Dual phosphorylation of protein phosphatase PPM1H promotes dephosphorylation of Smad1 in cellulo

• Published in: Biochemical and biophysical research communications 2020-09, Vol.530 (3), p.513-519
• Main Authors: Osawa, Jin, Akizuki, Kazutoshi, Kashimura, Akari, Ueta, Saki, Nakatani, Misato, Inui, Yuiko, Shigeri, Yasushi, Ishida, Atsuhiko, Kameshita, Isamu, Sueyoshi, Noriyuki
• Format: Article
• Language: English
• Subjects: Animals; Ca2+ signaling; Calcium-Calmodulin-Dependent Protein Kinase Type 1 - metabolism; cAMP signaling; Cell Line, Tumor; Cyclic AMP-Dependent Protein Kinases - metabolism; HEK293 Cells; Hierarchical phosphorylation; Humans; Mice; Phos-tag SDS-PAGE; Phosphorylation; PPM family; Protein Ser/Thr phosphatase; Smad1 Protein - metabolism
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2020.05.082

Protein phosphatase PPM1H is known to participate in various biological or pathophysiological mechanisms. However, little is known about the molecular mechanisms of its regulation. In this study, we investigated the protein kinases that directly phosphorylate PPM1H, identifying them as cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase I (CaMKI). In vitro and in silico analyses showed that the phosphorylation sites of PPM1H by PKA and CaMKI were Ser-123 and Ser-210, respectively. The phosphorylation state of PPM1H in cells exhibited the kinase activator- and inhibitor-dependent changes. In mouse neuroblastoma Neuro2a cells, phosphorylation of Ser-210 was much higher in the phospho-mimetic mutant (S123D) than in the non-phosphorylatable mutant (S123A) when they were treated with ionomycin. This suggests that a hierarchical phosphorylation, with initial phosphorylation of Ser-123 promoting subsequent phosphorylation of Ser-210, occurs in these neuron-like cells. Moreover, in cell-based assay a PPM1H(S123A/S210A) double mutant barely dephosphorylated Smad1, a transcription factor known as an endogenous substrate of PPM1H. These results suggest that cAMP and Ca2+/calmodulin regulate dephosphorylation of Smad1 through the dual phosphorylation of PPM1H at Ser-123 and Ser-210. •We investigated the mechanism regulating protein phosphatase PPM1H.•PPM1H was phosphorylated at Ser-123 by PKA and at Ser-210 by CaMKI.•In 293T cells, phosphorylated PPM1H(WT) dephosphorylated its substrate, Smad1.•However, non-phosphorylatable PPM1H(S123A/S210A) barely dephosphorylated Smad1.•In conclusion, dual phosphorylation of PPM1H promotes dephosphorylation of Smad1.