Ras-mutant Cancer Cells Display B-Raf Binding to Ras That Activates Extracellular Signal-regulated Kinase and Is Inhibited by Protein Kinase A Phosphorylation

The small G protein Ras regulates proliferation through activation of the mitogen-activated protein (MAP) kinase (ERK) cascade. The first step of Ras-dependent activation of ERK signaling is Ras binding to members of the Raf family of MAP kinase kinase kinases, C-Raf and B-Raf. Recently, it has been reported that in melanoma cells harboring oncogenic Ras mutations, B-Raf does not bind to Ras and does not contribute to basal ERK activation. For other types of Ras-mutant tumors, the relative contributions of C-Raf and B-Raf are not known. We examined non-melanoma cancer cell lines containing oncogenic Ras mutations and express both C-Raf and B-Raf isoforms, including the lung cancer cell line H1299 cells. Both B-Raf and C-Raf were constitutively bound to oncogenic Ras and contributed to Ras-dependent ERK activation. Ras binding to B-Raf and C-Raf were both subject to inhibition by the cAMP-dependent protein kinase PKA. cAMP inhibited the growth of H1299 cells and Ras-dependent ERK activation via PKA. PKA inhibited the binding of Ras to both C-Raf and B-Raf through phosphorylations of C-Raf at Ser-259 and B-Raf at Ser-365, respectively. These studies demonstrate that in non-melanocytic Ras-mutant cancer cells, Ras signaling to B-Raf is a significant contributor to ERK activation and that the B-Raf pathway, like that of C-Raf, is a target for inhibition by PKA. We suggest that cAMP and hormones coupled to cAMP may prove useful in dampening the effects of oncogenic Ras in non-melanocytic cancer cells through PKA-dependent actions on B-Raf as well as C-Raf.     

Rheb inhibits C-raf activity and B-raf/C-raf heterodimerization

The Ras-Raf-MEK signaling cascade is critical for normal development and is activated in many forms of cancer. We have recently shown that B-Raf kinase interacts with and is inhibited by Rheb, the target of the GTPase-activating domain of the tuberous sclerosis complex 2 gene product tuberin. Here, we demonstrate for the first time that activation of Rheb is associated with decreased B-Raf and C-Raf phosphorylation at residues Ser-446 and Ser-338, respectively, concomitant with a decrease in the activities of both kinases and decreased heterodimerization of B-Raf and C-Raf. Importantly, the impact of Rheb on B-Raf/C-Raf heterodimerization and kinase activity are rapamycin-insensitive, indicating that they are independent of Rheb activation of the mammalian target of rapamycin-Raptor complex. In addition, we found that Rheb inhibits the association of B-Raf with H-Ras. Taken together, these results support a central role of Rheb in the regulation of the Ras/B-Raf/C-Raf/MEK signaling network.     

Requirement of B-Raf,C-Raf,and A-Raf for the growth and survival of mouse embryonic stem cells

Extracellular signal-regulated kinases (ERKs) have been implicated to be dispensable for self-renewal of mouse embryonic stem (ES) cells, and simultaneous inhibition of both ERK signaling and glycogen synthase kinase 3 (GSK3) not only allows mouse ES cells to self-renew independent of extracellular stimuli but also enables more efficient derivation of naïve ES cells from mouse and rat strains. Interestingly, some ERKs stay active in mouse ES cells which are maintained in regular medium containing leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP). Yet, the upstream signaling for ERK activation and their roles in mouse ES cells, other than promoting or priming differentiation, have not been determined. Here we found that mouse ES cells express three forms of Raf kinases, A-Raf, B-Raf, and C-Raf. Knocking-down each single Raf member failed to affect the sustained ERK activity, neither did A-Raf and B-Raf double knockdown or B-Raf and C-Raf double knockdown change it in ES cells. Interestingly, B-Raf and C-Raf double knockdown, not A-Raf and B-Raf knockdown, inhibited the maximal ERK activation induced by LIF, concomitant with the slower growth of ES cells. On the other hand, A-Raf, B-Raf, and C-Raf triple knockdown markedly inhibited both the maximal and sustained ERK activity in ES cells. Moreover, Raf triple knockdown, similar to the treatment of U-0126, an MEK inhibitor, significantly inhibited the survival and proliferation of ES cells, thereby compromising the colony propagation of mouse ES cells. In summary, our data demonstrate that all three Raf members are required for ERK activation in mouse ES cells and are involved in growth and survival of mouse ES cells.     

