Molecular determinants of signal transduction in tropomyosin receptor kinases.

Cell Survival through Trk Neurotrophin Receptors Is Differentially Regulated by Ubiquitination

Endocytosis of Trk (tropomyosin-related kinase) receptors is critical for neurotrophin signal transduction and biological functions. However, the mechanism governing endocytosis of TrkB (tropomyosin-related kinase B) and the specific contributions of TrkB endocytosis to downstream signaling are unknown. In this study, we report that blocking clathrin, dynamin, or AP2 in cultured neurons of the central nervous system inhibited brain-derived neurotrophic factor (BDNF)-induced activation of Akt but not ERK. Treating neurons with the clathrin inhibitor monodansylcadaverine or a peptide that blocks dynamin function specifically abrogated Akt pathway activation in response to BDNF but did not affect the response of other downstream effectors or the up-regulation of immediate early genes neuropeptide Y and activity-regulated cytoskeleton-associated protein. Similar effects were found in neurons expressing small interfering RNA to silence AP2 or a dominant negative form of dynamin that inhibits clathrin-mediated endocytosis. In PC12 cells, ERK but not Akt activation required TrkA endocytosis following stimulation with nerve growth factor, whereas the opposite was true when TrkA-expressing neurons were stimulated with nerve growth factor in the central nervous system. Thus, the specific effects of internalized Trk receptors probably depend on the presence of cell type-specific modulators of neurotrophin signaling and not on differences inherent to Trk receptors themselves. Endocytosis-dependent activation of Akt in neurons was found to be critical for BDNF-supported survival and dendrite outgrowth. Together, these results demonstrate the functional requirement of clathrin- and dynamin-dependent endocytosis in generating the full intracellular response of neurons to BDNF in the central nervous system.

Neurotrophins, such as nerve growth factor and brain-derived neurotrophic factor, activate Trk receptor tyrosine kinases through receptor dimerization at the cell surface followed by autophosphorylation and recruitment of intracellular signaling molecules. The intracellular pathways used by neurotrophins share many common protein substrates that are used by other receptor tyrosine kinases (RTK), such as Shc, Grb2, FRS2, and phospholipase C-gamma. Here we describe a novel RTK mechanism that involves a 220-kilodalton membrane tetraspanning protein, ARMS/Kidins220, which is rapidly tyrosine phosphorylated in primary neurons after neurotrophin treatment. ARMS/Kidins220 undergoes multiple tyrosine phosphorylation events and also serine phosphorylation by protein kinase D. We have identified a single tyrosine (Tyr(1096)) phosphorylation event in ARMS/Kidins220 that plays a critical role in neurotrophin signaling. A reassembled complex of ARMS/Kidins220 and CrkL, an upstream component of the C3G-Rap1-MAP kinase cascade, is SH3-dependent. However, Tyr(1096) phosphorylation enables ARMS/Kidins220 to recruit CrkL through its SH2 domain, thereby freeing the CrkL SH3 domain to engage C3G for MAP kinase activation in a neurotrophin dependent manner. Accordingly, mutation of Tyr(1096) abolished CrkL interaction and sustained MAPK kinase activity, a response that is not normally observed in other RTKs. Therefore, Trk receptor signaling involves an inducible switch mechanism through an unconventional substrate that distinguishes neurotrophin action from other growth factor receptors.

Aminoglycoside-Induced Degeneration of Adult Spiral Ganglion Neurons Involves Differential Modulation of Tyrosine Kinase B and p75 Neurotrophin Receptor Signaling

PROTAC Compounds Targeting TRK for Use in Cancer Therapeutics.

Specificity in Trk Receptor:Neurotrophin Interactions: The Crystal Structure of TrkB-d5 in Complex with Neurotrophin-4/5

We have established that docosahexaenoic acid (DHA), the major polyunsaturated fatty acid in the retina, promotes survival of rat retina photoreceptors during early development in vitro and upon oxidative stress by activating the ERK/MAPK signaling pathway. Here we have investigated whether DHA turns on this pathway through activation of retinoid X receptors (RXRs) or by inducing tyrosine kinase (Trk) receptor activation. We also evaluated whether DHA release from phospholipids was required for its protective effect. Addition of RXR antagonists (HX531, PA452) to rat retinal neuronal cultures inhibited DHA protection during early development in vitro and upon oxidative stress induced with Paraquat or H2O2. In contrast, the Trk inhibitor K252a did not affect DHA prevention of photoreceptor apoptosis. These results imply that activation of RXRs was required for DHA protection whereas Trk receptors were not involved in this protection. Pretreatment with 4-bromoenol lactone, a phospholipase A2 inhibitor, blocked DHA prevention of oxidative stress-induced apoptosis of photoreceptors. It is noteworthy that RXR agonists (HX630, PA024) also rescued photoreceptors from H2O2-induced apoptosis. These results provide the first evidence that activation of RXRs prevents photoreceptor apoptosis and suggest that DHA is first released from phospholipids and then activates RXRs to promote the survival of photoreceptors.

