Biomedical subjects

Maria M Magiera

Publications and source records attributed to Maria M Magiera.

5 recordsLinked to original sources

PubMed2026

Microtubule posttranslational modifications provide unique recognition patterns for associated proteins.

Microtubules are key components of the eukaryotic cytoskeleton involved in vital functions in virtually every cell. Among the emerging molecular mechanisms to adapt microtubules to their diverse functions is the biochemical diversification of tubulin molecules by posttranslational modifications (PTMs) and differential gene expression, a concept known as the 'tubulin code'. A key question remains whether the tubulin code has the potential to selectively control microtubule interactions of different microtubule-associated proteins (MAPs) to act as a specific signalling system. To answer this question, we used a medium-throughput in vitro approach to screen 46 proteins for their binding preferences to microtubules with altered PTM or isotype composition. We demonstrate that subsets of these MAPs have unique sensitivities to PTMs, while other proteins are not affected. As a result, PTMs, or combinations of them, differentially attract or repulse individual MAPs to microtubules. Our findings offer mechanistic proof for a key hypothesis of the tubulin code-the capacity to selectively and differentially regulate MAP-microtubule interactions.

Journal Article↗

PubMed2005

Microtubule-associated protein 1B-light chain 1 enhances activation of Rap1 by exchange protein activated by cyclic AMP but not intracellular targeting.

We have previously demonstrated that EPAC1 interacts with light chain (LC) 2 of microtubule-associated protein (MAP) 1A. In the present study, we investigated whether the structurally related LC1 of MAP1B also interacts with EPAC1. We demonstrate that LC1 copurifies with EPAC1 from extracts of PC-12 cells, using cyclic AMP-agarose. Using recombinant LC1 and LC2 in pull-down and solid phase binding assays, we demonstrate direct interaction with a glutathione S-transferase-fusion of the cyclic AMP-binding (CAMP) domain of EPAC1. We also tested whether LC1 directed intracellular targeting of EPAC1 through its interaction with the CAMP domain. EPAC1 was found be in the soluble and particulate, nuclear/perinuclear fractions of cells. We found that the catalytic (CAT) domain of EPAC1, and not the CAMP domain, was responsible for recruitment to the nuclear/perinuclear fraction of cells. The targeting sequence responsible was located between amino acids 764 and 838 of EPAC1. Overexpresssion of an isolated CAT domain in COS1 cells was found to displace endogenous EPAC1 from the nuclear/perinuclear fraction, thereby inhibiting EPAC-activated Rap1 in this compartment. In contrast, LC1 was not able to compete for the binding of EPAC1 to this fraction. LC1, however, was able to enhance interaction of EPAC1 with cyclic AMP and heightened the ability of EPAC to activate Rap1. Antibody disruption of EPAC1/LC1 interaction in PC-12 cells ablated the ability of cyclic AMP to activate Rap1. LC1 is therefore not involved in intracellular targeting of EPAC1, but it is rather a molecular chaperone of EPAC activity toward Rap1.

Animals↗

PubMed2004

Exchange protein directly activated by cAMP (EPAC) interacts with the light chain (LC) 2 of MAP1A.

Using EPAC1 (exchange protein directly activated by cAMP 1) as bait in two-hybrid screens of foetal and adult human brain libraries, we identified the LC2 (light chain 2) of MAP1A (microtubule-associated protein 1A) as a protein capable of interaction with EPAC1. We applied an immunoprecipitation assay to demonstrate protein interaction between EPAC1 and LC2 in co-transfected human embryonic kidney 293 cells. EPAC2 also co-immunoprecipitated with LC2 from extracts of rat cerebellum. Immunolocalization in co-transfected human embryonic kidney 293 cells revealed that EPAC1 co-localizes with LC2 throughout the cell body. We found that endogenous EPAC2 is also immunolocalized with LC2 in PC12 cells. Immunolocalization of EPAC1 in transfected COS1 cells showed that EPAC1 is associated with the perinuclear region surrounding the nucleus and filamentous structures throughout the cell. Removal of the cAMP-binding domain of EPAC1 (DeltacAMP-EPAC1) appeared to disrupt targeting of EPAC1 in cells resulting in a more dispersed staining pattern. Using two-hybrid assay, we tested the ability of LC2 to interact with DeltacAMP-EPAC1 and DeltaDEP-EPAC1, which lacks a DEP domain (dishevelled, Egl-10 and pleckstrin homology domain). We found that deletion of the cAMP-binding domain inhibited interaction between EPAC1 and LC2 in a two-hybrid assay, but removal of the DEP domain had little effect. LC2 was found to interact with a glutathione-S-transferase-fusion protein of the cAMP-binding domain of EPAC1 in a pull-down assay, but not the DEP, REM (Ras exchange motif) or CAT (catalytic) domains. Together with our two-hybrid results, this suggests that the cAMP-binding domain of EPAC1 mediates interaction with LC2.

Adult↗

PubMed2004

A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes.

Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo in the yeast Saccharomyces cerevisiae. This strategy directs the amplified tags to the desired chromosomal loci due to flanking homologous sequences provided by the PCR-primers, thus enabling the selective introduction of any sequence at any place of a gene, e.g. for the generation of C-terminal tagged genes or for the exchange of the promoter and N-terminal tagging of a gene. To make this method most powerful we constructed a series of 76 novel cassettes, containing a broad variety of C-terminal epitope tags as well as nine different promoter substitutions in combination with N-terminal tags. Furthermore, new selection markers have been introduced. The tags include the so far brightest and most yeast-optimized version of the red fluorescent protein, called RedStar2, as well as all other commonly used fluorescent proteins and tags used for the detection and purification of proteins and protein complexes. Using the provided cassettes for N- and C-terminal gene tagging or for deletion of any given gene, a set of only four primers is required, which makes this method very cost-effective and reproducible. This new toolbox should help to speed up the analysis of gene function in yeast, on the level of single genes, as well as in systematic approaches.

Gene Targeting↗

PubMed2002

Targeting of cyclic AMP degradation to beta 2-adrenergic receptors by beta-arrestins.

Catecholamines signal through the beta2-adrenergic receptor by promoting production of the second messenger adenosine 3',5'-monophosphate (cAMP). The magnitude of this signal is restricted by desensitization of the receptors through their binding to beta-arrestins and by cAMP degradation by phosphodiesterase (PDE) enzymes. We show that beta-arrestins coordinate both processes by recruiting PDEs to activated beta2-adrenergic receptors in the plasma membrane of mammalian cells. In doing so, the beta-arrestins limit activation of membrane-associated cAMP-activated protein kinase by simultaneously slowing the rate of cAMP production through receptor desensitization and increasing the rate of its degradation at the membrane.

3',5'-Cyclic-AMP Phosphodiesterases↗