Critical Role of FLRT1 Phosphorylation in the Interdependent Regulation of FLRT1 Function and FGF Receptor Signalling
Figure 2
FLRT1 is not a SFK substrate but phosphorylation is FGFR-and SFK-dependent.
A) HEK 293T cells were transfected with control (pcDNA31) or FGFR1 and a panel of either full-length FLRT1-HA or tyrosine substitution contructs as indicated (see Materials & Methods) One sample was pre-treated with FGFR kinase inhibitor (SU5402, 50mM, 1 hr) where indicated Cell lysates were immunoprecipitated with anti-HA and subsequently blotted with anti-phosphotyrosine (IP: HA, Blot: pY) or anti-HA (IP: HA, Blot: HA) to examine phosphorylated FLRT1HA levels (pFLRT1) or total immunoprecipitated FLRT1-HA levels (FLRT1), respectively Whole cell lysate (WCL) fractions were probed with anti-FGFR1 (Blot: FGFR) to control for protein expression B) HEK 293T cells were transfected with pcDNA31, FGFR1 and FLRT1-HA as indicated Cells were pre-incubated (1hr) with pharmacological inhibitors (SU5402, 50mM; SU6656, 20mM) Cell lysates were immunoprecipitated with anti-HA and subsequently blotted with anti-phosphotyrosine (IP: HA, Blot: pY) for pFLRT1 and anti-HA (IP: HA, Blot: HA) for FLRT1 WCL fractions were probed with anti-FGFR1 (Blot: FGFR), anti-phospho-ERK (Blot: pERK) or anti-ERK (Blot: ERK) Data in A) and B) are representative of ≥3 independent experiments Densitometric analysis (mean ± sem, n = 3) is the ratio of pFLRT1:FLRT1 and normalised to FLRT1 phosphorylation in the absence of inhibitor in both cases (**p<001, *p<005 non-parametric one way ANOVA) C) HEK 293T cells were transfected with pcDNA31, FLRT1-HA alone or co-transfected with either a constitutively active (KA) or kinase dead (KD) c-Src construct Cells were serum starved for 1hr and cell lysates immunoprecipitated with anti-HA and subsequently blotted with anti-phosphotyrosine (IP: HA, Blot: pY) for pFLRT1 or anti-HA (IP: HA, Blot: HA) for FLRT1 WCL fractions were probed with anti-phospho-ERK (Blot: pERK) or anti-ERK (Blot: ERK) Data are representative of 3 independent experiments D) HEK 293T cells were transfected with either pcDNA31 vector, FLRT1-HA or Y3F-FLRT1-HA constructs Cells were co-stimulated with FGF2 (20ng/ml) and heparin (10mg/ml) for the indicated times Cell lysates were blotted for anti-phospho-ERK (WCL IB: pERK), membranes were stripped and re-probed for anti-ERK (WCL IB: ERK) Densitometric analysis has been adjusted for ERK loading and expressed as an arbitrary pERK:ERK ratio Data are representative of at least 4 independent experiments E) 293T cells transfected with Y3F-FLRT1-HA were serum-starved in the absence or presence of pharmacological inhibitors of FGFR1 (SU5402, 50mM) and SFKs (SU6656, 20mM) and whole cell lysates probed with antiphospho-ERK (IB: pERK), anti-ERK (IB: ERK) and anti-HA (Blot: HA) for Y3FFLRT1-HA.