Unique N-region determines low basal activity and limited inducibility of A-RAF kinase: the role of N-region in the evolutionary divergence of RAF kinase function in vertebrates

In mammals the RAF family of serine/threonine kinases consists of three members, A-, B-, and C-RAF. A prominent feature of RAF isoforms regards differences in basal and inducible kinase activities. To elucidate the nature of these differences, we studied the role of the nonconserved residues within the N-region (Negative-charge regulatory region). The nonconserved amino acids in positions -3 and +1 relative to the highly conserved serine 299 in A-RAF and serine 338 in C-RAF have so far not been considered as regulatory residues. Here we demonstrate the essential role of these residues in the RAF activation process. Substitution of tyrosine 296 in A-RAF to arginine led to a constitutively active kinase. In contrast, substitution of glycine 300 by serine (mimicking B- and C-RAF) acts in an inhibitory manner. Consistent with these data, the introduction of glycine in the analogous position of C-RAF (S339G mutant) led to a constitutively active C-RAF kinase. Based on the three-dimensional structure of the catalytic domain of B-RAF and using the sequences of the N-regions of A- and C-RAF, we searched by molecular modeling for the putative contact points between these two moieties. A tight interaction between the N-region residue serine 339 of C-RAF and arginine 398 of the catalytic domain was identified and proposed to inhibit the kinase activity of RAF proteins, because abrogation of this interaction contributes to RAF activation. Furthermore, tyrosine 296 in A-RAF favors a spatial orientation of the N-region segment, which enables a tighter contact to the catalytic domain, whereas a glutamine residue at this position in C-RAF abrogates this interaction. Considering this observation, we suggest that tyrosine 296, which is unique for A-RAF, is a major determinant of the low activating potency of this RAF isoform.     

Isoform-specific localization of A-RAF in mitochondria

RAF kinase is a family of isoforms including A-RAF, B-RAF, and C-RAF. Despite the important role of RAF in cell growth and proliferation, little evidence exists for isoform-specific function of RAF family members. Using Western analysis and immunogold labeling, A-RAF was selectively localized in highly purified rat liver mitochondria. Two novel human proteins, which interact specifically with A-RAF, were identified, and the full-length sequences are reported. These proteins, referred to as hTOM and hTIM, are similar to components of mitochondrial outer and inner membrane protein-import receptors from lower organisms, implicating their involvement in the mitochondrial transport of A-RAF. hTOM contains multiple tetratricopeptide repeat (TPR) domains, which function in protein-protein interactions. TPR domains are frequently present in proteins involved in cellular transport systems. In contrast, protein 14-3-3, an abundant cytosolic protein that participates in many facets of signal transduction, was found to interact with C-RAF but not with A-RAF N-terminal domain. This information is discussed in view of the important role of mitochondria in cellular functions involving energy balance, proliferation, and apoptosis and the potential role of A-RAF in regulating these systems.     

RAF protein-serine/threonine kinases: Structure and regulation

A-RAF, B-RAF, and C-RAF are a family of three protein-serine/threonine kinases that participate in the RAS-RAF-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including apoptosis, cell cycle progression, differentiation, proliferation, and transformation to the cancerous state. RAS mutations occur in 15-30% of all human cancers, and B-RAF mutations occur in 30-60% of melanomas, 30-50% of thyroid cancers, and 5-20% of colorectal cancers. Activation of the RAF kinases requires their interaction with RAS-GTP along with dephosphorylation and also phosphorylation by SRC family protein-tyrosine kinases and other protein-serine/threonine kinases. The formation of unique side-to-side RAF dimers is required for full kinase activity. RAF kinase inhibitors are effective in blocking MEK1/2 and ERK1/2 activation in cells containing the oncogenic B-RAF Val600Glu activating mutation. RAF kinase inhibitors lead to the paradoxical increase in RAF kinase activity in cells containing wild-type B-RAF and wild-type or activated mutant RAS. C-RAF plays a key role in this paradoxical increase in downstream MEK-ERK activation.     

