14-3-3 Is Not Essential for Raf-1 Function: Identification of Raf-1 Proteins That Are Biologically Activated in a 14-3-3- and Ras-Independent Manner

Abstract

Recent reports have demonstrated the in vivo association of Raf-1 with members of the 14-3-3 protein family. To address the significance of the Raf-1-14-3-3 interaction, we investigated the enzymatic activity and biological function of Raf-1 in the presence and absence of associated 14-3-3. The interaction between these two molecules was disrupted in vivo and in vitro with a combination of molecular and biochemical techniques. Biochemical studies demonstrated that the enzymatic activities of Raf-1 were equivalent in the presence and absence of 14-3-3. Furthermore, mixing of purified Raf-1 and 14-3-3 in vitro was not sufficient to activate Raf-1. With a molecular approach, Cys-165 and Cys-168 as well as Ser-259 were identified as residues of Raf-1 required for the interaction with 14-3-3. Cys-165 and Cys-168 are located within the conserved cysteine-rich region of the CR1 domain, and Ser-259 is a conserved site of serine phosphorylation found within the CR2 domain. Mutation of either Cys-165 and Cys-168 or Ser-259 prevented the stable interaction of Raf-1 with 14-3-3 in vivo. Consistent with the model in which a site of serine phosphorylation is involved in the Raf-1-14-3-3 interaction, dephosphorylated Raf-1 was unable to associate with 14-3-3 in vitro. Phosphorylation may represent a general mechanism mediating 14-3-3 binding, because dephosphorylation of the Bcr kinase (known to interact with 14-3-3) also eliminated its association with 14-3-3. Finally, mutant Raf-1 proteins unable to stably interact with 14-3-3 exhibited enhanced enzymatic activity in human 293 cells and Xenopus oocytes and were biologically activated, as demonstrated by their ability to induce meiotic maturation of Xenopus oocytes. However, in contrast to wild-type Raf-1, activation of these mutants was independent of Ras. Our results therefore indicate that interaction with 14-3-3 is not essential for Raf-1 function.

Interactions of c-Raf-1 with phosphatidylserine and 14-3-3

“…mean, n=6) are given relative to a control sample (100%) that was incubated without peptide. The results have been pooled from three experiments The e ect of PS/Ca is also quite distinct from the two documented e ects that detergents have on Raf-1: RIPA bu er displaces 14-3-3 from Raf-1 without a ecting kinase activity (Michaud et al, 1995), whereas Empigen-BB displaces 14-3-3 and inactivates Raf-1 (Thorson et al, 1998). Clearly the e ect of PS/ Ca mimics neither of these.…”

“…The isolated RafCRD binds to 14-3-3 in vitro (Clark et al, 1997) and C165S,C168S mutations that disrupt the zinc ®nger within the RafCRD (RafCC/SS), have been reported to abrogate all binding of 14-3-3 to full-length Raf-1 in vitro (Michaud et al, 1995). However, when anti-Flag immunoprecipitates prepared from the NP-40 soluble fraction of COS cells expressing FlagRafCC/SS were immunoblotted for 14-3-3 we found a substantial amount of 14-3-3 was present, comparable to the amount of 14-3-3 in immunoprecipitates prepared from cells expressing wild type FlagRaf (Figure 1).…”

“…These data indicate that an intact zinc ®nger is required for some aspect of Raf-1 activation that occurs at the plasma membrane. In addition to Ras, two other CRD ligands have been identi®ed: 14-3-3 proteins, which bind to two phosphoserine-containing sites on Raf-1 (Muslin et al, 1996) as well as to the RafCRD (Michaud et al, 1995;Clark et al, 1997), and phosphatidylserine (PS), a plasma membrane inner lea¯et phospholipid (Ghosh et al, 1994) that can activate Raf-1 in vitro (Force et al, 1994).…”

“…The e ect of 14-3-3 proteins on the kinase activity of Raf-1 is contentious, since in vitro they have been reported to either have no e ect on Raf-1 kinase activity (Fu et al, 1994;Michaud et al, 1995;Suen et al, 1995) or to activate Raf-1 (Fantl et al, 1994;Freed et al, 1994;Irie et al, 1994;Li et al, 1995). Genetic studies have identi®ed dominant negative 14-3-3 mutants that can inhibit signalling downstream of Ras and Raf-1 (Chang and Rubin, 1997) and have con®rmed that 14-3-3 proteins are required for Rasmediated Raf-1 activation (Kockel et al, 1997).…”

Increasing complexity of Ras signaling

“…This family of ubiquitously expressed, highly conserved proteins can exist as dimers and are known to complex with a variety of proteins (BCR, cdc25, A20, PKC) suggesting that they may act as an adaptor proteins (Braselmann and McCormick, 1995;Conklin et al, 1995;Vincenz and Dixit, 1996). The role of 14-3-3 in Raf-1 regulation remains controversial (Michaud et al, 1995;Suen et al, 1995;Li et al, 1995;Irie et al, 1994;Freed et al, 1994;Fantl et al, 1994;Chang and Rubin, 1997;Luo et al, 1996;Braselmann and McCormick, 1995;Shimizu et al, 1994). This may be due, at least in part, to the complex nature of the Raf-1/14-3-3 interaction which involves multiple binding sites, including the Raf-1 CRD and two sites containing a phospho-serine consensus sequence for 14-3-3 at positions 259 and 621 of Raf-1 (Clark et al, 1997b;Li et al, 1993).…”