Molecular Mechanism Of ERK Regulation By Metal-dependent Protein Phosphatase1A (PPM1A)

Protein phosphorylation is an important post-translational modification that regulates almost every aspect of signal transduction in cells. As an important regulatory factor of protein phosphorylation, activation of the MAPK family kinase, ERK, is a point of convergence for many cellular activities in response to external stimulation. With stimuli, ERK activity is significantly increased by the phosphorylation of Thr202and Tyr204at its activation loop. Downregulation of ERK phosphorylation at these two sites by several phosphatases, such as PP2A, HePTP and MKP3, is essential for maintaining appropriate ERK functions, such as regulate cell circulation, apoptosis, differentiation, and migration, in different cellular processes. As members of the family of phosphatases, however, it is unknown if PPM family phosphatases which contain16members directly dephosphorylate ERK. In previous study, PPM1A regulate MAPK signal pathway negatively in yeast and also shows reasonable phosphatase activity on mammalian cells doubly phosphorylated ERK in vitro. Therefore, the study whether PPM1A regulate signal transduction through dephosphatase ERK at the cellular level and clarified the catalytic molecular mechanism of PPM1A to ERK is of great significance.In this study, we report that PPM1A negatively regulated ERK by directly dephosphorylating its pT position early in EGF stimulation and PPM1A can forming complexes with ERK. Additional kinetics studies revealed that key residues participated in phospho-ERK recognition by PPM1A. Importantly, PPM1A preferred the phospho-ERK peptide sequence over a panel of other phosphopeptides through the interactions of basic residues in the active site of PPM1A with the pT-E-pY motif of ERK. While the Lys165and Arg33residues were required for efficient catalysis or phosphosubstrate binding of PPM1A, the Gln185and Argl86residues were determinants of PPM1A substrate specificity. The interaction between the Arg186 residue of PPM1A and pY204residues of phospho-ERK was identified as a hot-spot for PPM1A/phospho-ERK interaction. To sum up, we report that PPM1A negatively regulated ERK by directly dephosphorylating its pT202position early in EGF stimulation. Additional biochemical studies revealed that key site participated in phospho-ERK recognition by PPM1A.