Phactr1/PP1 phosphatase substrates in mouse NIH3T3 fibroblasts

It was previously shown that expression of an activated Phactr1 mutant (Phactr1XXX) that constitutively forms the Phactr1/PP1 phosphatase holoenzyme, induces F-actin rearrangements in NIH3T3 fibroblasts. Expression of the Phactr1 PP1-binding domain (C-terminal) alone is also sufficient to induce such cytoskeletal changes. In contrast, expression of Phactr1XXXC derivative, which lacks the PP1 binding sequences does not result in alteration of cytoskeletal morphology (Wiezlak et al., 2012). These observations suggest that Phactr1/PP1 dephosphorylates target proteins involved in cytoskeletal dynamics. To identify potential Phactr1/PP1 substrates, we used differential SILAC phosphoproteomics in NIH3T3 cells inducibly expressing Phactr1XXX, Phactr1XXXC constructs or vector alone. Over 3000 phosphorylation sites were quantified, among which we determined Phactr1/PP1 target dephosphorylation sites by comparative analysis.