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The Serine/threonine kinase Stk33 exhibits autophosphorylation and phosphorylates the intermediate filament protein Vimentin.

Brauksiepe B, Mujica AO, Herrmann H, Schmidt ER - BMC Biochem. (2008)

Bottom Line: In order to prove that Stk33 and vimentin are also in vivo associated proteins co-immunoprecipitation experiments were carried out.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular Genetics, Johannes Gutenberg-University, Mainz, Germany. brauksib@uni-mainz.de

ABSTRACT

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.

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Co-sedimentation assay of recombinant Stk33 and recombinant vimentin ΔN50 using anti-Stk33 for precipitation. A: Immunoblotting analysis of sedimentated proteins using anti-Stk33 for detection. Lane 1–3: samples of washing steps 1–3; lane 5: co-sedimentation sample; lane 7: recombinant Stk33; lane 4 and 6 were free of sample. The arrowheads point towards IgG contamination. Arrow = Stk33. B: Immunoblotting analysis of sedimentated proteins using anti-vimentin for detection. Lane 1–3: samples of washing steps 1–3; lane 5: co-sedimentation sample; lane 7: recombinant vimentin ΔN50; lane 4 and 6 were free of sample. The protein detected in lane 6 is due to protein of lane 7 spilled over. As expected no IgG contamination is visible since an anti-mouse IgG peroxidase conjugate was used for detection of anti-vimentin and utilized anti-Stk33 is produced in rabbit. This assay was confirmed twice.
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Figure 5: Co-sedimentation assay of recombinant Stk33 and recombinant vimentin ΔN50 using anti-Stk33 for precipitation. A: Immunoblotting analysis of sedimentated proteins using anti-Stk33 for detection. Lane 1–3: samples of washing steps 1–3; lane 5: co-sedimentation sample; lane 7: recombinant Stk33; lane 4 and 6 were free of sample. The arrowheads point towards IgG contamination. Arrow = Stk33. B: Immunoblotting analysis of sedimentated proteins using anti-vimentin for detection. Lane 1–3: samples of washing steps 1–3; lane 5: co-sedimentation sample; lane 7: recombinant vimentin ΔN50; lane 4 and 6 were free of sample. The protein detected in lane 6 is due to protein of lane 7 spilled over. As expected no IgG contamination is visible since an anti-mouse IgG peroxidase conjugate was used for detection of anti-vimentin and utilized anti-Stk33 is produced in rabbit. This assay was confirmed twice.

Mentions: To test whether Stk33 binds directly to vimentin, an in vitro co-sedimentation assay was performed. By analyzing the sedimented proteins via Western Blotting experiments we could show, that recombinant Stk33 precipitated (Figure 5A, lane 5) and that vimentin is successfully co-sedimentated (Figure 5B, lane 5) using anti-Stk33 for precipitation. This suggests that vimentin binds directly to Stk33 and that none intermediate protein mediates the association.


The Serine/threonine kinase Stk33 exhibits autophosphorylation and phosphorylates the intermediate filament protein Vimentin.

Brauksiepe B, Mujica AO, Herrmann H, Schmidt ER - BMC Biochem. (2008)

Co-sedimentation assay of recombinant Stk33 and recombinant vimentin ΔN50 using anti-Stk33 for precipitation. A: Immunoblotting analysis of sedimentated proteins using anti-Stk33 for detection. Lane 1–3: samples of washing steps 1–3; lane 5: co-sedimentation sample; lane 7: recombinant Stk33; lane 4 and 6 were free of sample. The arrowheads point towards IgG contamination. Arrow = Stk33. B: Immunoblotting analysis of sedimentated proteins using anti-vimentin for detection. Lane 1–3: samples of washing steps 1–3; lane 5: co-sedimentation sample; lane 7: recombinant vimentin ΔN50; lane 4 and 6 were free of sample. The protein detected in lane 6 is due to protein of lane 7 spilled over. As expected no IgG contamination is visible since an anti-mouse IgG peroxidase conjugate was used for detection of anti-vimentin and utilized anti-Stk33 is produced in rabbit. This assay was confirmed twice.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2567967&req=5

Figure 5: Co-sedimentation assay of recombinant Stk33 and recombinant vimentin ΔN50 using anti-Stk33 for precipitation. A: Immunoblotting analysis of sedimentated proteins using anti-Stk33 for detection. Lane 1–3: samples of washing steps 1–3; lane 5: co-sedimentation sample; lane 7: recombinant Stk33; lane 4 and 6 were free of sample. The arrowheads point towards IgG contamination. Arrow = Stk33. B: Immunoblotting analysis of sedimentated proteins using anti-vimentin for detection. Lane 1–3: samples of washing steps 1–3; lane 5: co-sedimentation sample; lane 7: recombinant vimentin ΔN50; lane 4 and 6 were free of sample. The protein detected in lane 6 is due to protein of lane 7 spilled over. As expected no IgG contamination is visible since an anti-mouse IgG peroxidase conjugate was used for detection of anti-vimentin and utilized anti-Stk33 is produced in rabbit. This assay was confirmed twice.
Mentions: To test whether Stk33 binds directly to vimentin, an in vitro co-sedimentation assay was performed. By analyzing the sedimented proteins via Western Blotting experiments we could show, that recombinant Stk33 precipitated (Figure 5A, lane 5) and that vimentin is successfully co-sedimentated (Figure 5B, lane 5) using anti-Stk33 for precipitation. This suggests that vimentin binds directly to Stk33 and that none intermediate protein mediates the association.

Bottom Line: In order to prove that Stk33 and vimentin are also in vivo associated proteins co-immunoprecipitation experiments were carried out.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular Genetics, Johannes Gutenberg-University, Mainz, Germany. brauksib@uni-mainz.de

ABSTRACT

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.

Show MeSH