Daniel A. Ritt

Impact of feedback phosphorylation and Raf heterodimerization on normal and mutant B-Raf signaling.

ABSTRACT 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.

Signaling dynamics of the KSR1 scaffold complex

Scaffold proteins contribute to the spatiotemporal control of MAPK signaling and KSR1 is an ERK cascade scaffold that localizes to the plasma membrane in response to growth factor treatment. To better understand the molecular mechanisms of KSR1 function, we examined the interaction of KSR1 with each of the ERK cascade components, Raf, MEK, and ERK. Here, we identify a hydrophobic motif within the proline-rich sequence (PRS) of MEK1 and MEK2 that is required for constitutive binding to KSR1 and find that MEK binding and residues in the KSR1 CA1 region enable KSR1 to form a ternary complex with B-Raf and MEK following growth factor treatment that enhances MEK activation. We also find that docking of active ERK to the KSR1 scaffold allows ERK to phosphorylate KSR1 and B-Raf on feedback S/TP sites. Strikingly, feedback phosphorylation of KSR1 and B-Raf promote their dissociation and result in the release of KSR1 from the plasma membrane. Together, these findings provide unique insight into the signaling dynamics of the KSR1 scaffold and reveal that through regulated interactions with Raf and ERK, KSR1 acts to both potentiate and attenuate ERK cascade activation, thus regulating the intensity and duration of ERK cascade signaling emanating from the plasma membrane during growth factor signaling.

Effects of Raf Dimerization and its Inhibition on Normal and Disease-associated Raf Signaling

Raf kinases are essential for normal Ras-Raf-MEK-ERK pathway signaling, and activating mutations in components of this pathway are associated with a variety of human cancers, as well as the related developmental disorders Noonan, LEOPARD, and cardiofaciocutaneous syndromes. Although the Raf kinases are known to dimerize during normal and disease-associated Raf signaling, the functional significance of Raf dimerization has not been fully elucidated. Here, using mutational analysis and a peptide inhibitor, we show that dimerization is required for normal Ras-dependent Raf activation and for the biological function of disease-associated Raf mutants with moderate, low, or impaired kinase activity. However, dimerization is not needed for the function of B-Raf mutants with high catalytic activity, such as V600E-B-Raf. Importantly, we find that a dimer interface peptide can effectively block Raf dimerization and inhibit Raf signaling when dimerization is required for Raf function, thus identifying the Raf dimer interface as a therapeutic target.

KSR2 Is a Calcineurin Substrate that Promotes ERK Cascade Activation in Response to Calcium Signals

Protein scaffolds have emerged as important regulators of MAPK cascades, facilitating kinase activation and providing crucial spatio/temporal control to their signaling outputs. Using a proteomics approach to compare the binding partners of the two mammalian KSR scaffolds, we find that both KSR1 and KSR2 interact with the kinase components of the ERK cascade and have a common function in promoting RTK-mediated ERK signaling. Strikingly, we find that the protein phosphatase calcineurin selectively interacts with KSR2 and that KSR2 uniquely contributes to Ca2+-mediated ERK signaling. Calcineurin dephosphorylates KSR2 on specific sites in response to Ca2+ signals, thus regulating KSR2 localization and activity. Moreover, we find that depletion of endogenous KSR2 impairs Ca2+-mediated ERK activation and ERK-dependent signaling responses in INS1 pancreatic beta-cells and NG108 neuroblastoma cells. These findings identify KSR2 as a Ca2+-regulated ERK scaffold and reveal a new mechanism whereby Ca2+ impacts Ras to ERK pathway signaling.

CK2 Is a component of the KSR1 scaffold complex that contributes to Raf kinase activation.

Kinase Suppressor of Ras (KSR) is a molecular scaffold that interacts with the core kinase components of the ERK cascade, Raf, MEK, and ERK and provides spatial and temporal regulation of Ras-dependent ERK cascade signaling. In this report, we identify the heterotetrameric protein kinase, casein kinase 2 (CK2), as a new KSR1-binding partner. Moreover, we find that the KSR1/CK2 interaction is required for KSR1 to maximally facilitate ERK cascade signaling and contributes to the regulation of Raf kinase activity. Binding of the CK2 holoenzyme is constitutive and requires the basic surface region of the KSR1 atypical C1 domain. Loss of CK2 binding does not alter the membrane translocation of KSR1 or its interaction with ERK cascade components; however, disruption of the KSR1/CK2 interaction or inhibition of CK2 activity significantly reduces the growth-factor-induced phosphorylation of C-Raf and B-Raf on the activating serine site in the negative-charge regulatory region (N-region). This decrease in Raf N-region phosphorylation further correlates with impaired Raf, MEK, and ERK activation. These findings identify CK2 as a novel component of the KSR1 scaffolding complex that facilitates ERK cascade signaling by functioning as a Raf family N-Region kinase.

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.

