Abstract
BackgroundColocalization 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.ResultsThe results of the kinase assays demonstrate that Stk33 is able to specifically phosphorylate the non-α-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.ConclusionWe hypothesize that Stk33 is involved in the in vivo dynamics of the intermediate filament cytoskeleton by phosphorylating vimentin.
Highlights
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
calmodulin-dependent kinase family (CAMK) II is abundantly expressed in the brain [7] and a major effector for calcium-dependent signaling in neurons
Our results show that the serine/threonine kinase Stk33 phosphorylates the intermediate filament protein vimentin in vitro in the vimentin head domain
Summary
Colocalization of Stk with vimentin by double immunofluorescence in certain cells indicated that vimentin might be a target for phosphorylation by the novel kinase Stk. For testing the enzymatic activity of immunoprecipitated Stk we incubated precipitated Stk with recombinant vimentin proteins. The Stk gene in the mouse (and STK33 in human) is expressed differentially in a number of specific tissues and cells like testes, lung epithelia, alveolar macrophages, and horizontal cells in the retina. Based on sequence comparison with other kinases the STK33/Stk protein was classified as a member of the Ca2+/calmodulin-dependent kinase family (CAMK) [1,3,4,5]. As a consequence CAMK II bound to cytoskeletal vimentin is translocated into the cytosol This targeting is essential for signaling in differentiated smooth muscle cells because prevention of CAMK II targeting by antisense knockdown of CAMK II γ G-2 leads to inhibition of ERK (extracellular signal-related kinase) activation as well as to inhibition of muscle contraction [13]. Anchoring CAMK II γ G-2 to vimentin in unstimulated cells is discussed as a prerequisite for optimal kinase activation or for spatial separation of the kinase and its substrate [13]
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