Membrane Localization, Oligomerization, and Phosphorylation Are Required for Optimal Raf Activation

Abstract

Activation of the serine/threonine kinase c-Raf-1 requires membrane localization, phosphorylation, and oligomerization. To study these mechanisms of Raf activation more precisely, we have used a membrane-localized fusion protein, myr-Raf-GyrB, which can be activated by coumermycin-induced oligomerization in NIH3T3 transfectants. By introducing a series of point mutations into the myr-Raf-GyrB kinase domain (S338A, S338A/Y341F, Y340F/Y341F, and T491A/S494A) we can separately study the role that membrane localization, phosphorylation, and oligomerization play in the process of Raf activation. We find that phosphorylation of Ser-338 plays a critical role in Raf activation and that this requires membrane localization but not oligomerization of Raf. Mutation of Tyr-341 had a limited effect, whereas mutation of both Ser-338 and Tyr-341 resulted in a synergistic loss of Raf activation following coumermycin-induced dimerization. Importantly, we found that membrane localization and phosphorylation of Ser-338 were not sufficient to activate Raf in the absence of oligomerization. Thus, our studies suggest that three key steps are required for optimal Raf activation: recruitment to the plasma membrane by GTP-bound Ras, phosphorylation via membrane-resident kinases, and oligomerization.The Ras/Raf/MEK 1 /MAPK signaling pathway plays a key role in regulating cell proliferation and differentiation in a variety of organisms (1). In the absence of stimulation, Ras is found in its inactive GDP-bound state. Recruitment of guanine nucleotide exchange factors to the plasma membrane by activated cytokine or growth factor receptors promotes the exchange of GDP for GTP; this converts Ras to its active GTP-bound state. Ras-GTP activates a number of effector molecules including the serine/ threonine kinase Raf-1. Subsequently, activated Raf-1 phosphorylates MEK, which in turn phosphorylates MAPK. Ultimately, this leads to activation of transcription factors in the nucleus that regulate downstream cellular processes.The mechanisms responsible for Ras, MEK, and MAPK activation are well characterized. In contrast, our understanding of Raf activation remains incomplete. Previous results show that artificial farnesylation of Raf results in its activation (2, 3). This has led to the suggestion that membrane localization is sufficient for Raf-1 activation. Another proposed mechanism for Raf activation involves phosphorylation by membrane resident kinases (4). For example, previous studies (5, 6) have demonstrated that phosphorylation on both serine and tyrosine residues in the Raf kinase domain cooperate to activate Raf-1. Specifically, mutation of Tyr-341 or Ser-338 compromises Raf activation. These residues have been shown to be phosphorylated by membrane resident kinases such as Src/Lck (Tyr-341) or Pak1 and/or Pak3 (Ser-338) (4,7,8). A more recent finding by Chong et al. (9) suggests that two additional sites, Thr-491 and Ser-494, are also important residues in the kinase domain of Raf that contribute to its activation.Although considerabl...

Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling

“…RAF dimerization-mediated signaling was first suggested by the observation that artificial dimerization activates RAF (17, 18). Next, immunoprecipitation (IP) suggested that formation of homo-and heterotypic RAF "dimers" is associated with active RAS (15,16,19). X-ray crystallography of the BRAF catalytic domain (CatB) identified critical residues postulated to enable CatB-CatB dimer formation (11).…”

Differential localization of A-Raf regulates MST2-mediated apoptosis during epithelial differentiation

“…[51][52][53][54] We therefore examined, whether dynamic changes of A-Raf localization impact on MST2 binding. For this purpose, we used the rapalogue (AP21967)-inducible heterodimerization system, which permits the efficient and selective cross-linking of FRB and FKBP-tagged proteins, 55,56 We co-expressed a flag-tagged FRB A-Raf Figure S3).…”

“…Upon activation through growth factors, dephosphorylation at the cell membrane by PP2A and PP1 phosphatases releases 14-3-3 from Raf thereby enabling Ras binding and membrane recruitment. 51,53,[65][66][67] However, due to the exchange of an arginine for a lysine at position 22 in its RBD and a non-conserved tyrosine 296 in the N-region, A-Raf is only weakly activated by oncogenic H-Ras. 9,68 In addition, several phosphorylation sites between amino acids 248 and 267 seem to facilitate A-Raf dissociation from the plasma membrane.…”

Raf kinases in signal transduction and interaction with translation machinery

“…The phosphorylation sites Ser 338 SYY 341 , are essential for full kinase activation and for interaction with the substrate MEK (40 -42) . Phosphorylation of Ser338 stimulated by growth factor is Ras-dependent and is necessary for maximal C-Raf activation (4,40,43,44) . A recent report indicated that Ser338 could be an autophosphorylation site induced by the dimerization of C-Raf or heterodimerization of C-Raf and B-Raf (45) or be a target for casein kinase 2 (CK2) recruited to Raf-1 and B-Raf by the scaffold kinase suppressor of Ras (KSR) (46) .…”

“…The kinases that can be involved in the phosphorylation of Tyr341 include the Src (47,17) and JAK family kinases (Janus Kinase) (48) . Phosphorylation of Tyr340/341 stimulated by growth factor requires Ras-dependent membrane recruitment and Src family tyrosine kinases (18,19,40,45) , and phosphorylation of Tyr340/341 and Ser338 is required for maximal Raf activity (4,8,43,44) . Mutation of Tyr341 severely compromises Raf-1 kinase activity (47,17) .…”