It is now well established that RKIP-1 is a member of a large, evolutionarily conserved group of proteins involved in MAP kinase (MAPK) signal transduction. This signaling machinery evolved to rapidly activate nuclear transcription factors in response to extracellular stimuli [5
and it can influence diverse cellular functions including cell proliferation, differentiation and apoptosis. MAP kinase pathways are a three component kinase module comprised of a MAP kinase kinase kinase (MKKK) that is able to phosphorylate and activate a MAP kinase kinase (MKK), which in turn phosphorylates and stimulates a MAP kinase (MAPK) [28
]. The terminal kinases in the pathway include the extracellular regulated kinases (ERKs) that can stimulate transcription factors such as AP1. MAP kinase signaling can be initiated by a number of stimuli including EGF, TPA and ultraviolet light.
The Raf kinases belong to the family of MAPKKKs [30
]. A-Raf, B-Raf, and Raf-1 (or c-Raf) are the three known isoforms of Raf in mammalian cells. While A and B-Raf display a tissue-specific pattern of expression, Raf-1 is widely expressed. The events that lead to Raf activation are only partially understood, but they almost certainly involve multiple phosphorylations and dephosphorylations [31
] that trigger conformational changes and the exposure of the N-terminal regulatory domain of the protein. Raf has the ability to interact with a large number of proteins, including Hsp90, PP2A and Cdc25 phosphatases, Akt, PKCs, Jac kinases, MAPK ERK5, SUR-8, CNK, KSR and Grb10 adaptors, 14-3-3 adapter protein, and RKIP-1 [30
]. This elaborate network of Raf-interacting proteins constitutes a key regulatory element of the MAP kinase cascade [34
Of all the Raf-binding proteins, RKIP-1 is the only known inhibitor of this MAP kinase pathway (). Using a two hybrid approach, Yeung et al.
demonstrated that RKIP-1 could bind Raf-1 [35
], and additional data from this group indicate that RKIP-1 also binds MEK and ERK. Interestingly, RKIP-1 does not interact with AP-1, and AP-1 activity induced by a constitutively active MEK is not affected by RKIP-1 [33
]. Furthermore, it appears that RKIP-1 cannot bind Raf-1 and MEK simultaneously since the binding sites for MEK and Raf-1 overlap on RKIP-1.
Fig. 1 RKIP-1 regulates RAF-1, GPCR, and NF-κB signaling pathways. (A) In its unphosphorylated state, RKIP-1 binds to and inhibits the activity of RAF-1. When phosphorylated by PKC, RKIP-1 dissociates form RAF-1 and binds to and inhibits GRK-2. (B) RKIP-1 (more ...)
Signaling downstream of MEK is attenuated when RKIP-1 blocks the interaction between Raf-1 and MEK [35
]. Recently, Eves et al.
demonstrated that RKIP-1 indirectly influences the Aurora B kinase and spindle checkpoints, and thus cell cycle, through its action on Raf-1 and the MAP kinase pathway [36
The action of RKIP-1 is not limited to a single signaling cascade. Yeung et al.
demonstrated that inhibition of RKIP-1 enhances NF-κB-mediated transcription, while over-expression of RKIP-1 reduces it [37
]. RKIP-1’s ability to control the transcriptional activity of NF-κB is due in part to its capacity to negatively regulate IKK, an activator of NF-κB transcription. RKIP-1 accomplishes this by controlling the upstream regulators of IKK, namely TAK-1 and NIK, and it has been suggested that TAK-1, NIK, IKKα, and IKKβ form part of the 700-kDa TNFα-induced IKK complex [38
] (). Thus, RKIP-1 can influence NF-κB-
regulated processes such as those that mediate the production of cytokines, cytokine receptors, cell adhesion molecules, and apoptotic effectors.
GPCR signaling is yet another intracellular control system that is influenced by this protein (). RKIP-1 controls GPCR activity by interacting with the amino-terminal part of the G-protein coupled receptor kinase-2 (GRK2) and inhibiting its ability to phosphorylate its target [39
]. In its active state, GRK2 phosphorylates GPCRs, uncoupling them from their associated G-proteins and marking them for degradation. By blocking the activity of GRK2, RKIP-1 stimulates signaling through the GPCRs and influences processes such as cardiac physiology.
RKIP-1is a phosphoprotein and its one known phosphorylation site (serine 153) has been shown to be a target of PKC [41
]. The phosphorylated form of RKIP-1 has been localized to the centrosomal and kinetochore regions of the prometaphase chromosome, where it may be involved in regulating spindle check point proteins and movement through the cell cycle [36
]. Protein Kinase C (PKC)-mediated phosphorylation of RKIP-1 decreases RKIP-1’s affinity for Raf-1 and increases its affinity for GRK2 [40
]. These data suggest that cell cycle progression and the activity of two biologically important cellular signaling systems are linked by the phosphorylation-dependent activity of the RKIP-1 protein.