Das Interaktom der SH2-Domänen der Proteintyrosinphosphatase SHP2 und ihren krankheitsrelevanten Mutanten

摘要

The protein tyrosine phosphatase SHP2 is a common regulator of cytokine, growth factor signaling and has been shown to influence cell survival, proliferation and differentiation by regulation of MAP Kinase, Jak/STAT and PI3K pathways. Structurally SHP2 consists of two SH2 domains, a catalytically active PTP domain and tyrosine residues in the C-terminal tail which upon phosphorylation interact with SH2 domain containing proteins. In addition to its enzymatic function, the protein is also capable of exerting functions as an adaptor molecule. An optional intramolecular interaction of the N-terminal SH2 with the phosphatase domain is able to block the catalytic cleft sterically. Thereby the entry of substrates can be prevented. Due to the auto-inhibition, and the variety of interactions with other proteins which also influence the activity of SHP2, its regulation is complex. Since little is known about the interaction partners of SHP2, this projects aims to identify novel interaction partners using stable-isotope labeling with amino acids in cell culture (SILAC) combined with mass spectrometry. SH2 domains were expressed as GST-fusion proteins in bacteria, subsequently coupled to magnetic beads and used for precipitation of interaction partners from cell lysates. The identified interactome comprises of more than 1000 proteins from different signaling pathways. Germline and somatic mutations in PTPN11 are known be involved in several diseases like Noonan or LEOPARD syndrome or leukemia. Several of these mutations show altered enzymatic activity due to changed auto-inhibition but not all disease patterns can be explained by this observation. Hence, this study also aims to analyze the binding capacities of different disease relevant SHP2 mutants in the SH2 domains. The residues affected in the investigated mutants are not only located in the area responsible for the interaction of the N-terminal SH2 domain with the PTP domain but also the peptide binding area of the SH2 domains. Surface plasmon resonance reveals that the latter mutants show different binding properties to receptor peptides when compared to wild type. Furthermore, the altered binding can also be observed when using proteins precipitated from lysates that are analyzed by mass spectrometry. These observed binding properties might directly translate to altered, recruitment-dependent enzyme activity on target proteins and thereby explain disease patterns.