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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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A: Amino acid sequence alignment of the non helical head domain (H) of vimentin from human (Hsa) and mouse (Mmu). Starting points of the truncated vimentin derivatives used in this study are indicated (a = Δ12, b = Δ 20, c = Δ 30, d = Δ 42, e = Δ 50, f = Δ H). The numbers indicate how many amino acids were deleted from the amino-terminus. Phosphorylation sites in the vimentin head domain are indicated as reviewed in [33]. Black dots represent phosphorylation sites on human vimentin as mentioned in [26,35]. Open circles indicate phosphorylation sites in the mouse vimentin head domain according to [34]. Vimentin phosphorylation sites found in the hamster are symbolized by black squares [21]. B: A hypothetical structural model for the human vimentin head domain. Amino acids are represented by circles or boxes, aromatic amino acids are boxed, basic ones are filled, and potential phosphorylation sites are dotted. Figure modified from [26].
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Figure 3: A: Amino acid sequence alignment of the non helical head domain (H) of vimentin from human (Hsa) and mouse (Mmu). Starting points of the truncated vimentin derivatives used in this study are indicated (a = Δ12, b = Δ 20, c = Δ 30, d = Δ 42, e = Δ 50, f = Δ H). The numbers indicate how many amino acids were deleted from the amino-terminus. Phosphorylation sites in the vimentin head domain are indicated as reviewed in [33]. Black dots represent phosphorylation sites on human vimentin as mentioned in [26,35]. Open circles indicate phosphorylation sites in the mouse vimentin head domain according to [34]. Vimentin phosphorylation sites found in the hamster are symbolized by black squares [21]. B: A hypothetical structural model for the human vimentin head domain. Amino acids are represented by circles or boxes, aromatic amino acids are boxed, basic ones are filled, and potential phosphorylation sites are dotted. Figure modified from [26].

Mentions: The recombinant Stk33 enzyme was expressed in E. coli and affinity purified [2]. It contains all canonical kinase subdomains and signatures [20] and also the epitope for the anti-Stk33 antibody used in this study, which is located N-terminal to the kinase domain (Figure 1). In addition to nearly full length enzyme, a naturally occurring splice variant of Stk33, Stk33δ was also tested in the assay. This splice variant was cloned and expressed as described for recombinant nearly full length Stk33 [2] [GenBank:AM056057]. In this splice variant, there are 27 base pairs within the kinase domain missing. Parts of the missing amino acid sequence include the DFG-triplet [20] (Figure 1C). It is known, these amino acids are responsible for anchoring the phosphate donor ATP [20]. So this splice variant should produce an inactive form of Stk33 kinase. The recombinantly expressed Stk33δ isoform was tested for both autophosphorylation and phosphorylation of substrates such as vimentin wildtype and casein. As a positive control PKA catalytic subunit was used because it readily phosphorylates casein. This PKA catalytic subunit is not able to phosphorylate itself. Recombinant vimentin wildtype protein and different deletion derivatives expressed in E. coli were used as major targets in the assay. One technical problem had to be circumvented: Recombinant Stk33 and wildtype vimentin have a very similar electrophoretic mobility, and as we could show Stk33 is able to perform autophosphorylation. Thus on normal PAGE a discrimination between autophosphorylated Stk33 and phosphorylated wildtype vimentin is hardly possible. Therefore, the vimentin wildtype monomer was treated with increasing concentrations of glutaraldehyde [21] to form crosslinked vimentin tetramers with an apparent molecular weight of approximately 180 kDa (Figure 2B). This tetramer form of vimentin was included in the phosphorylation assay. Furthermore a number of vimentin mutants with different molecular weights were used in the kinase assay (Figure 2A, lane 2–6 and Figure 3). The deletion variants of human vimentin used are Δ12, Δ 20, Δ 30, Δ 42 and Δ 50, the numbers indicate how many amino acids were deleted from the amino-terminus (Figure 3). In addition, a mutant vimentin missing the entire non-α-helical amino-terminal domain ("head") was employed.