Positive and negative regulation of Raf kinase activity and function by phosphorylation.

Activating and inhibitory phosphorylation mechanisms play an essential role in regulating Raf kinase activity. Here we demonstrate that phosphorylation of C-Raf in the kinase activation loop (residues T491 and S494) is necessary, but not sufficient, for activation. C-Raf has additional activating phosphorylation sites at S338 and Y341. Mutating all four of these residues to acidic residues, S338D/Y341D/T491E/S494D (DDED), in C-Raf results in constitutive activity. However, acidic residue substitutions at the corresponding activation loop sites in B-Raf are sufficient to confer constitutive activity. B-Raf and C-Raf also utilize similar inhibitory phosphorylation mechanisms to regulate kinase activity. B-Raf has multiple inhibitory phosphorylation sites necessary for full kinase inhibition where C-Raf requires only one. We examined the functional significance of these inhibitory and activating phosphorylations in Caenorhabditis elegans lin-45 Raf. Eliminating the inhibitory phosphorylation or mimicking activating phosphorylation sites is sufficient to confer constitutive activity upon lin-45 Raf and induce multi-vulva phenotypes in C.elegans. Our results demonstrate that different members of the Raf family kinases have both common and distinct phosphorylation mechanisms to regulate kinase activity and biological function.     

Topological analysis of MAPK cascade for kinetic ErbB signaling

Ligand-induced homo- and hetero-dimer formation of ErbB receptors results in different biological outcomes irrespective of recruitment and activation of similar effector proteins. Earlier experimental research indicated that cells expressing both EGFR (epidermal growth factor receptor) and the ErbB4 receptor (E1/4 cells) induced E1/4 cell-specific B-Raf activation and higher extracellular signal-regulated kinase (ERK) activation, followed by cellular transformation, than cells solely expressing EGFR (E1 cells) in Chinese hamster ovary (CHO) cells. Since our experimental data revealed the presence of positive feedback by ERK on upstream pathways, it was estimated that the cross-talk/feedback pathway structure of the Raf-MEK-ERK cascade might affect ERK activation dynamics in our cell system. To uncover the regulatory mechanism concerning the ERK dynamics, we used topological models and performed parameter estimation for all candidate structures that possessed ERK-mediated positive feedback regulation of Raf. The structure that reliably reproduced a series of experimental data regarding signal amplitude and duration of the signaling molecules was selected as a solution. We found that the pathway structure is characterized by ERK-mediated positive feedback regulation of B-Raf and B-Raf-mediated negative regulation of Raf-1. Steady-state analysis of the estimated structure indicated that the amplitude of Ras activity might critically affect ERK activity through ERK-B-Raf positive feedback coordination with sustained B-Raf activation in E1/4 cells. However, Rap1 that positively regulates B-Raf activity might be less effective concerning ERK and B-Raf activity. Furthermore, we investigated how such Ras activity in E1/4 cells can be regulated by EGFR/ErbB4 heterodimer-mediated signaling. From a sensitivity analysis of the detailed upstream model for Ras activation, we concluded that Ras activation dynamics is dominated by heterodimer-mediated signaling coordination with a large initial speed of dimerization when the concentration of the ErbB4 receptor is considerably high. Such characteristics of the signaling cause the preferential binding of the Grb2-SOS complex to heterodimer-mediated signaling molecules.     

RKIP regulates MAP kinase signaling in cells with defective B-Raf activity

MAP kinase (MAPK) signaling results from activation of Raf kinases in response to external or internal stimuli. Here, we demonstrate that Raf kinase inhibitory protein (RKIP) regulates the activation of MAPK when B-Raf signaling is defective. We used multiple models including mouse embryonic fibroblasts (MEFs) and primary keratinocytes from RKIP- or Raf-deficient mice as well as allografts in mice to investigate the mechanism. Loss of B-Raf protein or activity significantly reduces MAPK activation in these cells. We show that RKIP depletion can rescue the compromised ERK activation and promote proliferation, and this rescue occurs through a Raf-1 dependent mechanism. These results provide formal evidence that RKIP is a bona fide regulator of Raf-1. We propose a new model in which RKIP plays a key role in regulating the ability of cells to signal through Raf-1 to ERK in B-Raf compromised cells.     

