Large-Scale Discovery of ERK2 Substrates Identifies ERK-Mediated Transcriptional Regulation by ETV3

Scott M. Carlson, Candace R. Chouinard, Adam Labadorf, Carol J. Lam, Katrin Schmelzle, Ernest Fraenkel, Forest M. White*

*To whom correspondence should be addressed. E-mail: fwhite{at}mit.edu

This PDF file includes:

• Methods. Detailed protocol for ERK2 substrate labeling and identification.
• References
• Fig. S1. Additional sites phosphorylated by ERK2 on IRS2, CDC42EP1, and ETV3.
• Fig. S2. MS/MS spectrum used to identify ETV3 as a direct ERK2 target and the matching spectrum of a synthetic peptide.
• Fig. S3. HA-tagged ETV3 exhibits MEK-dependent gel shift, which is reduced in phosphosite mutants, and an anti-HA antibody captures both the phosphorylated and the nonphosphorylated protein in ChIP.
• Fig. S4. Immunofluorescence for HA-tagged ETV3 expressed in HEK 293T cells showing nuclear localization regardless of ERK activity.
• Fig. S5. Immunofluorescence for endogenous ETV3 in DLD1 cells showing primarily nuclear localization regardless of ERK activity.
• Fig. S6. ETV3 binding sites occur near known transcriptional start sites.
• Fig. S7. Phosphorylation by ERK2 does not affect the stability of ETV3.
• Spectra. Manually curated spectra used to identify all AS-ERK2 targets.

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Other Supplementary Material for this manuscript includes the following:

• Table S1 (Microsoft Excel format). AS-ERK2 substrates and signal-to-background ratios.
• Table S2 (Microsoft Excel format). MEK-dependent phosphorylation in peptides isolated by phosphorylation motif peptide immunoprecipitation.
• Table S3 (Microsoft Excel format). Overview statistics of ETV3 ChIP-Seq data.
• Table S4 (Microsoft Excel format). ETV3 binding sites from ChIP-Seq.
• Table S5 (Microsoft Excel format). Gene mappings for ETV3 ChIP-Seq binding sites.

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© 2011 American Association for the Advancement of Science