The Serine/threonine kinase Stk33 exhibits autophosphorylation and phosphorylates the intermediate filament protein Vimentin.
BACKGROUND
Colocalization of Stk33 with vimentin by double immunofluorescence in certain cells indicated that vimentin might be a target for phosphorylation by the novel kinase Stk33. We therefore tested in vitro the ability of Stk33 to phosphorylate recombinant full length vimentin and amino-terminal truncated versions thereof. In order to prove that Stk33 and vimentin are also in vivo associated proteins co-immunoprecipitation experiments were carried out. For testing the enzymatic activity of immunoprecipitated Stk33 we incubated precipitated Stk33 with recombinant vimentin proteins. To investigate whether Stk33 binds directly to vimentin, an in vitro co-sedimentation assay was performed.


RESULTS
The results of the kinase assays demonstrate that Stk33 is able to specifically phosphorylate the non-alpha-helical amino-terminal domain of vimentin in vitro. Furthermore, co-immunoprecipitation experiments employing cultured cell extracts indicate that Stk33 and vimentin are associated in vivo. Immunoprecipitated Stk33 has enzymatic activity as shown by successful phosphorylation of recombinant vimentin proteins. The results of the co-sedimentation assay suggest that vimentin binds directly to Stk33 and that no additional protein mediates the association.


CONCLUSIONS
We hypothesize that Stk33 is involved in the in vivo dynamics of the intermediate filament cytoskeleton by phosphorylating vimentin.
Brauksiepe
Bastienne
B
Institute of Molecular Genetics, Johannes Gutenberg-University, Mainz, Germany. brauksib@uni-mainz.de
Mujica
Alejandro O
AO
Herrmann
Harald
H
Schmidt
Erwin R
ER
D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D002478:Cells, Cultured; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism*; D014746:Vimentin / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism*
D014746:Vimentin; D017346:Protein Serine-Threonine Kinases; C505234:Stk33 protein, mouse