Protein phosphatase 4 (PP4) is an evolutionarily conserved and essential Ser/Thr phosphatase that regulates cell division, development and DNA repair in eukaryotes. The major form of PP4, present from yeast to human, is the PP4c-R2-R3 heterotrimeric complex. The R3 subunit is responsible for substrate-recognition via its EVH1 domain. In typical EVH1 domains, conserved phenylalanine, tyrosine and tryptophan residues form the specific recognition site for their target's proline-rich sequences. Here, we identify novel binding partners of the EVH1 domain of the Drosophila R3 subunit, Falafel, and demonstrate that instead of binding to proline-rich sequences this EVH1 variant specifically recognizes atypical ligands, namely the FxxP and MxPP short linear consensus motifs. This interaction is dependent on an exclusively conserved leucine that replaces the phenylalanine invariant of all canonical EVH1 domains. We propose that the EVH1 domain of PP4 represents a new class of the EVH1 family that can accommodate low proline content sequences, such as the FxxP motif. Finally, our data implicate the conserved Smk-1 domain of Falafel in target-binding. These findings greatly enhance our understanding of the substrate-recognition mechanisms and function of PP4.

蛋白磷酸酶 4 (PP4) 是一种进化上保守且必需的 Ser/Thr 磷酸酶，可调节真核生物的细胞分裂、发育和 DNA 修复。 PP4 从酵母到人类的主要形式是 PP4c-R2-R3 异三聚体复合物。 R3 亚基负责通过其 EVH1 结构域进行底物识别。在典型的 EVH1 结构域中，保守的苯丙氨酸、酪氨酸和色氨酸残基形成其靶标富含脯氨酸的序列的特异性识别位点。在这里，我们鉴定了果蝇R3 亚基 Falafel 的 EVH1 结构域的新结合伴侣，并证明该 EVH1 变体不是与富含脯氨酸的序列结合，而是特异性识别非典型配体，即 FxxP 和 MxPP 短线性共有基序。这种相互作用依赖于一种完全保守的亮氨酸，它取代了所有规范 EVH1 结构域的苯丙氨酸不变量。我们认为 PP4 的 EVH1 结构域代表了 EVH1 家族的一个新类别，可以容纳低脯氨酸含量的序列，例如 FxxP 基序。最后，我们的数据表明 Falafel 的保守 Smk-1 结构域参与靶点结合。这些发现极大地增强了我们对 PP4 底物识别机制和功能的理解。