Keratins 8 and 18 (K8/18) heteropolymers may regulate cell signaling via the known K18 association with 14-3-3 proteins and 14-3-3 association with Raf-1 kinase. al., 1982; Fuchs and Weber, 1994; Coulombe and Omary, 2002). Glandular or simple-type epithelial cells express preferentially the keratin pair K8/18, as cytoplasmic filamentous/oligomeric or soluble tetrameric heteropolymers that consist of two K8 and two K18 molecules (Quinlan et al., 1984; Omary et al., 1998). The best-characterized keratin function is usually to protect epithelial cells Rabbit Polyclonal to GPR132 from necrotic or apoptotic forms of injury that are induced by mechanical and nonmechanical stresses (Gilbert et al., 2001; Coulombe and Omary, 2002; Oshima, 2002). This function is usually supported by numerous animal model studies and by the phenotypes of several keratin mutation-associated human diseases (Fuchs and Cleveland, 1998; Irvine and McLean, 1999; Magin et al., 2000). The cytoprotective and other proposed keratin functions are likely to be regulated by keratin phosphorylation and AS-605240 manufacturer keratin-associated proteins (Coulombe and Omary, 2002). For example, K18 serine-33 (S33) phosphorylation regulates keratin binding to 14-3-3 proteins during mitosis or after exposure to phosphatase inhibitors in the context of intact tissues or cultured cells (Liao and Omary, 1996; Ku et al., 1998, 2002). The significance of keratinC14-3-3 conversation is related to the phosphorylation-dependent association of 14-3-3 proteins with a wide range of signaling molecules including Raf-1 kinase, the AS-605240 manufacturer pro-apoptotic protein Bad, and cdc25 phosphatase among others (Fu et al., 2000; Tzivion and Avruch, 2002; Yaffe, 2002). Direct or indirect keratinCRaf conversation is not known but given the established associations of Raf kinase with 14-3-3 proteins and K8/18 with 14-3-3 proteins, we sought to define the dynamics, significance, and molecular domains that define RafCkeratinC14-3-3 associations. Results and conversation We examined the interrelationship of the known keratinC14-3-3 association (Liao and Omary, 1996; Ku et al., 1998) with the potential for a keratinCRaf association, in human colonic AS-605240 manufacturer HT29 cells, given the established RafC14-3-3 conversation (Tzivion et al., 1998). K8/18/19 immunoprecipitates were obtained from cells, with or without pretreatment with the phosphatase inhibitor okadaic acid (OA), followed by immunoblotting with antibodies (Abs) to Raf or 14-3-3 proteins. 14-3-3 binding to K8/18 increased dramatically due to K18 S33 hyperphosphorylation (Ku et al., 1998), but surprisingly Raf kinase associated with K8/18 under basal conditions and this association was abolished by phosphatase inhibition (Fig. 1 A, lanes 1 and 2). RafCkeratin association was also noted using other antikeratin Abs (anti-K19; Fig. 1 B), thereby indicating that it is unrelated to cross-reaction of antikeratin Abdominal muscles with Raf. The keratinCRaf conversation was also abolished in mice upon intraperitoneal administration of the phosphatase inhibitor and hepatotoxin microcystin-LR (MLR; Fig. 1 A, lanes 3C6). Open in a separate window Physique 1. Keratin association with Raf-1 kinase. (A) HT29 cells (lanes 1 and 2) were cultured in the presence or absence of OA (1 g/ml, 2 h), then solubilized with 1% NP-40. Alternatively, transgenic mice (lanes 3C6) that overexpress human K18 (Ku et al., 2002) were injected with MLR (30 g/kg) in saline (+) or with saline alone (?). After 2 h, the livers were homogenized with 1% NP-40. K8/18 immunoprecipitates were obtained from HT29 and liver NP-40 lysates then separated by SDS-PAGE. Duplicate gels were stained with Coomassie blue or transferred then blotted with Ab to Raf or 14-3-3. Note heat shock protein 70 (hsp70) association with K8/18. (B) BHK cells were transfected with vector alone, Raf, K8/18, K8/19, Raf+K8/18, or Raf+K8/19 constructs. After 3 d, transfected cells were solubilized followed by precipitation of K8/18 or K8/19 using K18- or K19-specific mAb. Immunoprecipitates (i.p.) were analyzed as in A. Arrow situated between K18 and K19 AS-605240 manufacturer highlights a nonspecific band, and arrowheads AS-605240 manufacturer show previously characterized K18 fragments (Ku et al., 1997). (C) A total cell lysate and K8/18 precipitates were prepared from.