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The Serine/threonine kinase Stk33 exhibits autophosphorylation and phosphorylates the intermediate filament protein
Bastienne Brauksiepe1, Alejandro O Mujica, Harald Herrmann
1Institute of Molecular Genetics, Johannes Gutenberg-University, Mainz, Germany. brauksib@uni-mainz.de
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.
Conclusion:
We hypothesize that Stk33 is involved in the in vivo dynamics of the intermediate filament cytoskeleton by phosphorylating vimentin.
Insights
The novel kinase Stk33 phosphorylates vimentin, a key intermediate filament protein. This phosphorylation, occurring in the amino-terminal domain, suggests Stk33 influences cytoskeleton dynamics in vivo.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Colocalization suggested Stk33 (serine/threonine kinase 33) may phosphorylate vimentin.
- Vimentin is a crucial component of the intermediate filament cytoskeleton.
Purpose of the Study:
- To investigate the in vitro phosphorylation of vimentin by Stk33.
- To confirm the in vivo association between Stk33 and vimentin.
- To determine the specific domain of vimentin phosphorylated by Stk33.
Main Methods:
- In vitro kinase assays using recombinant vimentin.
- Co-immunoprecipitation experiments with cultured cell extracts.
- In vitro co-sedimentation assays.
Main Results:
- Stk33 specifically phosphorylates the amino-terminal domain of vimentin in vitro.
- Stk33 and vimentin form an associated complex in vivo.
- Vimentin directly binds to Stk33, independent of other proteins.
Conclusions:
- Stk33 is an active kinase that phosphorylates vimentin.
- Stk33 likely plays a role in regulating intermediate filament cytoskeleton dynamics through vimentin phosphorylation.
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