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. 2014 Dec 1;127(23):5052–5065. doi: 10.1242/jcs.156059

Fig. 4.

Fig. 4.

GSK3 phosphorylates S309 on CAP1. (A) Treatment of HEK293T cells with GSK3 inhibitors LiCl, 6-BIO and SB216763 all reduced CAP1 phosphorylation at S307/S309. Cells were treated with the inhibitors for 14 hrs, and cell lysates were prepared for western blotting with both CAP1 and phospho-CAP1 antibodies. (B) Signals from three independent experiments were measured using densitometry and analyzed using Student's t-test and plotted in the graph; error bars represent ±s.e.m; *P<0.05, ** P<0.01. <@?show=[to]?>(C) Knockdown of GSK3β with lentiviral shRNA in HeLa cells reduced S307/S309 phosphorylation. HEK293FT cells were transfected using Lentiviral Packaging Mix together with the shRNA constructs. Supernatant medium was collected 48 hrs post transfection and used to infect HeLa cells. The cells were harvested 48 hrs post infection, and lysates were prepared and used in western blotting. (D) CAP1 co-precipitates with GSK3β. Lysates from CAP1-knockdown HeLa cells re-expressing 6xHis-CAP1 or a control vector were incubated with Ni-NTA beads to precipitate 6xHis-CAP1. The precipitates were resolved on SDS-PAGE and blotted with both 6xHis and GSK3β antibodies. (E) Kinase assays mapping S309 as the GSK3 site. Peptides harboring an alanine mutation at either S307 or S309 with a phosphorylated or dephosphorylated T314 (a.a. 305–316, QTSPSPKPATKK) were used as substrates in the kinase assays to test their phosphorylation by GSK3β; the GSK3 substrate GSM peptide (RRRPASVPPSPSLSRHSSHQRR; the serine residue shown in bold is phosphorylated) was used as a positive control. Samples were resolved on a 10–20% Tris-Tricine gradient gel (Bio-Rad) and signals were detected by autoradiography. Asterisks indicate quantified signals from the phosphorylated peptide, the arrowhead indicates partially degraded substrate. (F) Kinase assay results supporting T314 as the priming site for S309. A dephosphorylated T314 abolished the phosphorylation by GSK3; also both GSK3α and GSK3β phosphorylated S309. In E and F, numbers below the gels indicate the intensity of the signals in relation to the positive control.