Dataset Information

Rapid and efficient purification of Src homology 2 domain-containing proteins: Fyn, Csk and phosphatidylinositol 3-kinase p85.

ABSTRACT: To analyse the regulation of Src family tyrosine kinases in vitro, we have purified Fyn and Csk, a kinase capable of regulating Fyn activity by phosphorylation, from baculovirus-infected insect cells. The proteins were purified by affinity purification over a phosphotyrosine column. Highly purified proteins were eluted from the resin by a salt gradient and further purified by ion-exchange chromatography. This purification scheme was successfully applied to a third, unrelated protein that also contains the Src homology 2 (SH2) domain, namely the 85 kDa subunit of phosphatidylinositol 3-kinase, indicating that this method is versatile and should prove applicable to any protein with an accessible SH2 domain. The binding of Csk to different phosphopeptides was tested, and specificity for the autophosphorylation site of Fyn was demonstrated. Pure Csk was used to phosphorylate Fyn and down-regulate its kinase activity, and the kinetic parameters of both the active and the repressed forms of Fyn were determined. Repression of Fyn activity by Csk reduced binding of Fyn to phosphopeptides to undetectable levels, supporting the model that predicts an intramolecular interaction of the Fyn SH2 domain with a C-terminal phosphotyrosine residue.

SUBMITTER: Koegl M

PROVIDER: S-EPMC1137293 | biostudies-other | 1994 Sep

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:BACKGROUND: Epidermal growth factor receptor (EGFR) signalling is frequently altered during glioblastoma de novo pathogenesis. An important downstream modulator of this signal cascade is SHP2 (Src homology domain-containing phosphatase 2). METHODS: We examined the The Cancer Genome Atlas (TCGA) database for SHP2 mutations. We also examined the expression of a further 191 phosphatases in the TCGA database and used principal component and comparative marker analysis available from the Broad Institute to recapitulate the TCGA-defined subgroups and identify the specific phosphatases defining each subgroup. We identified five siRNAs from two independent commercial sources that were reported by the vendor to be pre-optimised in their specificity of SHP2 silencing. The specificity and physiological effects of these siRNAs were tested using an in vitro glioma model. RESULTS: TCGA data demonstrate SHP2 to be mutated in 2% of the glioblastoma multiforme's studied. Both mutations identified in this study are likely to be activating mutations. We found that the four subgroups of GBM as defined by TCGA differ significantly with regard to the expression level of specific phosphatases as revealed by comparative marker analysis. Surprisingly, the four subgroups can be defined solely on the basis of phosphatase expression level by principal component analysis. This result suggests that critical phosphatases are responsible for the modulation of specific molecular pathways within each subgroup. Src homology domain-containing phosphatase 2 constitutes one of the 12 phosphatases that define the classical subgroup. We confirmed the biological significance by siRNA knockdown of SHP2. All five siRNAs tested reduced SHP2 expression by 70-100% and reduced glioblastoma cell line growth by up to 80%. Profiling the established molecular targets of SHP2 (ERK1/2 and STAT3) confirmed specificity of these siRNAs. The loss of cell viability induced by SHP2 silencing could not be explained by a significant increase in apoptosis alone as demonstrated by terminal deoxyribonucleotidyl transferase-mediated nick-end labelling and propidium iodide staining. Src homology domain-containing phosphatase 2 silencing, however, did induce an increase in β-galactosidase staining. Propidium iodide staining also showed that SHP2 silencing increases the population of glioblastoma cells in the G1 phase of the cell cycle and reduces the population of such cells in the G2/M- and S-phase. CONCLUSION: Src homology domain-containing phosphatase 2 promotes the growth of glioblastoma cells by suppression of cellular senescence, a phenomenon not described previously. Selective inhibitors of SHP2 are commercially available and may be considered as a strategy for glioblastoma therapy.

Dataset Information

Rapid and efficient purification of Src homology 2 domain-containing proteins: Fyn, Csk and phosphatidylinositol 3-kinase p85.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets