RESUMEN
During myelination in the central nervous system, proteins arising from the gene in the oligodendrocyte lineage (golli) participate in diverse events in signal transduction and gene regulation. One of the interacting partners of the Golli-isoform BG21 was discovered by yeast-2-hybrid means and was denoted the Golli-interacting-protein (GIP). In subsequent in vitro studies of recombinant murine GIP, it was not possible to produce a full-length version of recombinant murine rmGIP in functional form under native conditions, primarily because of solubility issues, necessitating the study of a hexahistidine-tagged, truncated form ΔN-rmGIP. This protein is an acidic phosphatase belonging to the family of RNA-polymerase-2, small-subunit, C-terminal phosphatases (SCP1), and studies of the human ortholog hSCP1 have also been performed on truncated forms. Here, a new SUMO-expression and purification protocol has been developed for the preparation of a functional, full-length mSCP1/GIP (our nomenclature henceforth), with no additional purification tags. Both full-length mSCP1/GIP and the truncated murine form (now denoted ΔN-rmSCP1/GIP) had similar melting temperatures, indicating that the integrity of the catalytic core per se was minimally affected by the N-terminus. Characterization of mSCP1/GIP activity with the artificial substrate p-NPP (p-nitrophenylphosphate) yielded kinetic parameters comparable to those of ΔN-rmSCP1/GIP and the truncated human ortholog ΔN-hSCP1. Similarly, mSCP1/GIP dephosphorylated a more natural CTD-peptide substrate (but not protein kinase C-phosphorylated BG21) with comparable kinetics to ΔN-hSCP1. The successful production of an active, full-length mSCP1/GIP will enable future evaluation of the functional role of its N-terminus in protein-protein interactions (e.g., BG21) that regulate its phosphatase activity.
Asunto(s)
Escherichia coli/metabolismo , Proteína Básica de Mielina/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Proteínas Recombinantes/genética , Animales , Sistema Nervioso Central/metabolismo , Cromatografía de Afinidad , Escherichia coli/genética , Expresión Génica , Regulación de la Expresión Génica , Ratones , Proteína Básica de Mielina/biosíntesis , Proteína Básica de Mielina/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/metabolismo , Nitrobencenos/metabolismo , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/metabolismo , Fosforilación , Fosforilcolina/análogos & derivados , Fosforilcolina/metabolismo , Isoformas de Proteínas/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal/genéticaRESUMEN
The gene in the oligodendrocyte lineage (golli) encodes a number of proteins essential for myelination, comprising Golli and classic isoforms that are expressed in a developmentally-regulated manner. The Golli-interacting-protein (GIP) was previously discovered in a search for potential interacting partners of the Golli-isoform BG21, and was realised to be an acidic phosphatase belonging to the family of RNA-polymerase-2, small-subunit, C-terminal phosphatases (viz., SCP1). Here, we refer to this protein as mSCP1/GIP. In subsequent in vitro studies of recombinant murine SCP1/GIP, the inability to produce an active full-length version of the protein under native conditions necessitated the study of a truncated form ΔN-rmSCP1/GIP, but with inconclusive results regarding its interaction with BG21 [13]. We have since developed a new SUMO-expression and purification protocol for the preparation of a functional, full-length mGIP/SCP1, with no additional purification tags. Here, the interaction between mSCP1/GIP (with intact N-terminus) and BG21 is shown to be different than for the truncation mutant studied previously. Specifically, this interaction shows a dual effect on the enzymatic activity of mSCP1/GIP by BG21: BG21 enhanced mSCP1/GIP phosphatase activity (Ka = 30 µM), whereas PKCα-phosphorylated BG21 inhibited its activity (Ki = 2.9 µM), suggesting a potential role of BG21 as a molecular switch ("quick-brake mechanism") on mSCP1/GIP. The successful production of an active, full-length mSCP1/GIP thus demonstrates a role for its N-terminus in regulation of phosphatase activity, in events such as the regulation of transcription in oligodendrocytes.