RESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Eyes absent (EYA) proteins are unusual proteins combining in a single polypeptide chain transactivation, threonine phosphatase, and tyrosine phosphatase activities. They play pivotal roles in organogenesis and are involved in a variety of physiological and pathological processes including innate immunity, DNA damage repair or cancer metastasis. The molecular targets of EYA tyrosine phosphatase activity are still elusive. Therefore, we sought to identify novel EYA substrates and also to obtain further insight into the tyrosine-dephosphorylating role of EYA proteins in various cellular processes. We show here that Src kinase phosphorylates tyrosine residues in two human EYA family members, EYA1 and EYA3. Both can autodephosphorylate these residues and their nuclear and cytoskeletal localization seems to be controlled by Src phosphorylation. Next, using a microarray of phosphotyrosine-containing peptides, we identified a phosphopeptide derived from WD-repeat-containing protein 1 (WDR1) that is dephosphorylated by EYA3. We further demonstrated that several tyrosine residues on WDR1 are phosphorylated by Src kinase, and are efficiently dephosphorylated by EYA3, but not by EYA1. The lack of phosphorylation generates major changes to the cellular actin cytoskeleton. We, therefore, conclude that WDR1 is an EYA3-specific substrate, which implies that EYA3 is a key modulator of the cytoskeletal reorganization.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Sequência de Aminoácidos , Biocatálise , Sequência Conservada , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Mutação , Fosforilação , Domínios Proteicos , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/genética , Quinases da Família src/metabolismoRESUMO
Protein tyrosine phosphatases (PTP) are a large group of enzymes which work together with protein tyrosine kinases to control the tyrosine phosphorylation of proteins, thus playing a major role in cellular signaling. Here, we provide detailed protocols for expression and purification of the catalytic domain of RPTPµ and full length Eya3 as well as the extracellular region of PTPBR7. Methods are described for evaluation of the purity of the recombinant proteins thus obtained. For the purified Eya3 phosphatase we provide protocols for enzyme activity assay using either chromogenic, fluorescent, or peptide substrates. Determination of kinetic parameters by different graphical and computer-based procedures is also described.
Assuntos
Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismo , Western Blotting/métodos , Domínio Catalítico , Cromatografia de Afinidade/métodos , Cromatografia em Gel/métodos , Cromatografia por Troca Iônica/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Escherichia coli/genética , Expressão Gênica , Humanos , Cinética , Espectrometria de Massas/métodos , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
TPST1 is a human tyrosylprotein sulfotransferase that uses 3'phosphoadenosine-5'phosphosulfate (PAPS) to transfer the sulfate moiety to proteins predominantly designated for secretion. To achieve a general understanding of the cellular role of human tyrosine-directed sulfotransferases, we investigated targeting, structure and posttranslational modification of TPST1. Golgi localisation of the enzyme in COS-7 and HeLa cells was visualised by fluorescence imaging techniques. PNGase treatment and mutational studies determined that TPST1 bears N-linked glycosyl residues exclusively at position Asn60 and Asn262. By alanine mutation of these asparagine residues, we could determine that the N-linked oligosaccharides do not have an influence on Golgi retention of TPST1. In concert with N and C-terminal flanking residues, the transmembrane domain of TPST1 was determined to act in targeting and retention of the enzyme to the trans-Golgi compartment. This domain exhibits a pronounced secondary structure in a lipid environment. Further in vivo FRET studies using the transmembrane domain suggest that the human tyrosylprotein sulfotransferase may be functional as homodimer/oligomer in the trans-Golgi compartment.
