Figure 2.
FER phosphorylates Y374-PKCδ to promote cell surface EGFR and EGFR stabilization. (a and b) FER deficiency reduces pY374-PKCδ. Western blot showing level of pY374-PKCδ-myc (p-PKCδ-myc) relative to PKCδ-myc expression in three different PTPN14-KO clones (c1, a, and o) overexpressing PKCδ-myc and transiently transfected with control (siCtrl) or a pool of four FER (siFER, Dharmacon Smartpool) siRNAs followed by stimulation with 20 ng/ml EGF for 15 min (a) and quantified data from the Western blot (b; mean ± SEM; n = 3 biological replicates; **, P < 0.01, Student’s t test), with pY374-PKCδ levels detected using the phospho-specific Ab to pY374-PKCδ. (c) FER deficiency represses EGFR cell surface expression to control levels, measured as mean fluorescence intensity in PTPN14-KO cells. Cell surface EGFR in pCas9Vec or PTPN14-KO cells transiently transfected with control (siCtrl) or a pool of four FER (siFER) siRNAs (means ± SEM quantified from n > 30 cells from one representative experiment of three; **, P < 0.01; ***, P < 0.001, Student’s t test). (d and e) FER deficiency restores EGFR degradation in PTPN14-KO cells. Representative Western blot of FER expression in pCas9Vec or PTPN14-KO cells that were transiently transfected with either siCtrl or two different FER siRNAs (siFER3 and siFER4) separately (d) and time course of EGFR expression after EGF stimulation (100 ng/ml), obtained by Western blotting, relative to total protein in pCas9Vec or PTPN14-KO cells that were transiently transfected with siCtrl, siFER3, or siFER4 (e). Cells were pretreated 30 min with cycloheximide before EGF stimulation. The t1/2 ± SD of EGFR was obtained using a nonlinear one-phase decay fit of the time course; **, P < 0.01; *, P < 0.05; Student’s t test from n = 4 independent experiments. (f and g) In vitro phosphorylation of recombinant GST-PKCδ on Y374 by recombinant FER. Western blot of the indicated amounts of GST-PKCδ after incubation with recombinant FER in the presence or absence of ATP, as indicated, and blotted with either the pY374-PKCδ phospho-specific Ab or total PKCδ Ab (f) and a colorimetric assay of pY374-PKCδ using constant amounts of recombinant PKCδ with varying amounts of recombinant FER and vice versa (g). Data representative of three independent experiments.

FER phosphorylates Y374-PKCδ to promote cell surface EGFR and EGFR stabilization. (a and b) FER deficiency reduces pY374-PKCδ. Western blot showing level of pY374-PKCδ-myc (p-PKCδ-myc) relative to PKCδ-myc expression in three different PTPN14-KO clones (c1, a, and o) overexpressing PKCδ-myc and transiently transfected with control (siCtrl) or a pool of four FER (siFER, Dharmacon Smartpool) siRNAs followed by stimulation with 20 ng/ml EGF for 15 min (a) and quantified data from the Western blot (b; mean ± SEM; n = 3 biological replicates; **, P < 0.01, Student’s t test), with pY374-PKCδ levels detected using the phospho-specific Ab to pY374-PKCδ. (c) FER deficiency represses EGFR cell surface expression to control levels, measured as mean fluorescence intensity in PTPN14-KO cells. Cell surface EGFR in pCas9Vec or PTPN14-KO cells transiently transfected with control (siCtrl) or a pool of four FER (siFER) siRNAs (means ± SEM quantified from n > 30 cells from one representative experiment of three; **, P < 0.01; ***, P < 0.001, Student’s t test). (d and e) FER deficiency restores EGFR degradation in PTPN14-KO cells. Representative Western blot of FER expression in pCas9Vec or PTPN14-KO cells that were transiently transfected with either siCtrl or two different FER siRNAs (siFER3 and siFER4) separately (d) and time course of EGFR expression after EGF stimulation (100 ng/ml), obtained by Western blotting, relative to total protein in pCas9Vec or PTPN14-KO cells that were transiently transfected with siCtrl, siFER3, or siFER4 (e). Cells were pretreated 30 min with cycloheximide before EGF stimulation. The t1/2 ± SD of EGFR was obtained using a nonlinear one-phase decay fit of the time course; **, P < 0.01; *, P < 0.05; Student’s t test from n = 4 independent experiments. (f and g) In vitro phosphorylation of recombinant GST-PKCδ on Y374 by recombinant FER. Western blot of the indicated amounts of GST-PKCδ after incubation with recombinant FER in the presence or absence of ATP, as indicated, and blotted with either the pY374-PKCδ phospho-specific Ab or total PKCδ Ab (f) and a colorimetric assay of pY374-PKCδ using constant amounts of recombinant PKCδ with varying amounts of recombinant FER and vice versa (g). Data representative of three independent experiments.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal