Mechanism of Suppression of the Raf/MEK/Extracellular Signal-Regulated Kinase Pathway by the Raf Kinase Inhibitor Protein

Abstract

In metazoans, the Ras/Raf-1/MEK/extracellular signal-regulated kinase (ERK) module is a ubiquitously expressed signaling pathway that conveys mitogenic and differentiation signals from the cell membrane to the nucleus (6). This kinase cascade appears to be spatially organized in a signaling complex nucleated by Ras proteins (15). The small G protein Ras is activated by many growth factor receptors and binds the Raf-1 kinase with high affinity when activated. This induces the recruitment of Raf-1 from the cytosol to the cell membrane and its subsequent activation by mechanisms which remain incompletely understood (16). Activated Raf-1 then phosphorylates and activates MEK, a kinase that in turn phosphorylates and activates ERK, the prototypic mitogen-activated protein kinase (MAPK) (13). Activated ERKs can translocate to the nucleus and regulate gene expression by the phosphorylation of transcription factors (19).Studies with yeasts have revealed the important role of scaffolding proteins which assemble the components of MAPK pathways and thereby ensure that the signal transfer is efficient and specific (5). Mammalian homologues of such scaffolding proteins have been postulated, but despite extensive efforts, only a few candidates have been identified. These include JIP-1, a scaffolding protein for the stress-activated MAPKs/ JNKs (24), as well as Ksr, a protein kinase identified in genetic screens (4), which could have a similar function in the ERK pathway. Ksr binds to Raf-1, MEK, and ERK, but as both activation and inhibition by Ksr were observed, the physiological role of Ksr remains enigmatic (3,10,14,23,25,27). Since scaffolding proteins are expected to function in a stoichiometric manner, these discrepancies may have arisen from situations of nonstoichiometric expression levels (20) but also could reflect additional regulatory properties of Ksr. These observations suggest that the Raf-1/MEK/ERK pathway is subject to an additional level of regulation exerted by associated proteins. This hypothesis was further confirmed by the cloning of MP-1, a MEK-1-binding protein that specifically enhances the activation of ERK-1 (21).Using the yeast two-hybrid system, we recently identified a protein which binds to MEK, and ERK in vitro and in vivo (26). This protein was dubbed the Raf kinase inhibitor protein (RKIP) because it interfered with the activation of the Raf3MEK3ERK signaling pathway in vitro and in vivo. RKIP overexpression suppressed the ERK pathway and, as a consequence, interfered with Raf-1-induced transformation and AP-1-dependent transcription, whereas the downregulation of RKIP had the opposite effect. Genetic evidence indicated that RKIP functions at the Raf-1/MEK interface, because it suppressed signaling by activated Raf-1 mutants but not by activated MEK alleles. Here we describe the molecular mechanism of how RKIP works to inhibit the ERK pathway. MATERIALS AND METHODSPlasmids and protein expression. RKIP expression plasmids have been previously described (26). Deletion mutants of pCMV5-HA-RKIP...

Pilocytic astrocytomas of the optic nerve and their relation to pilocytic astrocytomas elsewhere in the central nervous system

“…This raises the possibility that other alterations in BRAF may drive the activation of p-MAPK1 and CDKN2A in pilocytic astrocytoma of the optic nerve. Alternatively, alterations in negative regulatory factors of the MAPK pathway, such as the Sprouty family of proteins, Raf kinase inhibitor protein, impedes mitogenic signal propagation, and the (7) 10 (16) dual-specificity MAPK phosphatase [29][30][31][32][33][34][35][36] may be found in pilocytic astrocytoma of the optic nerve. Studies are currently under way to test for these possibilities.…”

Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions

“…It also did not prevent the phosphorylation of MEK by MEKK-1. The basis for this selectivity is that RKIP can disrupt the physical interaction between Raf-1 and MEK (Figure 5), behaving like a competitor for substrate [128]. The binding sites for Raf and MEK in RKIP overlap, making their binding mutually exclusive.…”

“…Both proteins interact with the catalytic domain of Raf-1, but the essential RKIP binding site lies at the beginning of the catalytic domain (subdomains I and II), while MEK association requires subdomains VI-VIII in the core of the kinase, suggesting that RKIP may reduce binding affinity by an allosteric mechanism. Alternatively, bound RKIP could pose a steric hindrance that is prohibitive to the interaction between Raf and MEK [128].…”

“…Stimulation induces the release of RKIP from Raf-1, allowing activation of MEK and ERK. Later, when ERK activity declines, RKIP re-associates with Raf-1 [127,128]. Since RKIP acts as a stoichiometric inhibitor, the RKIP expression level may set the threshold for activation of the ERK pathway.…”

Absence of RKIP expression is an independent prognostic biomarker for gastric cancer patients

“…Raf kinase inhibitor protein (RKIP; also known as PEBP, for phosphatidylethanolamine-binding protein) is a widely expressed and highly conserved protein (5-7), which was firstly identified as a MAP kinase pathway inhibitor by modulating the function of Raf-1 (8,9). Currently, is known that RKIP also suppresses the activation of the nuclear factor κB (NF-κB) (10) and the regulator of G-protein coupled receptors (GRK-2) (11), and may be involved in regulation of the cell cycle (12).…”