion that different criteria for selecting on-site devices for either emergency laboratories in hospitals or for police stations and prisons should be used. Since the overall specificity of any of the devices was not 100% and false positives were identified, we found it important to confirm any positive screening test result.

Myotilin, a novel sarcomeric protein with two Ig-like domains, is encoded by a candidate gene for limb-girdle muscular dystrophy.

The striated muscle sarcomeres are highly organized structures composed of actin (thin) and myosin (thick) filaments that slide past each other during contraction. The integrity of sarcomeres is controlled by a set of structural proteins, among which are titin, a giant molecule that contains several immunoglobulin (Ig)-like domains and associates with thin and thick filaments, and [alpha]-actinin, an actin cross-linking protein. Mutations in several sarcomeric and sarcolemmal proteins have been shown to result in muscular dystrophy and cardiomyopathy. On the other hand, the disease genes underlying several disease forms remain to be identified. Here we describe a novel 57 kDa cytoskeletal protein, myotilin. Its N-terminal sequence is unique, but the C-terminal half contains two Ig-like domains homologous to titin. Myotilin is expressed in skeletal and cardiac muscle, it co-localizes with [alpha]-actinin in the sarcomeric I--bands and directly interacts with [alpha]-actinin. The human myotilin gene maps to chromosome 5q31 between markers AFM350yB1 and D5S500. The locus of a dominantly inherited limb-girdle muscular dystrophy (LGMD1A) resides in an overlapping narrow segment, and a new type of distal myopathy with vocal cord and pharyngeal weakness (VCPMD) has been mapped to the same locus. The muscle specificity and apparent role as a sarcomeric structural protein raise the possibility that defects in the myotilin gene may cause muscular dystrophy.

Homotypic and heterotypic interaction of the neurofibromatosis 2 tumor suppressor protein merlin and the ERM protein ezrin.

Ezrin, radixin and moesin (ERM) are homologous proteins, which are linkers between plasma membrane components and the actin-containing cytoskeleton. The ERM protein family members associate with each other in a homotypic and heterotypic manner. The neurofibromatosis 2 (NF2) tumor suppressor protein merlin (schwannomin) is structurally related to ERM members. Merlin is involved in tumorigenesis of NF2-associated and sporadic schwannomas and meningiomas, but the tumor suppressor mechanism is poorly understood. We have studied the ability of merlin to self-associate and bind ezrin. Ezrin was coimmunoprecipitated with merlin from lysates of human U251 glioma cells and from COS-1 cells transfected with cDNA encoding for merlin isoform I. The interaction was further studied and the association domains were mapped with the yeast two-hybrid system and with blot overlay and affinity precipitation experiments. The heterotypic binding of merlin and ezrin and the homotypic association of merlin involves interaction between the amino- and carboxy-termini. The amino-terminal association domain of merlin involves residues 1-339 and has similar features with the amino-terminal association domain of ezrin. The carboxy-terminal association domain cannot be mapped as precisely as in ezrin, but it requires residues 585-595 and a more amino-terminal segment. Unlike ezrin, merlin does not require activation for self-association but native merlin molecules can interact with each other. Heterodimerization between merlin and ezrin, however, occurs only following conformational alterations in both proteins. These results biochemically connect merlin to the cortical cytoskeleton and indicate differential regulation of merlin from ERM proteins.

Association of ezrin with intercellular adhesion molecule-1 and -2 (ICAM-1 and ICAM-2). Regulation by phosphatidylinositol 4, 5-bisphosphate.

Ezrin is a cytoplasmic linker molecule between plasma membrane components and the actin-containing cytoskeleton. We studied whether ezrin is associated with intercellular adhesion molecule (ICAM)-1, -2, and -3. In transfected cells, ICAM-1 and ICAM-2 colocalized with ezrin in microvillar projections, whereas an ICAM-1 construct attached to cell membrane via a glycophosphatidylinositol anchor was uniformly distributed on the cell surface. An interaction of ICAM-2 and ezrin was seen by affinity precipitation, microtiter binding assay, coimmunoprecipitation, and surface plasmon resonance methods. The calculated KD value was 3.3 x 10(-7) M. Phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2) induced an interaction of ezrin and ICAM-1 and enhanced the interaction of ezrin and ICAM-2, but ICAM-3 did not bind ezrin even in the presence of PtdIns(4,5)P2. PtdIns(4, 5)P2 was shown to bind to cytoplasmic tails of ICAM-1 and ICAM-2, which are the first adhesion proteins demonstrated to interact with PtdIns(4,5)P2. The results indicate an interaction of ezrin with ICAM-1 and ICAM-2 and suggest a regulatory role of phosphoinositide signaling pathways in regulation of ICAM-ezrin interaction.

A novel MLL-AF10 fusion mRNA variant in a patient with acute myeloid leukemia detected by a new asymmetric reverse transcription PCR method.

A novel variant of the chimerical MLL-AF10 mRNA transcript was detected in a pediatric patient with acute myeloid leukemia (AML) by a new asymmetric reverse-transcription polymerase chain reaction (ART-PCR) method. Sequence analysis of the fusion region on the amplified cDNA fragment showed an in-frame joining of exon e5 of the MLL gene and position 1931 of the cDNA sequence of the AF10 gene, giving rise to a new MLL-AF10 transcript. The presence of the new chimerical mRNA product in a sample from the patient was confirmed by classical RT-PCR.

Center and surround excitation in the receptive fields of frog retinal ganglion cells.

We have reexamined the receptive fields of frog retinal ganglion cells focussing on their surround properties. Carefully excluding artifacts due to stimulation of the (Gaussian) RF center, we found that spiking responses can be elicited by step stimulation of any receptor type in the surrounds of all the classes 1-4 Maturana et al. (1960) (J. gen. Physiol. 43, 129-175). The surround responses are antagonized by the responsive center and suppressed by the inhibitory surround, but are seen because of their slower dynamics. The responsive surround differs spectrally from the center: in the latter, cones and green rods compete, in the former, their signals sum.