Unmasking determinants of specificity in the human kinome.

Protein kinases control cellular responses to environmental cues by swift and accurate signal processing. Breakdowns in this high-fidelity capability are a driving force in cancer and other diseases. Thus, our limited understanding of which amino acids in the kinase domain encode substrate specificity, the so-called determinants of specificity (DoS), constitutes a major obstacle in cancer signaling. Here, we systematically discover several DoS and experimentally validate three of them, named the αC1, αC3, and APE-7 residues. We demonstrate that DoS form sparse networks of non-conserved residues spanning distant regions. Our results reveal a likely role for inter-residue allostery in specificity and an evolutionary decoupling of kinase activity and specificity, which appear loaded on independent groups of residues. Finally, we uncover similar properties driving SH2 domain specificity and demonstrate how the identification of DoS can be utilized to elucidate a greater understanding of the role of signaling networks in cancer (Creixell et al., 2015 [this issue of Cell]).

Kinome-wide decoding of network-attacking mutations rewiring cancer signaling.

Cancer cells acquire pathological phenotypes through accumulation of mutations that perturb signaling networks. However, global analysis of these events is currently limited. Here, we identify six types of network-attacking mutations (NAMs), including changes in kinase and SH2 modulation, network rewiring, and the genesis and extinction of phosphorylation sites. We developed a computational platform (ReKINect) to identify NAMs and systematically interpreted the exomes and quantitative (phospho-)proteomes of five ovarian cancer cell lines and the global cancer genome repository. We identified and experimentally validated several NAMs, including PKCγ M501I and PKD1 D665N, which encode specificity switches analogous to the appearance of kinases de novo within the kinome. We discover mutant molecular logic gates, a drift toward phospho-threonine signaling, weakening of phosphorylation motifs, and kinase-inactivating hotspots in cancer. Our method pinpoints functional NAMs, scales with the complexity of cancer genomes and cell signaling, and may enhance our capability to therapeutically target tumor-specific networks.

Structure of a GRK5-Calmodulin Complex Reveals Molecular Mechanism of GRK Activation and Substrate Targeting.

The phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) facilitates arrestin binding and receptor desensitization. Although this process can be regulated by Ca2+-binding proteins such as calmodulin (CaM) and recoverin, the molecular mechanisms are poorly understood. Here, we report structural, computational, and biochemical analysis of a CaM complex with GRK5, revealing how CaM shapes GRK5 response to calcium. The CaM N and C domains bind independently to two helical regions at the GRK5 N and C termini to inhibit GPCR phosphorylation, though only the C domain interaction disrupts GRK5 membrane association, thereby facilitating cytoplasmic translocation. The CaM N domain strongly activates GRK5 via ordering of the amphipathic αN-helix of GRK5 and allosteric disruption of kinase-RH domain interaction for phosphorylation of cytoplasmic GRK5 substrates. These results provide a framework for understanding how two functional effects, GRK5 activation and localization, can cooperate under control of CaM for selective substrate targeting by GRK5.

Linear motif specificity in signaling through p38α and ERK2 mitogen-activated protein kinases.

Mitogen-activated protein kinase (MAPK) cascades are essential for eukaryotic cells to integrate and respond to diverse stimuli. Maintaining specificity in signaling through MAPK networks is key to coupling distinct inputs to appropriate cellular responses. Docking sites-short linear motifs found in MAPK substrates, regulators, and scaffolds-can promote signaling specificity through selective interactions, but how they do so remains unresolved. Here, we screened a proteomic library for sequences interacting with the MAPKs extracellular signal-regulated kinase 2 (ERK2) and p38α, identifying selective and promiscuous docking motifs. Sequences specific for p38α had high net charge and lysine content, and selective binding depended on a pair of acidic residues unique to the p38α docking interface. Finally, we validated a set of full-length proteins harboring docking sites selected in our screens to be authentic MAPK interactors and substrates. This study identifies features that help define MAPK signaling networks and explains how specific docking motifs promote signaling integrity.

Structural Basis for Noncanonical Substrate Recognition of Cofilin/ADF Proteins by LIM Kinases.