A Minimal Model of Signaling Network Elucidates Cell-to-Cell Stochastic Variability in Apoptosis

Background

Signaling networks are designed to sense an environmental stimulus and adapt to it. We propose and study a minimal model of signaling network that can sense and respond to external stimuli of varying strength in an adaptive manner. The structure of this minimal network is derived based on some simple assumptions on its differential response to external stimuli.

Methodology

We employ stochastic differential equations and probability distributions obtained from stochastic simulations to characterize differential signaling response in our minimal network model. Gillespie''s stochastic simulation algorithm (SSA) is used in this study.

Conclusions/Significance

We show that the proposed minimal signaling network displays two distinct types of response as the strength of the stimulus is decreased. The signaling network has a deterministic part that undergoes rapid activation by a strong stimulus in which case cell-to-cell fluctuations can be ignored. As the strength of the stimulus decreases, the stochastic part of the network begins dominating the signaling response where slow activation is observed with characteristic large cell-to-cell stochastic variability. Interestingly, this proposed stochastic signaling network can capture some of the essential signaling behaviors of a complex apoptotic cell death signaling network that has been studied through experiments and large-scale computer simulations. Thus we claim that the proposed signaling network is an appropriate minimal model of apoptosis signaling. Elucidating the fundamental design principles of complex cellular signaling pathways such as apoptosis signaling remains a challenging task. We demonstrate how our proposed minimal model can help elucidate the effect of a specific apoptotic inhibitor Bcl-2 on apoptotic signaling in a cell-type independent manner. We also discuss the implications of our study in elucidating the adaptive strategy of cell death signaling pathways.     

Knockdown of B-Raf impairs spindle formation and the mitotic checkpoint in human somatic cells

It is well established that B-Raf signaling through the MAP kinase (ERK) pathways plays a prominent role in regulating cell proliferation but how it does this is not completely understood. Here, we show that B-Raf serves a physiological role during mitosis in human somatic cells. Knockdown of B-Raf using short interfering RNA (siRNA) resulted in pleiotropic spindle abnormalities and misaligned chromosomes in over 80% of the mitotic cells analyzed. A second B-Raf siRNA gave similar results suggesting these effects are specific to down-regulating B-Raf protein. In agreement with these findings, a portion of B-Raf was detected at the spindle structures including the spindle poles and kinetochores. Knockdown of C-Raf (Raf-1) had no detectable effects on spindle formation or chromosome alignment. Activation of the spindle assembly checkpoint was found to be dependent on B-Raf as evident by the inability of checkpoint proteins Bub1 and Mad2 to localize to unattached kinetochores in HeLa cells treated with B-Raf siRNA. Consistent with this, live-cell imaging microscopy showed that B-Raf-depleted cells exited mitosis earlier than control non-depleted cells. Finally, we provide evidence that B-Raf signaling promotes phosphorylation and kinetochore localization of the mitotic checkpoint kinase Mps1. Blocking B-Raf expression, ERK activity, or phosphorylation at Ser-821 residue perturbed Mps1 localization at unattached kinetochores. Thus, our data implicates a mitotic role for B-Raf in regulating spindle formation and the spindle checkpoint in human somatic cells.     

Diacylglycerol Kinase �� Augments C-Raf Activity and B-Raf/C-Raf Heterodimerization