Nerve growth factor belongs to a small family of proteins whose binding at the Trk and p75(NTR) transmembrane receptors triggers a cascade of signaling events that give rise to neurotrophic responses in neuronal cells and in vivo. Following their robust effects in animal models of neurodegeneration, neurotrophins have been evaluated for therapy for several human neurodegenerative diseases. However, due mainly to the poor pharmacokinetic behavior of these proteins, they have largely met without success in the clinic, making it desirable to develop small molecule neurotrophin mimetics. A range of compounds is described that achieves some of the neurotrophic and neuroprotective effects attributed to neurotrophins through a variety of mechanisms. These small molecules are divided into the following functional categories: (1). compounds that activate Trk receptors directly; (2). compounds that potentiate the actions of neurotrophins on Trk receptors; (3). compounds that activate Trk indirectly; (4). compounds that influence neurotrophin expression or secretion; and (5). a broad class of compounds that act downstream of, or independently of, Trk receptors. Unfortunately, most of the compounds that have been reported suffer from either lack of specificity for the desired mechanism/effect(s) or lack of efficacy of the compounds in appropriate in vivo models, or both. This second limitation has been particularly severe for compounds designed to mimic the neurotrophins in their interaction with Trk receptors, an ongoing and formidable challenge. Nevertheless, a small subset of the compounds, acting on intracellular signaling pathways downstream of Trk receptors, shows promise for the future treatment of neurodegenerative diseases.

The TrkA and TrkB tyrosine kinases are members of the neurotrophin receptor family and mediate survival, differentiation, growth, and apoptosis of neurons in response to stimulation by their ligands, NGF and BDNF, respectively. Expression levels of TrkA/TrkB are important prognostic factors in a variety of embryonal tumors including neuroblastoma, the most common solid tumor of childhood. Because TrkA/TrkB exhibit a high level of sequence similarity and use overlapping pathways for signal transduction, the existence of specific effector molecules crucial for receptor and cell-type-specific response is likely. To identify these effectors by analyzing biological effects of TrkA and TrkB activation in a defined model, we performed a proteome study using the human neuroblastoma SY5Y cell line stably transfected with the TrkA or TrkB cDNA. The use of the recently introduced DIGE (fluorescence two-dimensional difference gel electrophoresis) system (Amersham Biosciences, Piscataway, NJ) allowed us to monitor differences in protein expression between samples in one gel. Proteomic changes were monitored in a time course of 0, 0.5, 1, 6, and 24 h following receptor activation. Using MALDI mass spectrometry, we identified, respectively, 22 and 9 differentially expressed proteins upon the addition of neurotrophin in SY5Y-TrkB and SY5Y-TrkA cells. Functional assignment revealed that the majority of these proteins are involved in organization and maintenance of cellular structures.

The tropomyosin-related tyrosine kinases or neurotrophic tyrosine kinase receptors are a group of tyrosine kinases that play a crucial role in regulating neuronal growth and development. Neurotrophins are a class of protein-secreting cells that serve as the primary ligand for the Trk receptors. The four primary neurotrophins are nerve growth factor (NGF), brain-derived nerve factor (BDNF), neurotrophin-3, and neurotrophin-4/5. Mounting evidence suggests that Trk receptors can be categorized into three types: TrkA, TrkB, and TrkC. These receptors play a crucial role in facilitating neuronal growth and development. Trk receptors influence the survival and differentiation of neurons via many signalling cascades. Neurotrophin interaction with Trk receptors triggers a signalling cascade involving PLC, PI3K/Akt, and Ras/MAPK signalling pathways. Emerging evidence suggests that diminished neurotrophic support, changes in Trk receptor expression, or disruptions in signalling cascades play a crucial role in the development of various neurological disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), autism spectrum disorder (ASD), and many more. This review specifically explores therapeutic approaches targeting Trk receptors, their ligands, and Trk signaling in the context of various brain disorders. We focus on the potential for modulating or inhibiting Trk receptors as a treatment strategy for brain diseases.