Cofilin/actin-depolymerizing factor (ADF) proteins are critical nodes that relay signals from protein kinase cascades to the actin cytoskeleton, in particular through site-specific phosphorylation at residue Ser3. This is important for regulation of the roles of cofilin in severing and stabilizing actin filaments. Consequently, cofilin/ADF Ser3 phosphorylation is tightly controlled as an almost exclusive substrate for LIM kinases. Here we determine the LIMK1:cofilin-1 co-crystal structure. We find an interface that is distinct from canonical kinase-substrate interactions. We validate this previously unobserved mechanism for high-fidelity kinase-substrate recognition by in vitro kinase assays, examination of cofilin phosphorylation in mammalian cells, and functional analysis in S. cerevisiae. The interface is conserved across all LIM kinases. Remarkably, we also observe both pre- and postphosphotransfer states in the same crystal lattice. This study therefore provides a molecular understanding of how kinase-substrate recognition acts as a gatekeeper to regulate actin cytoskeletal dynamics.

Limonoids from the Branches and Leaves of Aglaia lawii and Their Cytotoxic Activities.

Three undescribed limonoids (1-3), named aglaians G-I, and one new natural product azedaralide (4), together with nine known analogues (5-13) were isolated from the branches and leaves of Aglaia lawii by RP C18 column, silica gel column, Sephadex LH-20 column chromatography and preparative HPLC. The structures of the new compounds were elucidated by IR, HRESIMS, 1D, 2D NMR, electronic circular dichroism (ECD) calculations and X-ray crystallography diffraction analysis. The results of bioassay showed that the compound 12 exhibited potential inhibitory activity against six human tumor cell lines (MDA-MB-231, MCF-7, Ln-cap, A549, HeLa and HepG-2) with IC50 values as 8.0-18.6 µM.

Synthesis and in†vitro Anti-Inflammatory Activity of Novel Dendrobine Amide/Sulfonamide Derivatives.

A traditional Chinese medicine ingredient, dendrobine, has been demonstrated to have anti-inflammatory properties. However, due to its poor anti-inflammatory properties, its clinical use is limited. Consequently, we have designed and synthesized 32 new amide/sulfonamide dendrobine derivatives and screened their anti-inflammatory activities in vitro. Experiments showed that nitric oxide (NO) generation in lipopolysaccharide (LPS)-induced RAW264.7 cells was strongly reduced by derivative 14, with an IC50 of 2.96 µM. Western blot research revealed that 14 decreased the concentration-dependent expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (INOS). Molecular docking was used to predict the binding of the inflammation-associated proteins COX-2 and INOS to compound 14.

Structurally Diverse Coumarins from Peucedanum praeruptorum and their Anti-Inflammatory Activities via NF-κB Signaling Pathway.

The phytochemical study of Peucedanum praeruptorum led to the isolation of twenty-five coumarins (1-25). Of which, (±) praeruptol A (±1), one pair of previous undescribed seco-coumarin enantiomers were obtained. Their structures were established according to HR-ESI-MS, NMR, X-ray single crystal diffraction analysis, as well as ECD calculation. All compounds were tested for anti-inflammatory activity in the RAW264.7 macrophage model, and eight compounds (7-10, and 13-16) exhibited significant inhibitory effects with IC50 values ranging from 9.48 to 34.66 µM. Among them, compound 7 showed the strongest inhibitory effect, which significantly suppressed the production of IL-6, IL-1ß, and TNF-α, as well as iNOS and COX-2 in a concentration-dependent manner. Further investigated results showed that compound 7 exerted an anti-inflammatory effect via the NF-κB signaling pathway.

Small Molecule Inhibition of the Autophagy Kinase ULK1 and Identification of ULK1 Substrates.

Many tumors become addicted to autophagy for survival, suggesting inhibition of autophagy as a potential broadly applicable cancer therapy. ULK1/Atg1 is the only serine/threonine kinase in the core autophagy pathway and thus represents an excellent drug target. Despite recent advances in the understanding of ULK1 activation by nutrient deprivation, how ULK1 promotes autophagy remains poorly understood. Here, we screened degenerate peptide libraries to deduce the optimal ULK1 substrate motif and discovered 15 phosphorylation sites in core autophagy proteins that were verified as in vivo ULK1 targets. We utilized these ULK1 substrates to perform a cell-based screen to identify and characterize a potent ULK1 small molecule inhibitor. The compound SBI-0206965 is a highly selective ULK1 kinase inhibitor in vitro and suppressed ULK1-mediated phosphorylation events in cells, regulating autophagy and cell survival. SBI-0206965 greatly synergized with mechanistic target of rapamycin (mTOR) inhibitors to kill tumor cells, providing a strong rationale for their combined use in the clinic.

Comprehensive profiling of the STE20 kinase family defines features essential for selective substrate targeting and signaling output.

Specificity within protein kinase signaling cascades is determined by direct and indirect interactions between kinases and their substrates. While the impact of localization and recruitment on kinase-substrate targeting can be readily assessed, evaluating the relative importance of direct phosphorylation site interactions remains challenging. In this study, we examine the STE20 family of protein serine-threonine kinases to investigate basic mechanisms of substrate targeting. We used peptide arrays to define the phosphorylation site specificity for the majority of STE20 kinases and categorized them into four distinct groups. Using structure-guided mutagenesis, we identified key specificity-determining residues within the kinase catalytic cleft, including an unappreciated role for the kinase ß3-αC loop region in controlling specificity. Exchanging key residues between the STE20 kinases p21-activated kinase 4 (PAK4) and Mammalian sterile 20 kinase 4 (MST4) largely interconverted their phosphorylation site preferences. In cells, a reprogrammed PAK4 mutant, engineered to recognize MST substrates, failed to phosphorylate PAK4 substrates or to mediate remodeling of the actin cytoskeleton. In contrast, this mutant could rescue signaling through the Hippo pathway in cells lacking multiple MST kinases. These observations formally demonstrate the importance of catalytic site specificity for directing protein kinase signal transduction pathways. Our findings further suggest that phosphorylation site specificity is both necessary and sufficient to mediate distinct signaling outputs of STE20 kinases and imply broad applicability to other kinase signaling systems.

An AMPK-independent signaling pathway downstream of the LKB1 tumor suppressor controls Snail1 and metastatic potential.

The serine/threonine kinase LKB1 is a tumor suppressor whose loss is associated with increased metastatic potential. In an effort to define biochemical signatures of metastasis associated with LKB1 loss, we discovered that the epithelial-to-mesenchymal transition transcription factor Snail1 was uniquely upregulated upon LKB1 deficiency across cell types. The ability of LKB1 to suppress Snail1 levels was independent of AMPK but required the related kinases MARK1 and MARK4. In a screen for substrates of these kinases involved in Snail regulation, we identified the scaffolding protein DIXDC1. Similar to loss of LKB1, DIXDC1 depletion results in upregulation of Snail1 in a FAK-dependent manner, leading to increased cell invasion. MARK1 phosphorylation of DIXDC1 is required for its localization to focal adhesions and ability to suppress metastasis in mice. DIXDC1 is frequently downregulated in human cancers, which correlates with poor survival. This study defines an AMPK-independent phosphorylation cascade essential for LKB1-dependent control of metastatic behavior.

Identification of a major determinant for serine-threonine kinase phosphoacceptor specificity.

Eukaryotic protein kinases are generally classified as being either tyrosine or serine-threonine specific. Though not evident from inspection of their primary sequences, many serine-threonine kinases display a significant preference for serine or threonine as the phosphoacceptor residue. Here we show that a residue located in the kinase activation segment, which we term the "DFG+1" residue, acts as a major determinant for serine-threonine phosphorylation site specificity. Mutation of this residue was sufficient to switch the phosphorylation site preference for multiple kinases, including the serine-specific kinase PAK4 and the threonine-specific kinase MST4. Kinetic analysis of peptide substrate phosphorylation and crystal structures of PAK4-peptide complexes suggested that phosphoacceptor residue preference is not mediated by stronger binding of the favored substrate. Rather, favored kinase-phosphoacceptor combinations likely promote a conformation optimal for catalysis. Understanding the rules governing kinase phosphoacceptor preference allows kinases to be classified as serine or threonine specific based on their sequence.

Analysis of the screening results of 24040 potential sperm donors in a human sperm bank in Henan Province, China: a 14-year retrospective cohort study.