The Ras/B-Raf/C-Raf/MEK/ERK signaling cascade is critical for the control of many fundamental cellular processes, including proliferation, survival, and differentiation. This study demonstrated that small interfering RNA-dependent knockdown of diacylglycerol kinase η (DGKη) impaired the Ras/B-Raf/C-Raf/MEK/ERK pathway activated by epidermal growth factor (EGF) in HeLa cells. Conversely, the overexpression of DGKη1 could activate the Ras/B-Raf/C-Raf/MEK/ERK pathway in a DGK activity-independent manner, suggesting that DGKη serves as a scaffold/adaptor protein. By determining the activity of all the components of the pathway in DGKη-silenced HeLa cells, this study revealed that DGKη activated C-Raf but not B-Raf. Moreover, this study demonstrated that DGKη enhanced EGF-induced heterodimerization of C-Raf with B-Raf, which transmits the signal to C-Raf. DGKη physically interacted with B-Raf and C-Raf, regulating EGF-induced recruitment of B-Raf and C-Raf from the cytosol to membranes. The DGKη-dependent activation of C-Raf occurred downstream or independently of the already known C-Raf modifications, such as dephosphorylation at Ser-259, phosphorylation at Ser-338, and interaction with 14-3-3 protein. Taken together, the results obtained strongly support that DGKη acts as a novel critical regulatory component of the Ras/B-Raf/C-Raf/MEK/ERK signaling cascade via a previously unidentified mechanism.The Ras/Raf/MEK³/ERK signaling pathway is critical for the transduction of the extracellular signals to the nucleus, regulating diverse physiological processes such as cell proliferation, differentiation, and survival (1, 2). The binding of extracellular ligands, such as growth factors and cytokines, to cell surface receptors activates Ras. The Raf serine/threonine kinase transmits signals from activated Ras to the downstream protein kinases, MEK1 and MEK2, subsequently leading to activation of ERK1 and ERK2.In mammals, the Raf kinase consists of three isoforms, A-Raf, B-Raf, and C-Raf (Raf-1). It is clinically known that both B-Raf and C-Raf mutations are associated with human cancers (3–5). Knock-out mouse studies demonstrated that each individual Raf isoform has distinct functions, although the three Raf isoforms have high homology in the amino acid sequence (6). The mechanisms underlying C-Raf activation are complicated and thus are not completely understood (3). In response to extracellular signals, C-Raf is initially recruited from cytosol to the plasma membrane and undergo conformational changes by binding directly to the active Ras (7). In addition, other modifications and factors are required for the sufficient activation of C-Raf. For example, dephosphorylation of Ser-259 and phosphorylation of Ser-338, Tyr-341, Thr-491, and Ser-494 are critical for the activation of C-Raf (8–11). Feedback phosphorylation of C-Raf by ERK was also reported to be important for the modulation of C-Raf activity (12, 13). C-Raf activity is regulated by the interaction with 14-3-3 protein (14). Moreover, the heterodimerization of C-Raf with B-Raf, which transmits the signal to C-Raf, has been reported to play an essential role in the activation of the MEK-ERK signaling pathway (15–17). Although B-Raf and C-Raf are the central regulatory components in the Ras/B-Raf/C-Raf/MEK/ERK signaling cascade involved in a variety of pathophysiological events, the activation mechanisms of C-Raf by B-Raf are still unclear.Diacylglycerol kinase (DGK) catalyzes the phosphorylation of diacylglycerol to generate phosphatidic acid. DGK has been recently recognized as an emerging key regulator in a wide range of cell signaling systems (18–20). To date, 10 mammalian DGK isozymes have been identified. They characteristically contain two or three protein kinase C-like C1 domains and a catalytic region and are subdivided into five subtypes according to their structural features (18–20). Their structural variety and distinct expression patterns in tissues allow us to presume that each DGK isozyme has its own biological functions. Indeed, recent studies have revealed that individual DGK isozymes play distinct roles in cell functions through interactions with unique partner proteins such as protein kinase C (21, 22), Ras guanyl nucleotide-releasing protein (23, 24), phosphatidylinositol-4-phosphate 5-kinase (25), chimerins (26, 27), AP-2 (28), and PSD-95 (29).DGKη belongs to the type II DGKs containing a pleckstrin homology domain at the N terminus and the separated catalytic region (19, 30). Two alternative splicing products of DGKη have been identified as DGKη1 and -η2 (31). DGKη2 possesses a sterile α-motif (SAM) domain at the C terminus, whereas DGKη1 does not. This study demonstrated that the expression levels of DGKη1 and -η2 were regulated differently by glucocorticoid, and that they were translocated from the cytoplasm to endosomes in response to stress stimuli as osmotic shock and oxidative stress (31). However, the physiological roles of DGKη remain unknown.This study showed that siRNA-dependent knockdown of DGKη inhibits cell proliferation of the HeLa cells. In addition, DGKη is required for the Ras/B-Raf/C-Raf/MEK/ERK signaling cascade activated by epidermal growth factor (EGF). Intriguingly, DGKη regulates recruitment of B-Raf and C-Raf from cytosol to membranes and their heterodimerization. Moreover, this study demonstrated that DGKη activates C-Raf but not B-Raf in an EGF-dependent manner. The data show DGKη as a novel key regulator of the Ras/B-Raf/C-Raf/MEK/ERK signaling pathway.     

