Differences in phosphorylation of human and chicken stathmin by MAP kinase

Stathmin/Op18 is a highly conserved 19 kDa cytosolic phosphoprotein. Human and chicken stathmin share 93% identity with only 11 amino acid substitutio ns. One of the substituted amino acids is serine 25, which is a glycine in chicken stathmin. In human stathmin, serine 25 is the main phosphorylation site for MAP kinase. in this study, we have compared the phosphorylation of human and chicken stathmin. The proteins were expressed in Sf9 cells using the baculovirus expression system and purified for in vitro phosphorylatio n assays. Phosphorylation with MAP kinase showed that chicken stathmin was phosphorylated 10 times less than human stathmin. To identify the phosphory lation sites we used liquid chromatography/mass spectrometry (LC/MS/MS). Th e only amino acid found phosphorylated was serine 38, which corresponds to the minor phosphorylation site in human stathmin. Phosphorylation with p34( cdc2)- and cGMP-dependent protein kinases gave almost identical phosphoryla tion levels in the two stathmins. (C) 2001 Wiley-Liss, Inc.