Figure 4.

EGF induces phosphorylation of β4 only on S1356, S1360, and S1364, which reduces the affinity of β4 for the plectin-1A ABD. (A) In vivo phosphopeptide maps of β4 isolated from either serum-starved PA-JEB/β4^WT cells (A) or PA-JEB/β4^WT (B), PA-JEB/β4^3xA (C), or PA-JEB (an equivalent area of gel where β4 would run was excised, D) cells treated for 30 min with EGF in the presence of [³²P]orthophosphate. (E) A schematic diagram of the results shown in A–D. Gray ovals, peptides containing β4 specific phosphorylation sites. (B) PA-JEB/β4^WT cells were either serum-starved (lane 1) or treated with 50 ng/ml EGF for the indicated times (in minutes; lanes 2–6) before lysis in mPER buffer. WCLs were probed for the presence of β4 phosphorylated on residues S1356, S1360, and/or S1364 using our rabbit polyclonal phospho-specific antibody β4 pS-CS (top), and the expression level of the β4 proteins was detected using a rabbit polyclonal antibody raised against the first pair of FNIII repeats in β4 (second from top panel). To verify the activation of the EGF receptor and PKC isoforms, WCLs were probed using an antibody that recognizes phospho-tyrosine 845 on the activated EGF receptor (third from top panel) and antibodies raised against phosphorylated Thr 660 of PKCβ II (pan-pPKC; second from bottom panel). The expression levels of the EGF receptor and PKC isoforms were detected using an antibody against the EGF receptor (1005; third from bottom panel) and a mixture of antibodies against PKC α, β, and δ (bottom). Interestingly, the phospho-tyrosine 845 EGFR antibody recognizes a cleaved protein fragment of the full-length EGF receptor that contains the phosphorylated cytoplasmic domain. (C) COS-7 cells were transiently transfected with either IL2R-β4^WT (lanes 1, 2, and 5) or IL2R-β4^3xA (lanes 3, 4, and 6) cDNA constructs or a control plasmid (lane 7) or an expression construct for the HA-plectin-1A ABD^WT (lanes 1–4 and 7) or a control plasmid (lanes 5 and 6). The cells were left untreated (lanes 1, 3, and 5–7) or treated with EGF (lanes 2 and 4) 30 min before lysis in mPER buffer. HA-IPs were probed for the presence of IL2R-β4 (top) and the HA-tagged ABDs (second from top panel). WCLs were probed for the expression level of IL2R-β4 proteins (third from top panel) and the EGF receptor (second from bottom panel). The activation of the EGF receptor was visualized using phosphotyrosine antibodies (third from bottom panel; the location of the EGFR is denoted by an asterisk) and the classical PKCs using pan antibodies raised against phosphorylated Thr 660 of PKCβ II (bottom panel; the location of the phospho-PKC is denoted by two asterisks). Quantitation was done in ImageJ and is a ratio of the band intensity shown in the top panel to those in the second to top panel for each lane, relative to lane 1.