Impact of Feedback Phosphorylation and Raf Heterodimerization on Normal and Mutant B-Raf Signaling

The B-Raf kinase is a Ras pathway effector activated by mutation in numerous human cancers and certain developmental disorders. Here we report that normal and oncogenic B-Raf proteins are subject to a regulatory cycle of extracellular signal-regulated kinase (ERK)-dependent feedback phosphorylation, followed by PP2A- and Pin1-dependent dephosphorylation/recycling. We identify four S/TP sites of B-Raf phosphorylated by activated ERK and find that feedback phosphorylation of B-Raf inhibits binding to activated Ras and disrupts heterodimerization with C-Raf, which is dependent on the B-Raf pS729/14-3-3 binding site. Moreover, we find that events influencing Raf heterodimerization can alter the transforming potential of oncogenic B-Raf proteins possessing intermediate or impaired kinase activity but have no significant effect on proteins with high kinase activity, such as V600E B-Raf. Mutation of the feedback sites or overexpression of the Pin1 prolyl-isomerase, which facilitates B-Raf dephosphorylation/recycling, resulted in increased transformation, whereas mutation of the S729/14-3-3 binding site or expression of dominant negative Pin1 reduced transformation. Mutation of each feedback site caused increased transformation and correlated with enhanced heterodimerization and activation of C-Raf. Finally, we find that B-Raf and C-Raf proteins containing mutations identified in certain developmental disorders constitutively heterodimerize and that their signaling activity can also be modulated by feedback phosphorylation.The Ras, Raf, MEK, and extracellular signal-regulated kinase (ERK) proteins are core components of one of the major signaling cascades regulating normal cell proliferation—the Ras pathway. Not surprising, deregulation of Ras pathway signaling is a major contributor to human cancer and has recently been linked with several developmental disorders, such as Noonan''s, LEOPARD, and cardiofaciocutaneous (CFC) syndromes (28). Given its importance to both normal and disease states, much effort has been directed toward elucidating the mechanisms that modulate Ras pathway signaling. Of all the pathway components, regulation of the Raf proteins has proved to be the most complex, involving inter- and intramolecular interactions, a change in subcellular localization, and phosphorylation and dephosphorylation events (6, 32).In mammalian cells, there are three Raf family members: A-Raf, B-Raf, and C-Raf (12). In their inactive state, all Raf proteins are found in the cytosol, with the N-terminal regulatory domain acting as an autoinhibitor of the C-terminal kinase domain (4, 5, 13). 14-3-3 dimers bind to phosphorylation sites present in both the N- and C-terminal regions and stabilize the autoinhibited state (22). To activate the Raf proteins, autoinhibition mediated by the N terminus must be relieved and the kinase domain must adopt the active catalytic conformation (6, 31, 32). Under normal signaling conditions, Ras activation helps mediate these events by recruiting the Raf proteins to the plasma membrane, which induces the release of 14-3-3 from the N-terminal binding site and facilitates phosphorylation of the Raf kinase domain (19). For the C-Raf and A-Raf proteins, phosphorylation occurs in two regions of the kinase domain, the negative-charge regulatory region (N-region) and the activation segment (4). In contrast, the N-region of B-Raf exhibits a constitutive negative charge due to increased basal phosphorylation of an activating serine site and the presence of two aspartic acid residues (18); thus, only phosphorylation of the activation segment is required. Phosphorylation of the activation segment serves both to destabilize the “inactive” catalytic conformation maintained by hydrophobic interactions between the glycine-rich loop and the activation segment and to stabilize the “active” catalytic conformation, whereas the negative charge of the N-region helps to disrupt the autoinhibitory activity of the N-terminal domain (5, 30, 31).Because the N-region of B-Raf exhibits a constitutive negative charge, B-Raf possesses higher basal kinase activity than other family members and is more susceptible to mutational activation (9, 11, 17). In particular, B-Raf is a major contributor to human cancer: somatic mutations in the B-Raf gene are detected in ∼50% of malignant melanomas and many colorectal, ovarian, and papillary thyroid carcinomas (7). Of the oncogenic mutations identified in B-Raf, the vast majority cluster to the two regions of the kinase domain responsible for maintaining the inactive catalytic conformation—the glycine-rich loop and the activation segment (31). Based on enzymatic activity, the oncogenic B-Raf proteins have been divided into three groups: those with high activity (130- to 700-fold more active than wild-type [WT] B-Raf), those with intermediate activity (64- to 1.3-fold more active), and surprisingly, those with impaired catalytic activity (0.8 to 0.3 of WT B-Raf activity) (31). Further analysis has revealed that all oncogenic B-Raf proteins heterodimerize constitutively with C-Raf and activate C-Raf in a Ras-independent manner that requires an intact C-Raf activation segment as well as the binding of 14-3-3 to the C-terminal pS621 binding site on C-Raf (11). Importantly, for the oncogenic B-Raf proteins with impaired kinase activity, the binding and activation of C-Raf are required for ERK activation in vivo (31). Interestingly, heterodimerization of B-Raf and C-Raf also occurs under normal signaling conditions; however, in this case, heterodimerization is Ras dependent and occurs at the plasma membrane following mitogen stimulation (11, 27).Once activated, either by upstream signaling or by mutational events, all Raf proteins are capable of initiating the phosphorylation cascade that results in the sequential activation of MEK and ERK. ERK then phosphorylates targets in both the cytoplasm and the nucleus that are required for cell proliferation. Strikingly, the Raf proteins themselves are also substrates of activated ERK. In regard to C-Raf, ERK-dependent feedback phosphorylation has been shown to instigate a regulatory cycle whereby phosphorylation of the feedback sites down-modulates C-Raf signaling, after which the hyperphosphorylated C-Raf protein is dephosphorylated and returned to a signaling-competent state through dephosphorylation events involving protein phosphatase 2A (PP2A) and the Pin1 prolyl-isomerase (8). For B-Raf, two ERK-dependent feedback sites, S750 and T753, have been identified, and phosphorylation of these sites has been reported to have a negative regulatory effect (3).In this study, we have further investigated the impact of feedback phosphorylation and heterodimerization on B-Raf signaling. Here we find that both normal and oncogenic B-Raf proteins are phosphorylated on four S/TP sites (S151, T401, S750, and T753) by activated ERK. Through mutational analysis, we find that phosphorylation of B-Raf at S151 inhibits binding to activated Ras, whereas phosphorylation of each of the feedback sites contributes to the disruption of B-Raf/C-Raf heterodimers. Moreover, we find that events influencing B-Raf/C-Raf heterodimerization, such as feedback phosphorylation and 14-3-3 binding, can alter the signaling activity of oncogenic B-Raf proteins possessing intermediate or impaired kinase activity as well as that of B-Raf and C-Raf proteins containing mutations identified in CFC and Noonan''s syndromes, respectively.     

