Molecular characterization of human stathmin expressed in Escherichia coli: site-directed mutagenesis of two phosphorylatable serines (Ser-25 and Ser-63).
Stathmin, a probable relay protein possibly integrating multiple intracellular 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 similar to those of the native protein. It is a good substrate for both cyclic AMP-dependent protein kinase (PKA) and p34cdc2, on the same four sites as the native eukaryotic protein. As shown by m.s., the difference 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 sequence in an alpha-helical conformation, as also predicted for the sequence between residues 47 and 124 by computer analysis. Replacement of Ser-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. Replacement of Ser-25, the major site phosphorylated by mitogen-activated protein kinase in vitro and in vivo, by the charged amino acid glutamic acid reproduced, in conjunction with the phosphorylation of Ser-16 by PKA, the mobility shift on SDS/polyacrylamide gels induced by the phosphorylation of Ser-25. This result strongly suggests that glutamic acid in position 25 is able to mimic the putative interactions of phosphoserine-25 with phosphoserine-16, as well as the resulting conformational changes that are probably also related to the functional regulation of stathmin.
Curmi
P A
PA
INSERM U153, Paris, France.
Maucuer
A
A
Asselin
S
S
Lecourtois
M
M
Chaffotte
A
A
Schmitter
J M
JM
Sobel
A
A
D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006801:Humans; D013058:Mass Spectrometry; D008868:Microtubule Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D050777:Stathmin
D017931:DNA Primers; D008868:Microtubule Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; C493995:STMN1 protein, human; D050777:Stathmin; D012694:Serine