STUDY QUESTION: Is there a relation between the characteristics of potential sperm donors and the acceptance rate of these potential donors? SUMMARY ANSWER: A relatively higher acceptance rate was observed for potential sperm donors who were aged ≤ 35 years, were married, had children, and who had received higher education, and acceptance rates were also higher during spring and winter than summer and autumn. WHAT IS KNOWN ALREADY: Recruiting donors to a sperm bank program is difficult and slow owing to the high rates of rejection and dropout. STUDY DESIGN, SIZE, DURATION: A total of 24040 potential sperm donors were screened by the Henan Human Sperm Bank from 2006 to 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: Potential sperm donors were recruited using the following baseline requirement: height of 168 cm or taller; age 22-45 years; currently attending or had graduated from high school or above. Men who met the criteria for age, height, and education level were invited for semen quality screening. The acceptable criteria for semen samples included liquefaction time < 60min, volume ≥ 2mL, sperm concentration ≥ 60 × 106/mL, progressive motility ≥ 60%, post-thaw motility ≥ 40%, pre-freezing total motile sperm per vial > 30 × 106/mL, post-thaw total motile sperm per vial > 12 × 106/mL, and freeze-thaw survival rate ≥ 60%. Any potential sperm donors meeting the minimum criteria for acceptable semen quality on two consecutive semen samples were scheduled for clinical assessment, physical examination, and laboratory tests. The reasons for sperm donor rejection were analyzed. The characteristics of accepted and rejected donors were compared using the chi-square test, and multivariate logistic regression analyses were conducted to identify factors associated with the acceptance rate of potential sperm donors and the positive rate of sexually transmitted diseases (STDs). MAIN RESULTS AND THE ROLE OF CHANCE: Only 23.38% (5620/24040) of potential sperm donors were accepted. The top four reasons for rejection were suboptimal semen quality (90.27%), STDs (6.26%), dropped out (2.65%), and chromosomal abnormalities (0.35%). The most common reason for the rejection of donors with an STD was a positive test for mycoplasmas (49.05%), followed by hepatitis B virus (27.56%), Chlamydia trachomatis (4.68%), and Escherichia coli (3.03%). n this study, the acceptance rate for men aged ≤ 35 years was significantly higher than that for men aged >35 years (P < 0.05). The acceptance rates were also significantly higher for men with a higher education than for men with lower education, married men than unmarried men, and men with children than men without children (P < 0.05). Moreover, acceptance rates were significantly higher during spring and winter than during summer (P <0.05) but were not significantly higher during autumn than during summer (P >0.05). LIMITATIONS, REASONS FOR CAUTION: This study was not performed to analyze the effect of lifestyle habits, such as alcohol consumption and cigarette smoking, on the acceptance rate of potential sperm donors. WIDER IMPLICATIONS OF THE FINDINGS: Only a small proportion of potential sperm donors were accepted in this anonymous sperm donor program. New strategies for sperm donor recruitment may be required to improve the acceptance rate. In the future, we may have to target potential sperm donors who are aged ≤ 35 years and who received higher education in order to improve the acceptance rate. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Joint Construction Project of Henan Medical Science and Technology Research Plan under grant number LHGJ20190389. The authors report no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Anti-tobacco mosaic virus (TMV) activity of chemical constituents from the seeds of Sophora tonkinensis.

Two new compounds, including one new arylbenzofuran (1) and one new pterocarpanoid (2), along with nine known ones, were isolated from the seeds of Sophora tonkinensis. The structures of the new compounds were elucidated based on a comprehensive spectroscopic data analysis. Compounds 2 and 3 exhibited good anti-tobacco mosaic virus (TMV) activities with the protective inhibition rate of 69.62% and 68.72% respectively, at concentration of 100 µg/ml.

Polycyclic polyprenylated acylphloroglucinol derivatives with neuroprotective effects from Hypericum monogynum.

A new polycyclic polyprenylated acylphloroglucinol (PPAP), hypermonin C (1), along with nine known PPAPs (2-10) were obtained from the leaves and twigs of Hypericum monogynum. The structures of the isolates were determined on the basis of extensive spectroscopic analysis. The neuroprotective effects of the isolates against several chemical-induced injuries in SH-SY5Y and PC12 cells were assessed, and most of the compounds exhibited significant protective effects at 10 µg/ml. Especially, three compounds (1, 3, and 7) showed excellent neuroprotective activity with a cell viability of 92.4% ∼ 95.8% in KCl-induced SH-SY5Y cell injury. Their preliminary structure-activity relationship was also discussed and the configuration of substituent in furohyperforin may be critical for the neuroprotective activity of PPAP derivatives.

