Differential Role of Threonine and Tyrosine Phosphorylation in the Activation and Activity of the Yeast MAPK Slt2

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 threonine and tyrosine of conserved TXY motif within activation loop (T190/Y192 in Slt2). Since sites results full MAPKs, MAPK pathways monitored with antibodies that detect dually phosphorylated forms. However, most these also recognize monophosphorylated species, whose relative abundance functionality diverse. By using different phosphospecific phosphate-affinity (Phos-tag) analysis on distinct mutants, we determined Y192- T190-monophosphorylated species coexist biphosphorylated Slt2, although pool remains unphosphorylated following stress. Among forms, only T190 exhibited biological activity. Upon stimulation, first Y192, mainly MAPKK Mkk1, this important for subsequent phosphorylation. Similarly, dephosphorylation Dual Specificity Phosphatase (DSP) Msg5 ordered, depending previous Y192 dephosphorylation. Whereas enhances catalytic activity, essential T195 residue critical functionality. Mutations abolish activity result a high increase inactive Y192-monophosphorylated Slt2. coexistence phosphoforms diverse significance highlights importance precise detection status.