MOLECULAR CHARACTERIZATION OF HUMAN STATHMIN EXPRESSED IN ESCHERICHIA-COLI - SITE-DIRECTED MUTAGENESIS OF 2 PHOSPHORYLATABLE SERINES (SER-25 AND SER-63)

Stathmin, a probable relay protein possibly integrating multiple intra cellular regulatory signals [reviewed in Sobel (1991) Trends Biochem. Sci. 16, 301-305], was expressed in Escherichia coli at levels as high as 20 % of total bacterial protein. Characterization of the purified recombinant protein revealed that it had biochemical properties very s imilar to those of the native protein. It is a good substrate for both cyclic AMP-dependent protein kinase (PKA) and p34cdc2, on the same fo ur sites as the native eukaryotic protein. As shown by m.s., the diffe rence in isoelectric points from the native protein is probably due to the absence of acetylation of the protein produced in bacteria. C.d. studies indicate that stathmin probably contains about 45% of its sequ ence in an alpha-helical conformation, as also predicted for the seque nce between residues 47 and 124 by computer analysis. Replacement of S er-63 by alanine by in vitro mutagenesis resulted in a ten times less efficient phosphorylation of stathmin by PKA which occurred solely on Ser-16, confirming that Ser-63 is the major target of this kinase. Rep lacement of Ser-25, the major site phosphorylated by mitogen-activated protein kinase in vitro and in vivo, by the charged amino acid glutam ic acid reproduced, in conjunction with the phosphorylation of Ser-16 by PKA, the mobility shift on SDS/polyacrylamide gels induced by the p hosphorylation of Ser-25. This result strongly suggests that glutamic acid in position 25 is able to mimic the putative interactions of phos phoserine-25 with phosphoserine-16, as well as the resulting conformat ional changes that are probably also related to the functional regulat ion of stathmin.