The p97-UBXD8 complex destabilizes mRNA by promoting release of ubiquitinated HuR from mRNP.

The assembly and disassembly of ribonucleoproteins (RNPs) are dynamic processes that control every step of RNA metabolism, including mRNA stability. However, our knowledge of how RNP remodeling is achieved is largely limited to RNA helicase functions. Here, we report a previously unknown mechanism that implicates the ATPase p97, a protein-remodeling machine, in the dynamic regulation of mRNP disassembly. We found that p97 and its cofactor, UBXD8, destabilize p21, MKP-1, and SIRT1, three established mRNA targets of the RNA-binding protein HuR, by promoting release of HuR from mRNA. Importantly, ubiquitination of HuR with a short K29 chain serves as the signal for release. When cells are subjected to stress conditions, the steady-state levels of HuR ubiquitination change, suggesting a new mechanism through which HuR mediates the stress response. Our studies reveal a new paradigm in RNA biology: nondegradative ubiquitin signaling-dependent disassembly of mRNP promoted by the p97-UBXD8 complex to control mRNA stability.

Does the sex ratio of singleton births after frozen single blastocyst transfer differ in relation to blastocyst development?

PURPOSE: To investigate the associations between blastocyst development and the sex ratio (male:female) among singleton live births resulting from single-blastocyst frozen embryo transfer (FET) cycles. METHODS: Patients with singleton live births following the first autologous single FET of non- preimplantation genetic testing (PGT) blastocysts in a single reproductive medicine department between January 2015 and February 2019 were included in this retrospective study. The primary outcome measure was the singleton sex ratio. Multivariable logistic regression models were used to estimate the associations between blastocyst quality and singleton sex ratio after adjustment for some potential confounders. RESULTS: There were 638 high-quality and 572 poor-quality single blastocyst FETs, and the blastocysts were conceived via 855 IVF and 355 ICSI treatments. A total of 1210 singleton live births were assessed. High-quality single blastocyst FET resulted in a significantly higher sex ratio than did poor-quality single blastocyst FET (60% vs. 49.7%, P < 0.001). The infertility cause was not associated with sex ratio among singleton live births (P = 0.537). The results of a multivariate analysis revealed that a high-quality blastocyst has a 150% higher probability of being male than a poor-quality blastocyst (adjusted odds ratio (aOR) 1.57; 95% CI 1.24-2, P < 0.001). Among the three blastocyst morphological parameters, Grade B trophectoderm was significantly associated with a higher sex ratio than Grade C (aOR 1.71; 95% CI 1.33-2.21. P < 0.001). Neither expansion degree nor inner cell mass degree were significantly associated with the singleton sex ratio. CONCLUSIONS: A single high-quality blastocyst FET has a higher chance of resulting in a male infant than a female infant. The results demonstrate that grade B trophectoderm confers benefits in improving the implantation potential of male blastocysts.

Oxyresveratrol Inhibits IL-1ß-Induced Inflammation via Suppressing AKT and ERK1/2 Activation in Human Microglia, HMC3.

Oxyresveratrol (OXY), a major phytochemical component derived from several plants, has been proved to have several pharmacological properties. However, the role of OXY in regulating neuroinflammation is still unclear. Here, we focused mainly on the anti-neuroinflammatory effects at the cellular level of OXY in the interleukin-1 beta (IL-1ß)-stimulated HMC3 human microglial cell line. We demonstrated that OXY strongly decreased the release of IL-6 and MCP-1 from HMC3 cells stimulated with IL-1ß. Nevertheless, IL-1ß could not induce the secretion of TNF-α and CXCL10 in this specific cell line, and that OXY did not have any effects on reducing the basal level of these cytokines in the sample culture supernatants. The densitometry analysis of immunoreactive bands from Western blot clearly indicated that IL-1ß does not trigger the nuclear factor-kappa B (NF-κB) signaling. We discovered that OXY exerted its anti-inflammatory role in IL-1ß-induced HMC3 cells by suppressing IL-1ß-induced activation of the PI3K/AKT/p70S6K pathway. Explicitly, the presence of OXY for only 4 h could strongly inhibit AKT phosphorylation. In addition, OXY had moderate effects on inhibiting the activation of ERK1/2. Results from immunofluorescence study further confirmed that OXY inhibited the phosphorylation of AKT and ERK1/2 MAPK upon IL-1ß stimulation in individual cells. These findings suggest that the possible anti-inflammatory mechanisms of OXY in IL-1ß-induced HMC3 cells are mainly through its ability to suppress the PI3K/AKT/p70S6K and ERK1/2 MAPK signal transduction cascades. In conclusion, our study provided accumulated data that OXY is able to suppress IL-1ß stimulation signaling in human microglial cells, and we believe that OXY could be a probable pharmacologic agent for altering microglial function in the treatment of neuroinflammation.

