Abstract
Keratins 8 (K8) and 18 are the primary intermediate filaments of simple epithelia. Phosphorylation of keratins at specific sites affects their organization, assembly dynamics, and their interaction with signaling molecules. A number of keratin in vitro and in vivo phosphorylation sites have been identified. One example is K8 Ser-73, which has been implicated as an important phosphorylation site during mitosis, cell stress, and apoptosis. We show that K8 is strongly phosphorylated on Ser-73 upon stimulation of the pro-apoptotic cytokine receptor Fas/CD95/Apo-1 in HT-29 cells. Kinase assays showed that c-Jun N-terminal kinase (JNK) was also activated with activation kinetics corresponding to that of K8 phosphorylation. Furthermore, K8 was also phosphorylated on Ser-73 by JNK in vitro, yielding similar phosphopeptide maps as the in vivo phosphorylated material. In addition, co-immunoprecipitation studies revealed that part of JNK is associated with K8 in vivo, correlating with decreased ability of JNK to phosphorylate the endogenous c-Jun. Taken together, K8 is a new cytoplasmic target for JNK in Fas receptor-mediated signaling. The functional significance of this phosphorylation could relate to regulation of JNK signaling and/or regulation of keratin dynamics.
Highlights
Keratins 8 (K8) and 18 are the primary intermediate filaments of simple epithelia
K8 Is Phosphorylated on Ser-73 Upon Fas receptor (FasR) Stimulation, and This Phosphorylation Occurs within the Soluble Keratin Pool— Keratins have been shown to be hyperphosphorylated under various conditions, including heat stress, virus infection, and stress-induced apoptosis
To investigate whether this is true for death receptor-mediated signaling, we stimulated the FasR and followed the time course of K8 phosphorylation in HT-29 cells, which are insensitive to FasR-mediated apoptosis but can be sensitized to undergo apoptosis by CHX [14]
Summary
Vol 277, No 13, Issue of March 29, pp. 10767–10774, 2002 Printed in U.S.A. The Intermediate Filament Protein Keratin 8 Is a Novel Cytoplasmic Substrate for c-Jun N-terminal Kinase*. In cultured HT-29 cells, hyperphosphorylation of K8 at Ser-73 was associated with apoptosis induced by anisomycin or etoposide The kinase regulating this site has not been identified, but members of the proline-directed mitogen-activated protein kinase (MAPK) family have been suggested as candidates [9]. Very little is known about the possible cytoplasmic targets for JNK In this respect, the neuronal intermediate filament neurofilament-H (NF-H) has been implicated as a JNK substrate, with phosphorylation at repeated Lys-Ser(P)-Pro-X-Glu motifs within the C-terminal domain [25]. We used stimulation of the FasR in HT-29 cells as a model, because in these cells FasR results in strong activation of JNK, without leading to apoptosis By using this model, we observed that K8 is phosphorylated by JNK both in vitro and in vivo. The association of JNK with K8/18 polymers may have a role in regulating JNK signaling and in adaptation to both environmental and physiological stresses
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