Deciphering protein kinase specificity through large-scale analysis of yeast phosphorylation site motifs.

Phosphorylation is a universal mechanism for regulating cell behavior in eukaryotes. Although protein kinases target short linear sequence motifs on their substrates, the rules for kinase substrate recognition are not completely understood. We used a rapid peptide screening approach to determine consensus phosphorylation site motifs targeted by 61 of the 122 kinases in Saccharomyces cerevisiae.

Substrate discrimination among mitogen-activated protein kinases through distinct docking sequence motifs.

Mitogen-activated protein kinases (MAPKs) mediate cellular responses to a wide variety of extracellular stimuli. MAPK signal transduction cascades are tightly regulated, and individual MAPKs display exquisite specificity in recognition of their target substrates. All MAPK family members share a common phosphorylation site motif, raising questions as to how substrate specificity is achieved.

The role of membrane and vesicular monoamine transporters in the neurotoxic and hypothermic effects of 1-methyl-4-(2'-aminophenyl)-1,2,3,6-tetrahydropyridine (2'-NH(2)-MPTP).

The neurotoxin 1-methyl-4-(2'-aminophenyl)-1,2,3,6-tetrahydropyridine (2'-NH(2)-MPTP) damages forebrain serotonin (5-HT) and norepinephrine (NE) nerve terminals while sparing striatal dopaminergic innervation. Previous studies suggest that 2'-NH(2)-MPTP acts by a mechanism that involves uptake by the plasma membrane 5-HT and NE transporters. The present investigation further explores the molecular mechanism of 2'-NH(2)-MPTP with regard to cellular transport and effects on body temperature.

A faster way to make GFP-based biosensors: two new transposons for creating multicolored libraries of fluorescent fusion proteins.

Background: There are now several ways to generate fluorescent fusion proteins by randomly inserting DNA encoding the Green Fluorescent Protein (GFP) into another protein's coding sequence. These approaches can be used to map regions in a protein that are permissive for GFP insertion or to create novel biosensors. While remarkably useful, the current insertional strategies have two major limitations: (1) they only produce one kind, or color, of fluorescent fusion protein and (2) one half of all GFP insertions within the target coding sequence are in the wrong orientation.

A new way to rapidly create functional, fluorescent fusion proteins: random insertion of GFP with an in vitro transposition reaction.

Background: The jellyfish green fluorescent protein (GFP) can be inserted into the middle of another protein to produce a functional, fluorescent fusion protein. Finding permissive sites for insertion, however, can be difficult. Here we describe a transposon-based approach for rapidly creating libraries of GFP fusion proteins.