RAF inhibitor-induced KSR1/B-RAF binding and its effects on ERK cascade signaling

RAF kinase inhibitors can induce ERK cascade signaling by promoting dimerization of RAF family members in the presence of oncogenic or normally activated RAS. This interaction is mediated by a dimer interface region in the RAF kinase domain that is conserved in members of the ERK cascade scaffold family, kinase suppressor of RAS (KSR). In this study, we find that most RAF inhibitors also induce the binding of KSR1 to wild-type and oncogenic B-RAF proteins, including V600E B-RAF, but promote little complex formation between KSR1 and C-RAF. The inhibitor-induced KSR1/B-RAF interaction requires direct binding of the drug to B-RAF and is dependent on conserved dimer interface residues in each protein, but, unexpectedly, is not dependent on binding of B-RAF to activated RAS. Inhibitor-induced KSR/B-RAF complex formation can occur in the cytosol and is observed in normal mouse fibroblasts, as well as a variety of human cancer cell lines. Strikingly, we find that KSR1 competes with C-RAF for inhibitor-induced binding to B-RAF and, as a result, alters the effect of the inhibitors on ERK cascade signaling.     

Elimination of B-RAF in Oncogenic C-RAF-expressing Alveolar Epithelial Type II Cells Reduces MAPK Signal Intensity and Lung Tumor Growth

Tumors are often greatly dependent on signaling cascades promoting cell growth or survival and may become hypersensitive to inactivation of key components within these signaling pathways. Ras and RAF mutations found in human cancer confer constitutive activity to these signaling molecules thereby converting them into an oncogenic state. RAF dimerization is required for normal Ras-dependent RAF activation and is required for the oncogenic potential of mutant RAFs. Here we describe a new mouse model for lung tumor development to investigate the role of B-RAF in oncogenic C-RAF-mediated adenoma initiation and growth. Conditional elimination of B-RAF in C-RAF BxB-expressing embryonic alveolar epithelial type II cells did not block adenoma formation. However, loss of B-RAF led to significantly reduced tumor growth. The diminished tumor growth upon B-RAF inactivation was due to reduced cell proliferation in absence of senescence and increased apoptosis. Furthermore, B-RAF elimination inhibited C-RAF BxB-mediated activation of the mitogenic cascade. In line with these data, mutation of Ser-621 in C-RAF BxB abrogated in vitro the dimerization with B-RAF and blocked the ability to activate the MAPK cascade. Taken together these data indicate that B-RAF is an important factor in oncogenic C-RAF-mediated tumorigenesis.     

Positive regulation of A-RAF by phosphorylation of isoform-specific hinge segment and identification of novel phosphorylation sites

In mammals the RAF family of serine/threonine kinases consists of three members, A-, B-, and C-RAF. Activation of RAF kinases involves a complex series of phosphorylations. Although the most prominent phosphorylation sites of B- and C-RAF are well characterized, little is known about regulatory phosphorylation of A-RAF. Using mass spectrometry, we identified here a number of novel in vivo phosphorylation sites in A-RAF. In particular, we found that Ser-432 participates in MEK binding and is indispensable for A-RAF signaling. On the other hand, phosphorylation within the activation segment does not contribute to epidermal growth factor-mediated activation. Furthermore, we show that the potential 14-3-3 binding domains in A-RAF are phosphorylated independently of its activation status. Of importance, we identified a novel regulatory domain in A-RAF (referred to as IH-segment) positioned between amino acids 248 and 267 that contains seven putative phosphorylation sites. Three of these sites, serines 257, 262, and 264, regulate A-RAF activation in a stimulatory manner. The spatial model of the A-RAF fragment, including residues between Ser-246 and Glu-277, revealed a switch of charge at the molecular surface of the IH-region upon phosphorylation, suggesting a mechanism in which the high accumulation of negative charges may lead to an electrostatic destabilization of protein-membrane interaction resulting in depletion of A-RAF from the plasma membrane. Together, we provide here for the first time a detailed analysis of in vivo A-RAF phosphorylation status and demonstrate that regulation of A-RAF by phosphorylation exhibits unique features compared with B- and C-RAF.     

Isoform-specific interaction of C-RAF with mitochondria

The proteins of the RAF family (A-RAF, B-RAF, and C-RAF) are serine/threonine kinases that play important roles in development, mature cell regulation, and cancer. Although it is widely held that their localization on membranes is an important aspect of their function, there are few data that address this aspect of their mode of action. Here, we report that each member of the RAF family exhibits a specific distribution at the level of cellular membranes and that C-RAF is the only isoform that directly targets mitochondria. We found that the RAF kinases exhibit intrinsic differences in terms of mitochondrial affinity and that C-RAF is the only isoform that binds this organelle efficiently. This affinity is conferred by the C-RAF amino-terminal domain and does not depend on the presence of RAS GTPases on the surface of mitochondria. Finally, we analyzed the consequences of C-RAF activation on mitochondria and observed that this event dramatically changes their morphology and their subcellular distribution. Our observations indicate that: (i) RAF kinases exhibit different localizations at the level of cellular membranes; (ii) C-RAF is the only isoform that directly binds mitochondria; and (iii) through its functional coupling with MEK, C-RAF regulates the shape and the cellular distribution of mitochondria.     

