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J Proteomics. 2013 Jun 12;84:132-47. doi: 10.1016/j.jprot.2013.04.005. Epub 2013 Apr 11.

Protein tyrosine phosphatase SHP2/PTPN11 mistargeting as a consequence of SH2-domain point mutations associated with Noonan Syndrome and leukemia.

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1
Department of Biochemistry, RWTH Aachen University, Pauwelsstrasse 30, D-52074 Aachen, Germany.

Abstract

SHP2/PTPN11 is a key regulator of cytokine, growth factor and integrin signaling. SHP2 influences cell survival, proliferation and differentiation by regulating major signaling pathways. Mutations in PTPN11 cause severe diseases like Noonan, LEOPARD syndrome or leukemia. Whereas several of these mutations result in altered enzymatic activity due to impaired auto-inhibition, not all disease patterns can be explained by this mechanism. In this study we analyzed altered binding properties of disease-related SHP2-mutants bearing point mutations within the SH2-domain (T42A, E139D, and R138Q). Mutants were chosen according to SPR assays, which revealed different binding properties of mutated SH2 towards phosphorylated receptor peptides. To analyze global changes in mutant binding properties we applied quantitative mass spectrometry (SILAC). Using an in vitro approach we identified overall more than 1000 protein candidates, which specifically bind to the SH2-domain of SHP2. We discovered that mutations in the SH2-domain selectively affected protein enrichment by altering the binding capacity of the SH2-domain. Mutation-dependent, enhanced or reduced exposure of SHP2 to its binding partners could have an impact on the dynamics of signaling networks. Thus, disease-associated mutants of SHP2 should not only be discussed in the context of deregulated auto-inhibition but also with respect to deregulated protein targeting of the SHP2 mutants.

BIOLOGICAL SIGNIFICANCE:

Using quantitative mass spectrometry based proteomics we provided evidence that disease related mutations in SHP2 domains of SHP2 are able to influence SHP2 recruitment to its targets in mutation dependent manner. We discovered that mutations in the SH2-domain selectively affected protein enrichment ratios suggesting altered binding properties of the SH2-domain. We demonstrated that mutations within SHP2, which had been attributed to affect the enzymatic activity (i.e. affect the open/close status of SHP2), also differ in respect to binding properties. Our study indicates that SHP2 mutations need to be discussed not only in terms of deregulated auto-inhibition but also with respect to deregulated protein targeting properties of the SHP2 mutants. Discovery of the new binding partners for disease-related SHP2 mutants might provide a fruitful foundation for developing strategies targeting Noonan-associated leukemia.

PMID:
23584145
DOI:
10.1016/j.jprot.2013.04.005
[Indexed for MEDLINE]
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