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Biomedical subjects

Donna J Webb

Publications and source records attributed to Donna J Webb.

23 records · Page 2Linked to original sources
PubMed2002

GIT1 functions in a motile, multi-molecular signaling complex that regulates protrusive activity and cell migration.

GIT1 is a multidomain protein that is thought to function as an integrator of signaling pathways controlling vesicle trafficking, adhesion and cytoskeletal organization. It regulates ARF GTPases and has binding domains for paxillin and PIX, which is a PAK-binding protein and an exchange factor for Rac. We show that GIT1 cycles between at least three distinct subcellular compartments, including adhesion-like structures, the leading edge and cytoplasmic complexes. The cytoplasmic structures, which also contain paxillin, PAK and PIX, do not detectably co-localize with endosomal Golgi or membrane markers, suggesting that they represent a novel supramolecular complex. The GIT1 cytoplasmic complexes are motile and tended to move toward the cell periphery where they joined existing adhesions. In retracting regions of the cells, the GIT1 complexes moved away from the disassembling adhesions toward the cell body. Using deletion mutants, we have identified domains that target GIT1 to each of the compartments. Localization to adhesions and the leading edge requires the paxillin-binding domain, which comprises the C-terminal 140 residues (cGIT1), whereas targeting to the cytoplasmic complexes requires the central region that contains ankyrin repeats and the PIX-binding domain. Expression of GIT1 or cGIT, but not nGIT1 in which the paxillin-binding domain is deleted, increases the rate of migration and the size and number of protrusions. The latter are inhibited when GIT1 is co-expressed with a kinase-dead PAK, suggesting that the GIT1 interaction with PAK is required for enhanced migration and protrusive activity. Furthermore, GIT1 targets constitutively activated PAK to adhesions and the leading edge via its interaction with paxillin. Since expression of cGIT targets endogenous GIT1 to the leading edge, it appears that the leading edge is the location of GIT1 responsible for these activities. Thus, GIT1 is a component of a motile, multimolecular complex that traffics a set of signaling components to specific locations in the cell where they regulate localized activities.

Animals↗
PubMed2002

Distinct binding sites in the structure of alpha 2-macroglobulin mediate the interaction with beta-amyloid peptide and growth factors.

Alpha(2)-macroglobulin (alpha(2)M) and its receptor, low density lipoprotein receptor-related protein (LRP), function together to facilitate the cellular uptake and degradation of beta-amyloid peptide (Abeta). In this study, we demonstrate that Abeta binds selectively to alpha(2)M that has been induced to undergo conformational change by reaction with methylamine. Denatured alpha(2)M subunits, which were immobilized on polyvinylidene difluoride membranes, bound Abeta, suggesting that alpha(2)M tertiary and quaternary structure are not necessary. To determine whether a specific sequence in alpha(2)M is responsible for Abeta binding, we prepared and analyzed defined alpha(2)M fragments and glutathione S-transferase-alpha(2)M peptide fusion proteins. A single sequence, centered at amino acids (aa) 1314-1365, was identified as the only major Abeta-binding site. Importantly, Abeta did not bind to the previously characterized growth factor-binding site (aa 718-734). Although the Abeta binding sequence is adjacent to the binding site for LRP, the results of experiments with mutated fusion proteins indicate that the two sites are distinct. Furthermore, a saturating concentration of Abeta did not inhibit LRP-mediated clearance of alpha(2)M-MA in mice. Using various methods, we determined that the K(D) for the interaction of Abeta with its binding site in the individual alpha(2)M subunit is 0.7-2.4 microm. The capacity of alpha(2)M to bind Abeta and deliver it to LRP may be greater than that predicted by the K(D), because each alpha(2)M subunit may bind Abeta and the bound Abeta may multimerize. These studies suggest a model in which alpha(2)M has three protein interaction sites with distinct specificities, mediating the interaction with Abeta, growth factors, and LRP.

Amyloid beta-Peptides↗
PubMed2002

Adhesion assembly, disassembly and turnover in migrating cells -- over and over and over again.

Cell migration is an integrated process that requires the continuous, coordinated formation and disassembly of adhesions. These processes are complex and require a regulated interaction of numerous molecules, and the activation of specific signalling pathways. Even though understanding these processes is challenging, important insights are beginning to emerge, and the technology to facilitate significant advances in this area is now in place.

Animals↗
PubMedDate not supplied

Methods for the detection of paxillin post-translational modifications and interacting proteins by mass spectrometry.

Methods for the simultaneous identification of interacting proteins and post-translational modifications of the focal adhesion adapter protein, paxillin, are presented. The strategy includes (1) lower-level, transient transfection of FLAG-GFP-Paxillin into HEK293 cells, (2) incubation of cells with phosphatase inhibitors prior to lysis, (3) purification of paxillin by anti-FLAG immunoprecipitation, (4) analysis of peptides generated from on-beads digestion using LTQ-FT or LTQ-ETD mass spectrometry, and (5) enrichment of phosphopeptide methyl esters with IMAC. Using the above strategies, we identify 29 phosphorylation sites (19 novel and 10 previously reported) and a novel glycosylation site on Ser 74. Furthermore, with this method, we simultaneously detect 10 co-purifying proteins which are present in focal adhesion complexes. Extension of these methods to other substrates should facilitate generation of global phosphorylation maps and protein-protein interactions for any protein of interest.

Amino Acid Sequence↗