B- and C-RAF display essential differences in their binding to Ras: the isotype-specific N terminus of B-RAF facilitates Ras binding

Recruitment of RAF kinases to the plasma membrane was initially proposed to be mediated by Ras proteins via interaction with the RAF Ras binding domain (RBD). Data reporting that RAF kinases possess high affinities for particular membrane lipids support a new model in which Ras-RAF interactions may be spatially restricted to the plane of the membrane. Although the coupling features of Ras binding to the isolated RAF RBD were investigated in great detail, little is known about the interactions of the processed Ras with the functional and full-length RAF kinases. Here we present a quantitative analysis of the binding properties of farnesylated and nonfarnesylated H-Ras to both full-length B- and C-RAF in the presence and absence of lipid environment. Although isolated RBD fragments associate with high affinity to both farnesylated and nonfarnesylated H-Ras, the full-length RAF kinases revealed fundamental differences with respect to Ras binding. In contrast to C-RAF that requires farnesylated H-Ras, cytosolic B-RAF associates effectively and with significantly higher affinity with both farnesylated and nonfarnesylated H-Ras. To investigate the potential farnesyl binding site(s) we prepared several N-terminal fragments of C-RAF and found that in the presence of cysteine-rich domain only the farnesylated form of H-Ras binds with high association rates. The extreme N terminus of B-RAF turned out to be responsible for the facilitation of lipid independent Ras binding to B-RAF, since truncation of this region resulted in a protein that changed its kinase properties and resembles C-RAF. In vivo studies using PC12 and COS7 cells support in vitro results. Co-localization measurements using labeled Ras and RAF documented essential differences between B- and C-RAF with respect to association with Ras. Taken together, these data suggest that the activation of B-RAF, in contrast to C-RAF, may take place both at the plasma membrane and in the cytosolic environment.     

The tumor suppressor DiRas3 forms a complex with H-Ras and C-RAF proteins and regulates localization, dimerization, and kinase activity of C-RAF

The maternally imprinted Ras-related tumor suppressor gene DiRas3 is lost or down-regulated in more than 60% of ovarian and breast cancers. The anti-tumorigenic effect of DiRas3 is achieved through several mechanisms, including inhibition of cell proliferation, motility, and invasion, as well as induction of apoptosis and autophagy. Re-expression of DiRas3 in cancer cells interferes with the signaling through Ras/MAPK and PI3K. Despite intensive research, the mode of interference of DiRas3 with the Ras/RAF/MEK/ERK signal transduction is still a matter of speculation. In this study, we show that DiRas3 associates with the H-Ras oncogene and that activation of H-Ras enforces this interaction. Furthermore, while associated with DiRas3, H-Ras is able to bind to its effector protein C-RAF. The resulting multimeric complex consisting of DiRas3, C-RAF, and active H-Ras is more stable than the two protein complexes H-Ras·C-RAF or H-Ras·DiRas3, respectively. The consequence of this complex formation is a DiRas3-mediated recruitment and anchorage of C-RAF to components of the membrane skeleton, suppression of C-RAF/B-RAF heterodimerization, and inhibition of C-RAF kinase activity.     

Wild-type and mutant B-RAF activate C-RAF through distinct mechanisms involving heterodimerization

The protein kinase B-RAF is mutated in approximately 7% of human cancers. Most mutations are activating, but, surprisingly, a small number have reduced kinase activity. However, the latter can still stimulate cellular signaling through the MEK-ERK pathway because they activate the related family member C-RAF. We examine the mechanism underlying C-RAF activation by B-RAF. We show that C-RAF is activated in the cytosol in a RAS-independent manner that requires activation segment phosphorylation and binding of 14-3-3 to C-RAF. We show that wild-type B-RAF forms a complex with C-RAF in a RAS-dependent manner, whereas the mutants bind independently of RAS. Importantly, we show that wild-type B-RAF can also activate C-RAF. Our data suggest that B-RAF activates C-RAF through a mechanism involving 14-3-3 mediated heterooligomerization and C-RAF transphosphorylation. Thus, we have identified a B-RAF-C-RAF-MEK-ERK cascade that signals not only in cancer but also in normal cells.     

Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF

Over 30 mutations of the B-RAF gene associated with human cancers have been identified, the majority of which are located within the kinase domain. Here we show that of 22 B-RAF mutants analyzed, 18 have elevated kinase activity and signal to ERK in vivo. Surprisingly, three mutants have reduced kinase activity towards MEK in vitro but, by activating C-RAF in vivo, signal to ERK in cells. The structures of wild type and oncogenic V599EB-RAF kinase domains in complex with the RAF inhibitor BAY43-9006 show that the activation segment is held in an inactive conformation by association with the P loop. The clustering of most mutations to these two regions suggests that disruption of this interaction converts B-RAF into its active conformation. The high activity mutants signal to ERK by directly phosphorylating MEK, whereas the impaired activity mutants stimulate MEK by activating endogenous C-RAF, possibly via an allosteric or transphosphorylation mechanism.     

BAD contributes to RAF-mediated proliferation and cooperates with B-RAF-V600E in cancer signaling

BAD (Bcl-2 antagonist of cell death) belongs to the proapoptotic BH3-only subfamily of Bcl-2 proteins. Physiological activity of BAD is highly controlled by phosphorylation. To further analyze the regulation of BAD function, we investigated the role of recently identified phosphorylation sites on BAD-mediated apoptosis. We found that in contrast to the N-terminal phosphorylation sites, the serines 124 and 134 act in an antiapoptotic manner because the replacement by alanine led to enhanced cell death. Our results further indicate that RAF kinases represent, besides PAK1, BAD serine 134 phosphorylating kinases. Importantly, in the presence of wild type BAD, co-expression of survival kinases, such as RAF and PAK1, leads to a strongly increased proliferation, whereas substitution of serine 134 by alanine abolishes this process. Furthermore, we identified BAD serine 134 to be strongly involved in survival signaling of B-RAF-V600E-containing tumor cells and found that phosphorylation of BAD at this residue is critical for efficient proliferation in these cells. Collectively, our findings provide new insights into the regulation of BAD function by phosphorylation and its role in cancer signaling.     

p21-Activated Kinase 1 (PAK1) Can Promote ERK Activation in a Kinase-independent Manner

PAK1 plays an important role in proliferation and tumorigenesis, at least partially by promoting ERK phosphorylation of C-RAF (Ser-338) or MEK1 (Ser-298). We observed how that overexpression of a kinase-dead mutant form of PAK1 increased phosphorylation of MEK1/2 (Ser-217/Ser-221) and ERK (Thr-202/Tyr-204), although phosphorylation of B-RAF (Ser-445) and C-RAF (Ser-338) remained unchanged. Furthermore, increased activation of the PAK1 activator Rac1 induced the formation of a triple complex of Rac1, PAK1, and MEK1 independent of the kinase activity of PAK1. These data suggest that PAK1 can stimulate MEK activity in a kinase-independent manner, probably by serving as a scaffold to facilitate interaction of C-RAF.     

A 3-phosphoinositide-dependent protein kinase-1 (PDK1) docking site is required for the phosphorylation of protein kinase Czeta (PKCzeta ) and PKC-related kinase 2 by PDK1

Members of the AGC subfamily of protein kinases including protein kinase B, p70 S6 kinase, and protein kinase C (PKC) isoforms are activated and/or stabilized by phosphorylation of two residues, one that resides in the T-loop of the kinase domain and the other that is located C-terminal to the kinase domain in a region known as the hydrophobic motif. Atypical PKC isoforms, such as PKCzeta, and the PKC-related kinases, like PRK2, are also activated by phosphorylation of their T-loop site but, instead of possessing a phosphorylatable Ser/Thr in their hydrophobic motif, contain an acidic residue. The 3-phosphoinositide-dependent protein kinase (PDK1) activates many members of the AGC subfamily of kinases in vitro, including PKCzeta and PRK2 by phosphorylating the T-loop residue. In the present study we demonstrate that the hydrophobic motifs of PKCzeta and PKCiota, as well as PRK1 and PRK2, interact with the kinase domain of PDK1. Mutation of the conserved residues of the hydrophobic motif of full-length PKCzeta, full-length PRK2, or PRK2 lacking its N-terminal regulatory domain abolishes or significantly reduces the ability of these kinases to interact with PDK1 and to become phosphorylated at their T-loop sites in vivo. Furthermore, overexpression of the hydrophobic motif of PRK2 in cells prevents the T-loop phosphorylation and thus inhibits the activation of PRK2 and PKCzeta. These findings indicate that the hydrophobic motif of PRK2 and PKCzeta acts as a "docking site" enabling the recruitment of PDK1 to these substrates. This is essential for their phosphorylation by PDK1 in cells.     

