Departamento de Microbiología y Parasitología, Facultad de Farmacia, Instituto Ramón y Cajal de Investigaciones Sanitarias (IRYCIS), Universidad Complutense de Madrid, Plaza de Ramón y Cajal s/n, 28040 Madrid, Spain.
Int J Mol Sci. 2021 Jan 23;22(3):1110. doi: 10.3390/ijms22031110.
The Mitogen-Activated Protein Kinase (MAPK) Slt2 is central to signaling through the yeast Cell Wall Integrity (CWI) pathway. MAPKs are regulated by phosphorylation at both the threonine and tyrosine of the conserved TXY motif within the activation loop (T190/Y192 in Slt2). Since phosphorylation at both sites results in the full activation of MAPKs, signaling through MAPK pathways is monitored with antibodies that detect dually phosphorylated forms. However, most of these antibodies also recognize monophosphorylated species, whose relative abundance and functionality are diverse. By using different phosphospecific antibodies and phosphate-affinity (Phos-tag) analysis on distinct Slt2 mutants, we determined that Y192- and T190-monophosphorylated species coexist with biphosphorylated Slt2, although most of the Slt2 pool remains unphosphorylated following stress. Among the monophosphorylated forms, only T190 exhibited biological activity. Upon stimulation, Slt2 is first phosphorylated at Y192, mainly by the MAPKK Mkk1, and this phosphorylation is important for the subsequent T190 phosphorylation. Similarly, dephosphorylation of Slt2 by the Dual Specificity Phosphatase (DSP) Msg5 is ordered, with dephosphorylation of T190 depending on previous Y192 dephosphorylation. Whereas Y192 phosphorylation enhances the Slt2 catalytic activity, T190 is essential for this activity. The conserved T195 residue is also critical for Slt2 functionality. Mutations that abolish the activity of Slt2 result in a high increase in inactive Y192-monophosphorylated Slt2. The coexistence of different Slt2 phosphoforms with diverse biological significance highlights the importance of the precise detection of the Slt2 phosphorylation status.
丝裂原活化蛋白激酶（MAPK）Slt2 是酵母细胞壁完整性（CWI）信号通路的核心。MAPK 通过其激活环中保守的 TXY 基序中的苏氨酸和酪氨酸的磷酸化来调节（Slt2 中的 T190/Y192）。由于两个位点的磷酸化导致 MAPK 的完全激活，因此通过 MAPK 途径的信号传递是通过检测双重磷酸化形式的抗体来监测的。然而，大多数这些抗体也识别单磷酸化物种，其相对丰度和功能是多样化的。通过使用不同的磷酸特异性抗体和磷酸亲和（Phos-tag）分析不同的 Slt2 突变体，我们确定 Y192 和 T190 单磷酸化物种与双磷酸化 Slt2 共存，尽管在应激后大多数 Slt2 池仍然未磷酸化。在单磷酸化形式中，只有 T190 表现出生物活性。在受到刺激后，Slt2 首先被 MAPKK Mkk1 在 Y192 处磷酸化，这种磷酸化对于随后的 T190 磷酸化很重要。同样，Slt2 由双特异性磷酸酶（DSP）Msg5 去磷酸化是有序的，T190 的去磷酸化取决于先前的 Y192 去磷酸化。虽然 Y192 磷酸化增强了 Slt2 的催化活性，但 T190 对于这种活性是必不可少的。保守的 T195 残基对于 Slt2 的功能也是至关重要的。使 Slt2 失去活性的突变导致无活性的 Y192 单磷酸化 Slt2 大量增加。具有不同生物学意义的不同 Slt2 磷酸化形式的共存突出了精确检测 Slt2 磷酸化状态的重要性。
