Retaining of the assembly capability of vimentin phosphorylated by mitogen‐activated protein kinase‐activated protein kinase‐2

Abstract

Intermediate filament (IF) networks can be regulated by phosphorylation of unit proteins, such as vimentin, by specific kinases leading to reorganization of the IF filamentous structure. Recently, we identified mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP kinase-2) as a vimentin kinase (Cheng and Lai [1998] J. Cell. Biochem. 71:169-181). Herein we describe the results of further in vitro studies investigating the effects of MAPKAP kinase-2 phosphorylation on vimentin and the effects of the phosphorylation on the filamentous structure. We show that MAPKAP kinase-2 mainly phosphorylates vimentin at Ser-38, Ser-50, Ser-55, and Ser-82, residues all located in the head domain of the protein. Surprisingly, and in stark contrast to phosphorylation by most other kinases, phosphorylation of vimentin by MAPKAP kinase-2 has no discernable effect on its assembly. It suggested that structure disassembly is not the only obligated consequence of phosphorylated vimentin as regulated by other kinases. Finally, a mutational analysis of each of the phosphorylated serine residues in vimentin suggested that no single serine site was primarily responsible for structure maintenance, implying that the retention of filamentous structure may be the result of the coordinated action of several phosphorylated serine sites. This also shed new lights on the functional task(s) of vimentin that is intermediate filament proteins might provide a phosphate reservoir to accommodate the phosphate surge without any structural changes.

The Intermediate Filament Protein Vimentin Is a New Target for Epigallocatechin Gallate

“…We used the JB6 mouse epidermal cell line, a system that has been used extensively as an in vitro model for tumor promotion studies (12), to identify novel proteins that bind with EGCG. The results indicated that EGCG binds with the intermediate filament (IF) protein, vimentin with high affinity (K d ϭ 3.3 nM).…”

“…Bacterial Expression and Purification of the GST-Vimentin Fusion Protein-The GST-vimentin fusion protein was expressed in Escherichia coli BL21 and purified as described (12). The GST-vimentin was used for affinity binding and in vitro kinase assays.…”

Leukocyte transmigration is modulated by chemokine‐mediated PI3Kγ‐dependent phosphorylation of vimentin

“…Cells precisely regulate dynamic equilibrium between unpolymerized subunits and polymerized filaments that can form insoluble, stable structures [12]. These changes, causing extension or shortening of fibers, can be triggered by specific phosphorylation events, particularly on serine residues within the vimentin N-terminal non-helical domain [10,13,14]. Consistently, treatment with protein phosphatase inhibitors, such as calyculin-A [15] or okadaic acid [16], results in a dose-and time-dependent increase in vimentin phosphorylation, causing rapid disassembly of pre-existing filaments, delay of fiber assembly and increased levels of soluble vimentin tetramers [15,17,18].…”

“…Consistently, treatment with protein phosphatase inhibitors, such as calyculin-A [15] or okadaic acid [16], results in a dose-and time-dependent increase in vimentin phosphorylation, causing rapid disassembly of pre-existing filaments, delay of fiber assembly and increased levels of soluble vimentin tetramers [15,17,18]. These phosphorylation events are triggered by a wide range of protein kinases, such as PKA and PKC [15,19], PKG [20], Rho-kinase and Aurora B [21], PAK [22,23], MAPKAP K-2 [10], Cdc2 kinase [24] and CaMKII [25] and involve a large number of serine residues (e.g. Ser 4,[6][7][8][9]25,33,38,41,50,55,65,71,72,82) with a complex pattern of overlapping specificities [15,26,27].…”

“…Among these, the class three intermediate filament vimentin is abundantly expressed by leukocytes and plays multiple roles in cell shape organization. Vimentin filaments are assembled from elongated and thin rod-like dimers that form tetramers through coiled-coil interactions [10]. These tetramers represent the first assembly step, as they are the smallest soluble oligomers found in vivo [11].…”