New signaling pathway for parathyroid hormone and cyclic AMP action on extracellular-regulated kinase and cell proliferation in bone cells. Checkpoint of modulation by cyclic AMP

cAMP signaling, activated by extracellular stimuli such as parathyroid hormone, has cell type-specific effects important for cellular proliferation and differentiation in bone cells. Recent evidence of a second enzyme target for cAMP suggests divergent effects on extracellular-regulated kinase (ERK) activity depending on Epac/Rap1/B-Raf signaling. We investigated the molecular mechanism of the dual functionality of cAMP on cell proliferation in clonal bone cell types. MC3T3-E1 and ATDC5, but not MG63, express a 95-kDa isoform of B-Raf. cAMP stimulated Ras-independent and Rap1-dependent ERK phosphorylation and cell proliferation in B-Raf-expressing cells, but inhibited growth in B-Raf-lacking cells. The mitogenic action of cAMP was blocked by the ERK pathway inhibitor PD98059. In B-Raf-transduced MG63 cells, cAMP stimulated ERK activation and cell proliferation. Thus, B-Raf is the dominant molecular switch that permits differential cAMP-dependent regulation of ERK with important implications for cell proliferation in bone cells. These findings might explain the dual functionality of parathyroid hormone on osteoblastic cell proliferation.     

The GPCR–β-arrestin complex allosterically activates C-Raf by binding its amino terminus

G protein–coupled receptors (GPCRs) convert external stimuli into cellular signals through heterotrimeric guanine nucleotide-binding proteins (G-proteins) and β-arrestins (βarrs). In a βarr-dependent signaling pathway, βarrs link GPCRs to various downstream signaling partners, such as the Raf–mitogen-activated protein kinase extracellular signal–regulated kinase–extracellular signal-regulated kinase cascade. Agonist-stimulated GPCR–βarr complexes have been shown to interact with C-Raf and are thought to initiate the mitogen-activated protein kinase pathway through simple tethering of these signaling partners. However, recent evidence shows that in addition to canonical scaffolding functions, βarrs can allosterically activate downstream targets, such as the nonreceptor tyrosine kinase Src. Here, we demonstrate the direct allosteric activation of C-Raf by GPCR–βarr1 complexes in vitro. Furthermore, we show that βarr1 in complex with a synthetic phosphopeptide mimicking the human V2 vasopressin receptor tail that binds and functionally activates βarrs also allosterically activates C-Raf. We reveal that the interaction between the phosphorylated GPCR C terminus and βarr1 is necessary and sufficient for C-Raf activation. Interestingly, the interaction between βarr1 and C-Raf was considerably reduced in the presence of excess activated H-Ras, a small GTPase known to activate C-Raf, suggesting that H-Ras and βarr1 bind to the same region on C-Raf. Furthermore, we found that βarr1 interacts with the Ras-binding domain of C-Raf. Taken together, these data suggest that in addition to canonical scaffolding functions, GPCR–βarr complexes directly allosterically activate C-Raf by binding to its amino terminus. This work provides novel insights into how βarrs regulate effector molecules to activate downstream signaling pathways.     

Rit contributes to nerve growth factor-induced neuronal differentiation via activation of B-Raf-extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades

Rit is one of the original members of a novel Ras GTPase subfamily that uses distinct effector pathways to transform NIH 3T3 cells and induce pheochromocytoma cell (PC6) differentiation. In this study, we find that stimulation of PC6 cells by growth factors, including nerve growth factor (NGF), results in rapid and prolonged Rit activation. Ectopic expression of active Rit promotes PC6 neurite outgrowth that is morphologically distinct from that promoted by oncogenic Ras (evidenced by increased neurite branching) and stimulates activation of both the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase signaling pathways. Furthermore, Rit-induced differentiation is dependent upon both MAP kinase cascades, since MEK inhibition blocked Rit-induced neurite outgrowth, while p38 blockade inhibited neurite elongation and branching but not neurite initiation. Surprisingly, while Rit was unable to stimulate ERK activity in NIH 3T3 cells, it potently activated ERK in PC6 cells. This cell type specificity is explained by the finding that Rit was unable to activate C-Raf, while it bound and stimulated the neuronal Raf isoform, B-Raf. Importantly, selective down-regulation of Rit gene expression in PC6 cells significantly altered NGF-dependent MAP kinase cascade responses, inhibiting both p38 and ERK kinase activation. Moreover, the ability of NGF to promote neuronal differentiation was attenuated by Rit knockdown. Thus, Rit is implicated in a novel pathway of neuronal development and regeneration by coupling specific trophic factor signals to sustained activation of the B-Raf/ERK and p38 MAP kinase cascades.     

Rafs constitute a nodal point in the regulation of embryonic endothelial progenitor cell growth and differentiation

Mouse embryonic endothelial progenitor cells (eEPCs) acquire a mature phenotype after treatment with cyclic adenosine monophosphate (cAMP), suggesting an involvement of Raf serine/threonine kinases in the differentiation process. To test this idea, we investigated the role of B-Raf and C-Raf in proliferation and differentiation of eEPCs by expressing fusion proteins consisting of the kinase domains from Raf molecules and the hormone binding site of the estrogen receptor (ER), or its variant, the tamoxifen receptor. Our findings show that both B- and C-Raf kinase domains, when lacking adjacent regulatory parts, are equally effective in inducing eEPC differentiation. In contrast, the C-Raf kinase domain is a more potent stimulator of eEPC proliferation than B-Raf. In a complimentary approach, we used siRNA silencing to knockdown endogenously expressed B-Raf and C-Raf in eEPCs. In this experimental setting, we found that eEPCs lacking B-Raf failed to differentiate, whereas loss-of C-Raf function primarily slowed cell growth without impairing cAMP-induced differentiation. These findings were further corroborated in B-Raf null eEPCs, isolated from the corresponding knockout embryos, which failed to differentiate in vitro. Thus, gain- and loss-of-function experiments point to distinct roles of B-Raf and C-Raf in regulating growth and differentiation of endothelial progenitor cells, which may harbour therapeutic implications.     