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)

A: Amino acid sequence alignment of the non helical head domain (H) of vimentin from human (Hsa) and mouse (Mmu). Starting points of the truncated vimentin derivatives used in this study are indicated (a = Δ12, b = Δ 20, c = Δ 30, d = Δ 42, e = Δ 50, f = Δ H). The numbers indicate how many amino acids were deleted from the amino-terminus. Phosphorylation sites in the vimentin head domain are indicated as reviewed in [33]. Black dots represent phosphorylation sites on human vimentin as mentioned in [26,35]. Open circles indicate phosphorylation sites in the mouse vimentin head domain according to [34]. Vimentin phosphorylation sites found in the hamster are symbolized by black squares [21]. B: A hypothetical structural model for the human vimentin head domain. Amino acids are represented by circles or boxes, aromatic amino acids are boxed, basic ones are filled, and potential phosphorylation sites are dotted. Figure modified from [26].
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Figure 3: A: Amino acid sequence alignment of the non helical head domain (H) of vimentin from human (Hsa) and mouse (Mmu). Starting points of the truncated vimentin derivatives used in this study are indicated (a = Δ12, b = Δ 20, c = Δ 30, d = Δ 42, e = Δ 50, f = Δ H). The numbers indicate how many amino acids were deleted from the amino-terminus. Phosphorylation sites in the vimentin head domain are indicated as reviewed in [33]. Black dots represent phosphorylation sites on human vimentin as mentioned in [26,35]. Open circles indicate phosphorylation sites in the mouse vimentin head domain according to [34]. Vimentin phosphorylation sites found in the hamster are symbolized by black squares [21]. B: A hypothetical structural model for the human vimentin head domain. Amino acids are represented by circles or boxes, aromatic amino acids are boxed, basic ones are filled, and potential phosphorylation sites are dotted. Figure modified from [26].
Mentions: The recombinant Stk33 enzyme was expressed in E. coli and affinity purified [2]. It contains all canonical kinase subdomains and signatures [20] and also the epitope for the anti-Stk33 antibody used in this study, which is located N-terminal to the kinase domain (Figure 1). In addition to nearly full length enzyme, a naturally occurring splice variant of Stk33, Stk33δ was also tested in the assay. This splice variant was cloned and expressed as described for recombinant nearly full length Stk33 [2] [GenBank:AM056057]. In this splice variant, there are 27 base pairs within the kinase domain missing. Parts of the missing amino acid sequence include the DFG-triplet [20] (Figure 1C). It is known, these amino acids are responsible for anchoring the phosphate donor ATP [20]. So this splice variant should produce an inactive form of Stk33 kinase. The recombinantly expressed Stk33δ isoform was tested for both autophosphorylation and phosphorylation of substrates such as vimentin wildtype and casein. As a positive control PKA catalytic subunit was used because it readily phosphorylates casein. This PKA catalytic subunit is not able to phosphorylate itself. Recombinant vimentin wildtype protein and different deletion derivatives expressed in E. coli were used as major targets in the assay. One technical problem had to be circumvented: Recombinant Stk33 and wildtype vimentin have a very similar electrophoretic mobility, and as we could show Stk33 is able to perform autophosphorylation. Thus on normal PAGE a discrimination between autophosphorylated Stk33 and phosphorylated wildtype vimentin is hardly possible. Therefore, the vimentin wildtype monomer was treated with increasing concentrations of glutaraldehyde [21] to form crosslinked vimentin tetramers with an apparent molecular weight of approximately 180 kDa (Figure 2B). This tetramer form of vimentin was included in the phosphorylation assay. Furthermore a number of vimentin mutants with different molecular weights were used in the kinase assay (Figure 2A, lane 2–6 and Figure 3). The deletion variants of human vimentin used are Δ12, Δ 20, Δ 30, Δ 42 and Δ 50, the numbers indicate how many amino acids were deleted from the amino-terminus (Figure 3). In addition, a mutant vimentin missing the entire non-α-helical amino-terminal domain ("head") was employed.

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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