Substrate priming enhances phosphorylation by the budding yeast kinases Kin1 and Kin2.

Multisite phosphorylation of proteins is a common mechanism for signal integration and amplification in eukaryotic signaling networks. Proteins are commonly phosphorylated at multiple sites in an ordered manner, whereby phosphorylation by one kinase primes the substrate by generating a recognition motif for a second kinase. Here we show that substrate priming promotes phosphorylation by Saccharomyces cerevisiae Kin1 and Kin2, kinases that regulate cell polarity, exocytosis, and the endoplasmic reticulum (ER) stress response. Kin1/Kin2 phosphorylated substrates within the context of a sequence motif distinct from those of their most closely related kinases. In particular, the rate of phosphorylation of a peptide substrate by Kin1/Kin2 increased >30-fold with incorporation of a phosphoserine residue two residues downstream of the phosphorylation site. Recognition of phosphorylated substrates by Kin1/Kin2 was mediated by a patch of basic residues located in the region of the kinase αC helix. We identified a set of candidate Kin1/Kin2 substrates reported to be dually phosphorylated at sites conforming to the Kin1/Kin2 consensus sequence. One of these proteins, the t-SNARE protein Sec9, was confirmed to be a Kin1/Kin2 substrate both in vitro and in vivo Sec9 phosphorylation by Kin1 in vitro was enhanced by prior phosphorylation at the +2 position. Recognition of primed substrates was not required for the ability of Kin2 to suppress the growth defect of secretory pathway mutants but was necessary for optimal growth under conditions of ER stress. These results suggest that at least some endogenous protein substrates of Kin1/Kin2 are phosphorylated in a priming-dependent manner.

Neurofibromatosis type 1 alternative splicing is a key regulator of Ras/ERK signaling and learning behaviors in mice.

Appropriate activation of the Ras/extracellular signal-regulated kinase (ERK) protein signaling cascade within the brain is crucial for optimal learning and memory. One key regulator of this cascade is the Nf1 Ras GTPase activating protein (RasGAP), which attenuates Ras/ERK signaling by converting active Ras is bound to guanosine triphosphate, activating Ras into inactive Ras is bound to guanosine diphosphate, inactivating Ras. A previous study using embryonic stem cells and embryonic stem cell-derived neurons indicated that Nf1 RasGAP activity is modulated by the highly regulated alternative splicing of Nf1 exon 23a. In this study, we generated Nf123aIN/23aIN mice, in which the splicing signals surrounding Nf1 exon 23a were manipulated to increase exon inclusion. Nf123aIN/23aIN mice are viable and exon 23a inclusion approaches 100% in all tissues, including the brain, where the exon is normally almost completely skipped. Ras activation and phosphorylation of ERK1/2 downstream of Ras are both greatly increased in Nf123aIN/23aIN mouse brain lysates, confirming that exon 23a inclusion inhibits Nf1 RasGAP activity in vivo as it does in cultured cells. Consistent with the finding of altered Ras/ERK signaling in the brain, Nf123aIN/23aIN mice showed specific deficits in learning and memory compared with Nf1+/+ mice. Nf123aIN/23aIN mice performed poorly on the T-maze and Morris water maze tests, which measure short- and long-term spatial memory, respectively. In addition, Nf123aIN/23aIN mice showed abnormally elevated context-dependent fear and a diminished ability to extinguish a cued fear response, indicating defective associative fear learning. Therefore, the regulated alternative splicing of Nf1 is an important mechanism for fine-tuning Ras/ERK signaling as well as learning and memory in mice.