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. 2021 Jan 7;220(2):e201902073. doi: 10.1083/jcb.201902073

Figure 2.

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.