Functional analysis of C‐TAK1 substrate binding and identification of PKP2 as a new C‐TAK1 substrate

Cdc25C‐associated kinase 1 (C‐TAK1) has been implicated in cell cycle regulation and Ras signaling through its interactions with two putative substrates, the Cdc25C phosphatase and the MAPK scaffold KSR1. Here, we identify sequence motifs required for stable C‐TAK1 association and substrate phosphorylation. Using a mutational approach to disrupt binding of C‐TAK1 to KSR1 and Cdc25C, we demonstrate that C‐TAK1 contributes to the regulation of these proteins in vivo through the generation of 14‐3‐3‐binding sites. KSR1 proteins defective in C‐TAK1 binding had severely reduced phosphorylation at the 14‐3‐3‐binding site in vivo, were constitutively localized to the plasma membrane and had increased biological activity. Disruption of the Cdc25C–C‐TAK1 interaction resulted in reduced 14‐3‐3‐binding site phosphorylation and nuclear accumulation of Cdc25C in interphase cells. Finally, utilizing the acquired C‐TAK1 binding and substrate phosphorylation data, we identify plakophilin 2 (PKP2) as a novel C‐TAK1 substrate. Phosphorylation of PKP2 by C‐TAK1 also generates a 14‐3‐3‐binding site that influences PKP2 localization. These findings underscore the importance of C‐TAK1 as a regulator of 14‐3‐3 binding and protein localization.

The importance of Raf dimerization in cell signaling.

The Raf family of protein kinases are key signaling intermediates, acting as a central link between the membrane-bound Ras GTPases and the downstream kinases MEK and ERK. Raf kinase regulation is well-known for its complexity but only recently has it been realized that many of the mechanisms involved in Raf regulation also modulate Raf dimerization, now acknowledged to be a required step for Raf signaling in multiple cellular contexts. Recent studies have shown that Raf dimerization is necessary for normal Ras-dependent Raf kinase activation and contributes to the pathogenic function of disease-associated mutant Raf proteins with all but high intrinsic kinase activity. Raf dimerization has also been found to alter therapeutic responses and disease progression in patients treated with ATP-competitive Raf inhibitors as well as certain other kinase-targeted drugs. This demonstration of clinical significance has stimulated the recent development of biosensor assays that can monitor inhibitor-induced Raf dimerization as well as studies demonstrating the therapeutic potential of blocking Raf dimerization.

The CNK2 scaffold interacts with vilse and modulates Rac cycling during spine morphogenesis in hippocampal neurons.

Protein scaffolds play an important role in signal transduction, functioning to facilitate protein interactions and localize key pathway components to specific signaling sites. Connector enhancer of KSR-2 (CNK2) is a neuronally expressed scaffold recently implicated in nonsyndromic, X-linked intellectual disability (NS-XLID) [1-3]. NS-XLID patients have deficits in cognitive function and their neurons often exhibit dendritic spine abnormalities [4], suggesting a role for CNK2 in synaptic signaling and/or spine formation. To gain insight regarding how CNK2 might contribute to these processes, we used mass spectrometry to identify proteins that interact with the endogenous CNK2 scaffold. Here, we report that the major binding partner of CNK2 is Vilse/ARHGAP39 and that CNK2 complexes are enriched for proteins involved in Rac/Cdc42 signaling, including Rac1 itself, α-PIX and β-PIX, GIT1 and GIT2, PAK3 and PAK4, and members of the cytohesin family. Binding between CNK2 and Vilse was found to be constitutive, mediated by the WW domains of Vilse and a proline motif in CNK2. Through mutant analysis, protein depletion and rescue experiments, we identify CNK2 as a spatial modulator of Rac cycling during spine morphogenesis and find that the interaction with Vilse is critical for maintaining RacGDP/GTP levels at a balance required for spine formation.

A CC-SAM, for coiled coil-sterile α motif, domain targets the scaffold KSR-1 to specific sites in the plasma membrane.

A previously unknown module that mediates membrane binding is identified in the scaffold KSR. Another Helping Hand to the Membrane Growth factor stimulation activates the mitogen-activated protein kinase module, in which Raf activates MEK, which in turn activates ERK. Kinase suppressor of Ras-1 (KSR-1) requires a phospholipid-binding atypical C1 domain to associate with the plasma membrane to promote assembly of this module. Koveal et al. identified a sterile α motif (SAM) adjacent to a coiled coil (CC) domain in KSR-1. Structural and biochemical analysis of just the CC and SAM regions showed that CC-SAM formed a single, structural module. CC-SAM bound directly to micelles and bicelles in vitro and was targeted to the plasma membrane in growth factor–treated cells. Structure-based point mutations in helix α3 of the CC motif prevented the binding of CC-SAM to micelles and bicelles. Full-length KSR-1 with these mutations did not localize to the plasma membrane or interact with B-Raf when expressed in growth factor–treated cells. Thus, KSR-1 requires the atypical C1 and CC-SAM domains to associate with the plasma membrane. Kinase suppressor of Ras-1 (KSR-1) is an essential scaffolding protein that coordinates the assembly of the mitogen-activated protein kinase (MAPK) module, consisting of the MAPK kinase kinase Raf, the MAPK kinase MEK (mitogen-activated or extracellular signal–regulated protein kinase kinase), and the MAPK ERK (extracellular signal–regulated kinase) to facilitate activation of MEK and thus ERK. Although KSR-1 is targeted to the cell membrane in part by its atypical C1 domain, which binds to phospholipids, other domains may be involved. We identified another domain in KSR-1 that we termed CC-SAM, which is composed of a coiled coil (CC) and a sterile α motif (SAM). The CC-SAM domain targeted KSR-1 to specific signaling sites at the plasma membrane in growth factor–treated cells, and it bound directly to various micelles and bicelles in vitro, indicating that the CC-SAM functioned as a membrane-binding module. By combining nuclear magnetic resonance spectroscopy and experiments in cultured cells, we found that membrane binding was mediated by helix α3 of the CC motif and that mutating residues in α3 abolished targeting of KSR-1 to the plasma membrane. Thus, in addition to the atypical C1 domain, the CC-SAM domain is required to target KSR-1 to the plasma membrane.