Brain-derived neurotrophic factor signaling via its receptor tropomyosin receptor kinase B regulates several crucial physiological processes. It has been shown to act in the brain, promoting neuronal survival, growth, and plasticity as well as in the rest of the body where it is involved in regulating for instance aspects of the metabolism. Due to its crucial and very pleiotropic activity, reduction of brain-derived neurotrophic factor levels and alterations in the brain-derived neurotrophic factor/tropomyosin receptor kinase B signaling have been found to be associated with a wide spectrum of neurological diseases. However, because of its poor bioavailability and pharmacological properties, brain-derived neurotrophic factor itself has a very low therapeutic value. Moreover, the concomitant binding of exogenous brain-derived neurotrophic factor to the p75 neurotrophin receptor has the potential to elicit several unwanted and deleterious side effects. Therefore, developing tools and approaches to specifically promote tropomyosin receptor kinase B signaling has become an important goal of translational research. Among the newly developed tools are different categories of tropomyosin receptor kinase B receptor agonist molecules. In this review, we give a comprehensive description of the different tropomyosin receptor kinase B receptor agonist drugs developed so far and of the results of their application in animal models of several neurological diseases. Moreover, we discuss the main benefits of tropomyosin receptor kinase B receptor agonists, concentrating especially on the new tropomyosin receptor kinase B agonist antibodies. The benefits observed both in vitro and in vivo upon application of tropomyosin receptor kinase B receptor agonist drugs seem to predominantly depend on their general neuroprotective activity and their ability to promote neuronal plasticity. Moreover, tropomyosin receptor kinase B agonist antibodies have been shown to specifically bind the tropomyosin receptor kinase B receptor and not p75 neurotrophin receptor. Therefore, while, based on the current knowledge, the tropomyosin receptor kinase B receptor agonists do not seem to have the potential to reverse the disease pathology per se, promoting brain-derived neurotrophic factor/tropomyosin receptor kinase B signaling still has a very high therapeutic relevance.

In Vivo Restoration of Physiological Levels of Truncated TrkB.T1 Receptor Rescues Neuronal Cell Death in a Trisomic Mouse Model

Neurotrophin signaling plays important roles in regulating the survival, differentiation, and maintenance of neurons in the nervous system. Binding of neurotrophins to their cognate receptors Trks induces transactivation and phosphorylation of the receptor at several tyrosine residues. These phosphorylated tyrosine residues then serve as crucial docking sites for adaptor proteins containing a Src homology 2 or phosphotyrosine binding domain, which upon association with the receptor initiates multiple signaling events to mediate the action of neurotrophins. Here we report the identification of a Src homology 2 domain-containing molecule, SLAM-associated protein (SAP), as an interacting protein of TrkB in a yeast two-hybrid screen. SAP was initially identified as an adaptor molecule in SLAM family receptor signaling for regulating interferon-gamma secretion. In the current study, we found that SAP interacted with TrkA, TrkB, and TrkC receptors in vitro and in vivo. Binding of SAP required Trk receptor activation and phosphorylation at the tyrosine 674 residue, which is located in the activation loop of the kinase domain. Overexpression of SAP with Trk attenuated tyrosine phosphorylation of the receptors and reduced the binding of SH2B and Shc to TrkB. Moreover, overexpression of SAP in PC12 cells suppressed the nerve growth factor-dependent activation of extracellular signal-regulated kinases 1/2 and phospholipase Cgamma, in addition to inhibiting neurite outgrowth. In summary, our findings demonstrated that SAP may serve as a negative regulator of Trk receptor activation and downstream signaling.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide that acts through G protein-coupled receptors, exerts neuroprotective effects upon many neuronal populations. However, the intracellular signaling mechanisms that account for PACAP's trophic effects are not well characterized. Here we have tested the possibility that PACAP uses neurotrophin signaling pathways. We have found that PACAP treatment resulted in an increase in TrkA tyrosine kinase activity in PC12 cells and TrkB activity in hippocampal neurons. The activation of TrkA receptors by PACAP required at least 1 h of treatment and did not involve binding to nerve growth factor. Moreover, PACAP induced an increase in activated Akt through a Trk-dependent mechanism that resulted in increased cell survival after trophic factor withdrawal. The increases in Trk and Akt were blocked by K252a, an inhibitor of Trk receptor activity. In addition, transactivation of TrkA receptors by PACAP could be inhibited with PP1, an inhibitor of Src family kinases or BAPTA/AM, (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester), an intracellular calcium chelator. Therefore, PACAP can exert trophic effects through a mechanism involving Trk receptors and utilization of tyrosine kinase signaling. This ability may explain several neuroprotective actions of PACAP upon neuronal populations after injury, nerve lesion, or neurotrophin deprivation.