Structure-function similarities between a plant receptor-like kinase and the human interleukin-1 receptor-associated kinase-4

Phylogenetic analysis has previously shown that plant receptor-like kinases (RLKs) are monophyletic with respect to the kinase domain and share an evolutionary origin with the animal interleukin-1 receptor-associated kinase/Pelle-soluble kinases. The lysin motif domain-containing receptor-like kinase-3 (LYK3) of the legume Medicago truncatula shows 33% amino acid sequence identity with human IRAK-4 over the kinase domain. Using the structure of this animal kinase as a template, homology modeling revealed that the plant RLK contains structural features particular to this group of kinases, including the tyrosine gatekeeper and the N-terminal extension α-helix B. Functional analysis revealed the importance of these conserved features for kinase activity and suggests that kinase activity is essential for the biological role of LYK3 in the establishment of the root nodule nitrogen-fixing symbiosis with rhizobia bacteria. The kinase domain of LYK3 has dual serine/threonine and tyrosine specificity, and mass spectrometry analysis identified seven serine, eight threonine, and one tyrosine residue as autophosphorylation sites in vitro. Three activation loop serine/threonine residues are required for biological activity, and molecular dynamics simulations suggest that Thr-475 is the prototypical phosphorylated residue that interacts with the conserved arginine in the catalytic loop, whereas Ser-471 and Thr-472 may be secondary sites. A threonine in the juxtamembrane region and two threonines in the C-terminal lobe of the kinase domain are important for biological but not kinase activity. We present evidence that the structure-function similarities that we have identified between LYK3 and IRAK-4 may be more widely applicable to plant RLKs in general.     

Glycogen Synthase Kinase 3�� Interaction Protein Functions as an A-kinase Anchoring Protein

A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinase 3β interaction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3β (glycogen synthase kinase 3β). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3β by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3β and thereby provides a mechanism for the integration of PKA and GSK3β signaling pathways.     

Hydrophobic motif phosphorylation is not required for activation loop phosphorylation of p70 ribosomal protein S6 kinase 1 (S6K1)

p70 ribosomal protein S6 kinase 1 (S6K1) is regulated by multiple phosphorylation events. Three of these sites are highly conserved among AGC kinases (cAMP dependent Protein Kinase, cGMP dependent Protein Kinase, and Protein Kinase C subfamily): the activation loop in the kinase domain, and two C-terminal sites, the turn motif and the hydrophobic motif. The common dogma has been that phosphorylation of the hydrophobic motif primes S6K1 for the phosphorylation at the activation loop by phosphoinositide-dependent protein kinase 1 (PDK1). Here, we show that the turn motif is, in fact, phosphorylated first, the activation loop second, and the hydrophobic motif is third. Specifically, biochemical analyses of a construct of S6K1 lacking the C-terminal autoinhibitory domain as well as full-length S6K1, reveals that S6K1 is constitutively phosphorylated at the turn motif when expressed in insect cells and becomes phosphorylated in vitro by purified PDK1 at the activation loop. Only the species phosphorylated at the activation loop by PDK1 gets phosphorylated at the hydrophobic motif by mammalian target of rapamycin (mTOR) in vitro. These data are consistent with a previous model in which constitutive phosphorylation of the turn motif provides the key priming step in the phosphorylation of S6K1. The data provide evidence for regulation of S6K1, where hydrophobic motif phosphorylation is not required for PDK1 to phosphorylate S6K1 at the activation loop, but instead activation loop phosphorylation of S6K1 is required for mTOR to phosphorylate the hydrophobic motif of S6K1.     

