Publikationsserver der RWTH Aachen University
Charakterisierung der Proteinkinase DYRK1A : Substrat- und Autophosphorylierung
Abstract
dc:descriptionThe Dual Specificity YAK1p-Related Kinase1A (DYRK1A) encodes an evolutionary highly conserved protein kinase. Due to different animal models and its genomic localisation on human chromosome 21, is it considered a candidate gene in mental retardation of the Down-syndrome. Little was known about the cellular function of DYRK1A, but comparison with related kinases and its nuclear localisation gave evidence for its regulatory involvement in mitosis and cell differentiation. Studying the catalysed reactions of phosphorylation is one way to get more information about the molecular function of protein kinases. DYRK1A phosphorylates substrates on serine/threonine residues, whereas autophosphorylation of serine/threonine residues as well as of tyrosine residues is possible. Due to former results, DYRK1A seemed to autophosphorylate its N-terminal segment. Thus, one topic of this work was to study the autophosphorylation of the N-terminal segment. After recombinant expression in bacteria DYRK1A could be shown to be autophosphorylated on tyrosine-111, whereas tyrosine-104 seemed to be involved in this phosphorylation. Mutation analysis of both residues showed no effect on its enzymatic activity in vitro. It is noticeable that tyrosine-111 lies within a possible recognition site of PTB-domains. Thus, tyrosine-111 could be involved in the control of interactions between DYRK1A and PTB-domains of other proteins. In addition tyrosine-111 lies just N-terminal of a biparted nuclear localisation sequence of DYRK1A and thus, involvement in the subcellular localisation of DYRK1A can be hypothesized. DYRK1A specifically phosphorylates a couple of protein substrates in vitro. One of them is histone H3, a typical substrate of in vitro phosphorylation reactions. The second topic of this work was to do further study this phosphorylation reaction. First, a new method to produces pure and unmodified recombinant histone H3 was developed. Homogeneity was 95%. Protein sequencing detected no posttranslational modifications except N-terminal cleavage of the first methionine. In-vitro phosphorylation reactions showed that recombinant Histone H3 is specifically phosphorylated on threonine-45 by DYRK1A. The synthetic oligopeptide H338-52 around threonine-45 was shown to be an efficient substrate of DYRK1A. Two remarkable residues lie within this sequence, namely an arginine and a proline, which might be considered to be part of the recognition sequence of DYRK1A substrates. This could be confirmed by mutation analysis, which moreover showed that inserting a proline directly N-terminal of the acceptor residue further increases the efficiency of substrate phosphorylation. Proline-directed substrate phosphorylation can also be seen in other related kinases of the CMGC-group, whereas an additional arginine within the recognition sequence of substrates is a typical feature of kinases of the DYRK-family.
Degree
thesis:*- Grantor dc:publisher
- Publikationsserver der RWTH Aachen University
- Year dc:date
- 2005
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Himpel, Sunke
- Contributors dc:contributor
-
- Becker, Walter
Subjects
dc:subject × 8Rights
dc:rights- Statement dc:rights
-
- info:eu-repo/semantics/openAccess
- Language dc:language
- ger
Identifiers
dc:identifier.*- OAI identifier oai:identifier
- oai:publications.rwth-aachen.de:60083