KINETIC-STUDY ON DEPHOSPHORYLATION OF MYELIN BASIC-PROTEIN BY SOME PROTEIN PHOSPHATASES

The dephosphorylation specificity of protein phosphatase 2A (PP2A), ca lcineurin (PP2B) and protein phosphatase 2C (PP2C) were studied in vit ro using myelin basic protein (MBP) as a model substrate which was ful ly phosphorylated at multiple sites by protein kinase C (PKC) or cycli c AMP-dependent protein kinase (PKA). In order to determine the site s pecificity of phosphates in myelin basic protein, the protein was dige sted with trypsin and the radioactive phosphopeptide fragments were is olated by high performance liquid chromatography (HPLC) on reversed-ph ase column. Subsequent analysis and/or sequential manual Edman degrada tion of the purified phosphopeptides revealed that Thr-65 and Ser-115 were most extensively phophorylated by PKA and Ser-55 by PKC. For the dephosphorylation kinetics, the phosphorylated MBP was treated with ca lcineurin or PP2C with various time intervals and the reaction was ter minated by direct tryptic digest. Both Thr-65 and Ser-115 residues wer e dephosphorylated more rapidly than any other ones by phosphatases. H owever it can be differentiated further by first-order kinetics that t he PP2B dephosphorylated both Thr-65 and Ser-115 with almost same mann er, whereas PP2C dephosphorylated somewhat preferentially the Ser-115.