Signal transducers and activators of transcription-3 binding to the fibroblast growth factor receptor is activated by receptor amplification.

저자: Dudka AA, Sweet SM, Heath JK.

저널: Cancer Res. 2010 Apr 15;70(8):3391-401

사용한 제품:  Dynabeads Streptavidin MyOne T1 (Cat# 65601, 65602)

Abstract

Fibroblast growth factor receptors (FGFR) are cell surface tyrosine kinases that function in cell proliferation and differentiation. Aberrant FGFR signaling occurs in diverse cancers due to gene amplification, but the associated oncogenic mechanisms are poorly understood. Using a proteomics approach, we identified signal transducers and activators of transcription-3 (STAT3) as a receptor-binding partner that is mediated by Tyr677 phosphorylation on FGFR. Binding to activated FGFR was essential for subsequent tyrosine phosphorylation and nuclear translocation of STAT3, along with activation of its downstream target genes. Tyrosine phosphorylation of STAT3 was also dependent on concomitant FGFR-dependent activity of SRC and JAK kinases. Lastly, tyrosine (but not serine) phosphorylation of STAT3 required amplified FGFR protein expression, generated either by enforced overexpression or as associated with gene amplification in cancer cells. Our findings show that amplified FGFR expression engages the STAT3 pathway, and they suggest therapeutic strategies to attack FGFR-overexpressing cancers.

제품 사용법

Peptide pull-down experiment.

Dynabeads MyOne Streptavidin T1 (Invitrogen) were washed and resuspended in PBST. Desthiobiotinylated FGFR1 peptide (1 μmol/L) was added to Dynabeads, incubated for 1 h and washed with PBST before the addition of HEK293T whole cell lysate. After 1 h, whole cell lysate was removed and beads were extensively washed with PBST. Following the addition of 2× reduced SDS sample buffer, proteins were resolved by SDS-PAGE and analyzed by Western blotting.