Regulation of the Ste20-like kinase, SLK: involvement of activation segment phosphorylation

Expression and activation of the Ste20-like kinase, SLK, is increased during kidney development and recovery from ischemic acute kidney injury. SLK promotes apoptosis, and it may regulate cell survival during injury or repair. This study addresses the role of phosphorylation in the regulation of kinase activity. We mutated serine and threonine residues in the putative activation segment of the SLK catalytic domain and expressed wild type (WT) and mutant proteins in COS-1 or glomerular epithelial cells. Compared with SLK WT, the T183A, S189A, and T183A/S189A mutants showed reduced in vitro kinase activity. SLK WT, but not mutants, increased activation-specific phosphorylation of c-Jun N-terminal kinase (JNK) and p38 kinase. Similarly, SLK WT stimulated activator protein-1 reporter activity, but activation of activator protein-1 by the three SLK mutants was ineffective. To test if homodimerization of SLK affects phosphorylation, the cDNA encoding SLK amino acids 1-373 (which include the catalytic domain) was fused with a cDNA for a modified FK506-binding protein, Fv (Fv-SLK 1-373). After transfection, the addition of AP20187 (an FK506 analog) induced regulated dimerization of Fv-SLK 1-373. AP20187-stimulated dimerization enhanced the kinase activity of Fv-SLK 1-373 WT. In contrast, kinase activity of Fv-SLK 1-373 T183A/S189A was weak and was not enhanced after dimerization. Finally, apoptosis was increased after expression of Fv-SLK 1-373 WT but not T183A/S189A. Thus, phosphorylation of Thr-183 and Ser-189 plays a key role in the activation and signaling of SLK and could represent a target for novel therapeutic approaches to renal injury.     

Splice variants of the dual specificity tyrosine phosphorylation-regulated kinase 4 (DYRK4) differ in their subcellular localization and catalytic activity

Dual specificity tyrosine phosphorylation-regulated kinases, DYRKs, are a family of conserved protein kinases that play key roles in the regulation of cell differentiation, proliferation, and survival. Of the five mammalian DYRKs, DYRK4 is the least studied family member. Here, we show that several splice variants of DYRK4 are expressed in tissue-specific patterns and that these variants have distinct functional capacities. One of these variants contains a nuclear localization signal in its extended N terminus that mediates its interaction with importin α3 and α5 and that is capable of targeting a heterologous protein to the nucleus. Consequently, the nucleocytoplasmic mobility of this variant differs from that of a shorter isoform in live cell imaging experiments. Other splicing events affect the catalytic domain, including a three-amino acid deletion within subdomain XI that markedly reduces the enzymatic activity of DYRK4. We also show that autophosphorylation of a tyrosine residue within the activation loop is necessary for full DYRK4 kinase activity, a defining feature of the DYRK family. Finally, by comparing the phosphorylation of an array of 720 peptides, we show that DYRK1A, DYRK2, and DYRK4 differ in their target recognition sequence and that preference for an arginine residue at position P -3 is a feature of DYRK1A but not of DYRK2 and DYRK4. Therefore, we highlight the use of subcellular localization as an important regulatory mechanism for DYRK proteins, and we propose that substrate specificity could be a source of functional diversity among DYRKs.     

RAF kinase activity regulates neuroepithelial cell proliferation and neuronal progenitor cell differentiation during early inner ear development

Background

Early inner ear development requires the strict regulation of cell proliferation, survival, migration and differentiation, coordinated by the concerted action of extrinsic and intrinsic factors. Deregulation of these processes is associated with embryonic malformations and deafness. We have shown that insulin-like growth factor I (IGF-I) plays a key role in embryonic and postnatal otic development by triggering the activation of intracellular lipid and protein kinases. RAF kinases are serine/threonine kinases that regulate the highly conserved RAS-RAF-MEK-ERK signaling cascade involved in transducing the signals from extracellular growth factors to the nucleus. However, the regulation of RAF kinase activity by growth factors during development is complex and still not fully understood.

Methodology/Principal Findings

By using a combination of qRT-PCR, Western blotting, immunohistochemistry and in situ hybridization, we show that C-RAF and B-RAF are expressed during the early development of the chicken inner ear in specific spatiotemporal patterns. Moreover, later in development B-RAF expression is associated to hair cells in the sensory patches. Experiments in ex vivo cultures of otic vesicle explants demonstrate that the influence of IGF-I on proliferation but not survival depends on RAF kinase activating the MEK-ERK phosphorylation cascade. With the specific RAF inhibitor Sorafenib, we show that blocking RAF activity in organotypic cultures increases apoptosis and diminishes the rate of cell proliferation in the otic epithelia, as well as severely impairing neurogenesis of the acoustic-vestibular ganglion (AVG) and neuron maturation.

Conclusions/Significance

We conclude that RAF kinase activity is essential to establish the balance between cell proliferation and death in neuroepithelial otic precursors, and for otic neuron differentiation and axonal growth at the AVG.