Phosphorylation of keratin intermediate filaments by protein kinase C, by calmodulin‐dependent protein kinase and by cAMP‐dependent protein kinase

Abstract

Keratins, constituent proteins of intermediate filaments of epithelial cells, are phosphoproteins containing phosphoserine and phosphothreonine. We examined the in vitro phosphorylation of keratin filaments by CAMPdependent protein kinase, protein kinase C and Ca2 +/calmodulin-dependent protein kinase 11. When rat liver keratin filaments reconstituted by type I keratin 18 (molecular mass 47 kDa; acidic type) and type I1 keratin 8 (molecular mass 55 kDa; basic type) in a 1 : 1 ratio were used as substrates, all the protein kinases phosphorylated both of the constituent proteins to a significant rate and extent, and disassembly of the keratin filament structure occurred. Kinetic analysis suggested that all these protein kinases preferentially phosphorylate keratin 8, compared to keratin 18. The amino acid residues of keratins 8 and 18 phosphorylated by CAMP-dependent protein kinase or protein kinase C were almost exclusively serine, while those phosphorylated by Ca2 c calmoddin-dependent protein kinase I1 were serine and threonine. Peptide mapping analysis indicated that these protein kinases phosphorylate keratins 8 and 18 in a different manner. These observations gave the way for in vivo studies of the role of phosphorylation in the reorganization of keratin filaments.

Specific in vivo phosphorylation sites determine the assembly dynamics of vimentin intermediate filaments

“…While the bulk (70-80% of the total) of wild-type vimentin was disassembled into the soluble tetrameric form following in vitro phosphorylation with PKA, only a small disassembled pool (15-20% of total) resulted from the same treatment of S38:A/S72:A vimentin. This result is in line with previous studies showing that phosphorylation with PKA leads to an increased solubility of IF proteins (Inagaki et al, 1988;Inagaki et al, 1990;Yano et al, 1991), but also confirms that of all PKA sites, these two in particular are important in determining the assembly state of vimentin. The first in vivo study of PKA-mediated phosphorylation on vimentin structure was that by Lamb et al (Lamb et al, 1989), showing marked effects on the organization of vimentin.…”

“…Surprisingly, CaM kinase II did not show any obvious matches with the interphase phosphopeptides. In previous studies, CaM kinase has been shown to both phosphorylate vimentin in vitro and in vivo (Ogawara et al, 1995;Yano et al, 1994;Yano et al, 1991). As the generated CaM kinase-specific sites could not be observed in this study, these sites do not seem to belong to the sites that display active turnover, but are rather restricted to specific functions under certain physiological conditions and and/or in specific cell types.…”

A novel mechanism of keratin cytoskeleton organization through casein kinase Iα and FAM83H in colorectal cancer

“…Phosphorylation of keratins induces the disassembly of keratin filaments. Keratin filaments reconstituted using rat keratin 8 and keratin 18 were disassembled by in vitro phosphorylation of the keratins, but reassembled by their dephosphorylation (Yano et al, 1991). Upon treatment of epithelial cells with the protein phosphatase inhibitors okadaic acid and orthovanadate, phosphorylation levels of keratins were enhanced and keratin filaments were concomitantly disassembled (Strnad et al, 2001;Strnad et al, 2002); however, only a few kinases such as p38 MAPK and PKCf were reported to be involved in keratin filament disassembly in different situations (Sivaramakrishnan et al, 2009;Wöll et al, 2007).…”

Induction of rapid and reversible cytokeratin filament network remodeling by inhibition of tyrosine phosphatases

“…Accordingly, increased levels of phosphorylated CK polypeptides were detected in mitotic cells (Celis et al, 1983;Liao et al, 1997;Omary et al, 1998) and also during meiosis (Klymkowsky et al, 1991). Phosphorylation may affect CK organization in a number of different ways: it leads to a shift in the equilibrium between the soluble and filamentous state toward the soluble form ; it may directly induce filament disassembly as suggested by in vitro experiments (Yano et al, 1991); and it alters the interaction with IFAPs, as demonstrated for the binding to the signaling 14-3-3 proteins, which act as solubility factors for CKs (Liao and Omary, 1996;Ku et al, 1998). Furthermore, phosphorylation of the cytoskeletal crosslinker plectin may affect CK organisation, as has been shown for plectin-lamin-B and plectin-vimentin interactions (Foisner et al, 1991;Foisner et al, 1996).…”