(A, B) In vitro kinase assay of (A) recombinant FL xCPEB4 or (B) fragments (1, 2 and 3) with metaphase II oocyte extracts treated with specific kinase inhibitors (roscovitine, Cdk inhibitor; SL0101, p90Rsk inhibitor; BI-2536, Plk1 inhibitor; U0126, MEK inhibitor; and FR180204, ERK inhibitor). DMSO was used as a negative control. The percentage of phosphorylation compared to DMSO is indicated (autoradiography, 32P). A representative experiment from three independent biological replicates is shown. See also Figure 2—figure supplement 1–2. (C) Two-dimensional phosphopeptide maps of xCPEB4 fragments (1, 2 and 3) phosphorylated with metaphase II (MII) oocyte extracts or with recombinant ERK2 or Cdk1/cyclin B. Phosphopeptides were resolved by thin-layer electrophoresis (TLE) followed by thin-layer chromatography (TLC). Arrows indicate sample origin. Phosphopeptides detected in MII were numbered. Asterisks (*) indicate phosphopeptides generated with recombinant kinases not present in MII. A representative experiment from three independent biological replicates is shown. See also Figure 2—figure supplement 3. (D) Disorder tendency (PONDR VL-TX predictor) and mass spectrometry phosphorylation site identification of xCPEB4. Asterisks (*) indicate phosphosites identified with MII extracts. Bold letters indicate phosphosites identified with either ERK2 or Cdk1/cyclin B. Green indicates ERK2 phosphorylation sites, while purple indicates Cdk1 phosphorylation sites. Red lines represent large disordered regions. The xCPEB4 fragments used are outlined. See also Figure 2—figure supplement 4.
DOI:
http://dx.doi.org/10.7554/eLife.19298.004