Revealing A-Raf functions through its interactome

A-Raf is a member of the Raf kinase family. Unlike B-Raf and C-Raf, the functions of A-Raf remain obscure. To gain more insight into the biological functions of A-Raf, we investigated the A-Raf interactome using proteomics. We found 132 proteins that interact with A-Raf and confirmed the interaction of 12 of these proteins with A-Raf by western blotting. Our data suggested that A-Raf regulates apoptosis, RNA catabolism, GTPase activity, and cell adhesion by interacting with proteins located in different cellular compartments. We identified all ten hallmarks of cancer in these interacting proteins, suggesting that A-Raf is involved in carcinogenesis. Our results also indicated that A-Raf may play a role in different diseases and signaling pathways. These findings have identified potential regulators of A-Raf and provide a systemic insight into its biological functions.     

Cytokine-induced signaling networks prioritize dynamic range over signal strength

Signaling networks respond to diverse stimuli, but how the state of the signaling network is relayed to downstream cellular responses is unclear. We modeled how incremental activation of signaling molecules is transmitted to control apoptosis as a function of signal strength and dynamic range. A linear relationship between signal input and response output, with the dynamic range of signaling molecules uniformly distributed across activation states, most accurately predicted cellular responses. When nonlinearized signals with compressed dynamic range relay network activation to apoptosis, we observe catastrophic, stimulus-specific prediction failures. We develop a general computational technique, "model-breakpoint analysis," to analyze the mechanism of these failures, identifying new time- and stimulus-specific roles for Akt, ERK, and MK2 kinase activity in apoptosis, which were experimentally verified. Dynamic range is rarely measured in signal-transduction studies, but our experiments using model-breakpoint analysis suggest it may be a greater determinant of cell fate than measured signal strength.     

beta 2-adrenergic receptor activates extracellular signal-regulated kinases (ERKs) via the small G protein rap1 and the serine/threonine kinase B-Raf

G protein-coupled receptors can induce cellular proliferation by stimulating the mitogen-activated protein (MAP) kinase cascade. Heterotrimeric G proteins are composed of both alpha and betagamma subunits that can signal independently to diverse intracellular signaling pathways including those that activate MAP kinases. In this study, we examined the ability of isoproterenol, an agonist of the beta(2)-adrenergic receptor (beta(2)AR), to stimulate extracellular signal-regulated kinases (ERKs). Using HEK293 cells, which express endogenous beta(2)AR, we show that isoproterenol stimulates ERKs via beta(2)AR. This action of isoproterenol requires cAMP-dependent protein kinase and is insensitive to pertussis toxin, suggesting that Galpha(s) activation of cAMP-dependent protein kinase is required. Interestingly, beta(2)AR activates both the small G proteins Rap1 and Ras, but only Rap1 is capable of coupling to Raf isoforms. beta(2)AR inhibits the Ras-dependent activation of both Raf isoforms Raf-1 and B-Raf, whereas Rap1 activation by isoproterenol recruits and activates B-Raf. beta(2)AR activation of ERKs is not blocked by expression of RasN17, an interfering mutant of Ras, but is blocked by expression of either RapN17 or Rap1GAP1, both of which interfere with Rap1 signaling. We propose that isoproterenol can activate ERKs via Rap1 and B-Raf in these cells.     

Phospholipase activity of phospholipase C-gamma1 is required for nerve growth factor-regulated MAP kinase signaling cascade in PC12 cells

Phospholipase C-gamma1 (PLC-gamma1) hydrolyzes phosphatidylinositol 4,5-bisphosphate to the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (DAG). PLC-gamma1 is implicated in a variety of cellular signalings and processes including mitogenesis and calcium entry. However, numerous studies demonstrate that the lipase activity is not required for PLC-gamma1 to mediate these events. Here, we report that the phospholipase activity of PLC-gamma1 plays an essential role in nerve growth factor (NGF)-triggered Raf/MEK/MAPK pathway activation in PC12 cells. Employing PC12 cells stably transfected with an inducible form of wild-type PLC-gamma1 or lipase inactive PLC-gamma1 with histidine 335 mutated into glutamine in the catalytic domain, we show that NGF provokes robust activation of MAP kinase in wild-type but not in lipase inactive cells. Both Ras/C-Raf/MEK1 and Rap1/B-Raf/MEK1 pathways are intact in the wild-type cells. By contrast, these signaling cascades are diminished in the mutant cells. Pretreatment with cell permeable DAG analog 1-oleyl-2-acetylglycerol rescues the MAP kinase pathway activation in the mutant cells. These observations indicate that the lipase activity of PLC-gamma1 mediates NGF-regulated MAPK signaling upstream of Ras/Rap1 activation probably through second messenger DAG-activated Ras and Rap-GEFs.