The RAS (mitogen-activated protein kinase) MAPK pathway is commonly deregulated in human cancer, including childhood acute myeloid leukemias (AMLs). Canonical strong activating somatic mutation of RAS-MAPK pathway genes are recurrently found in AMLs. They affect integral components of the pathway, and upstream activators, and include NRAS, KRAS, BRAF, PTPN11, and FMS-related tyrosine kinase 3 (FLT3). Irrespective of etiology, the oncogenic RAS signal is frequently potentiated as leukemia progress; unknown mechanisms may contribute to this emerging aspect of leukemia evolution. It is well established that RAS activation can trigger compensatory feedback mechanisms that dampen signaling output. Suppression of these negative regulators may result in an enhancement of RAS signaling driving AML progression. Recent mathematical modeling and subsequent experiments revealed that mutations of the tumor suppressor gene NF1 (neurofibromin 1) can amplify the effects of other RAS pathway mutations, including weakly activating, non canonical RAS mutants. Combinations of RAS-MAPK pathway mutations (including mutations of RAS-MAPK regulators) could therefore serve the role of leukemia driver [1]. SPRED1 and SPRED2 are members of the evolutionarily conserved SPROUTY/SPRED family of membrane-associated negative regulators of the RAS MAPK pathway. SPRED1 can interact with neurofibromin (the NF1 gene product) by inducing the plasma membrane localization of the latter and the subsequent down-regulation of RAS-GTP levels [2]. Recently, we looked for SPRED1 mutations in 230 cases of pediatric acute leukemia. We found 2% of patients carrying SPRED1 germline mutations coexpressed with known lesions activating the RAS-MAPK pathway. Strikingly, SPRED1 transcription was profoundly decreased in almost the totality of AMLs with consequent downregulation at the protein level. SPRED1 lowest levels were observed in FLT3-ITD mutated leukemia. Our study revealed a new general mechanism which contributes to deregulate RAS-MAPK pathway in pediatric AMLs [3]. Sprouty 4 (SPRY4), another RAS-MAPK inhibitor, was recently functionally validated as a tumor suppressor in AMLs [4]. Zhao et al. showed that in mice, premalignant myeloid cells harboring a Kras G12D allele, retained low levels of RAS signaling owing to negative feedback involving Spry4 that prevented transformation. SPRY4 is located on human chromosome 5q, a region affected by large heterozygous deletions that are associated with aggressive disease in which gain-of-function mutations in the RAS pathway are rare. AML cells harbor losses of multiple RAS signaling negative regulatory genes that can functionally cooperate to achieve high levels of RAS pathway activation. Acquisition of RAS-MAPK pathway activation in certain AML subtype may therefore be driven by loss of negative regulators, like SPRED or SPRY proteins. As the level of RAS signaling output is critical for the transforming processes, concomitant loss of several of these genes may be needed to overcome the critical threshold of RAS activation. The loss of inhibitory proteins may indeed represent a decisive step to escape oncogene induced senescence triggered by RAS activating lesions and promote full cellular malignancy [4–5]. Given the number of potential negative regulators in this pathway, the foreseeable combinations that may replace oncogenic RAS (or others RAS activators) are seemingly very high. It is tempting to speculate that emerging actors, like DUSP (Dual Specific Phosphatases) or RAS-GAP (RAS GTPase activating proteins) genes might also be involved in RAS oncogenic signaling in AMLs. Constitutional loss-of-function mutations in the SPRED1 gene cause a rare phenotype referred as neurofibromatosis type 1 (NF1)-like syndrome or Legius syndrome, consisting in multiple cafe-au-lait macules, axillary freckling, learning disabilities, and macrocephaly. NF1 and Legius syndrome belong to the neuro-cardio-facio-cutaneous syndromes that are caused by deregulating constitutional mutations of the RAS-MAPK signaling pathway [2]. These RASopathies that include Noonan syndrome, LEOPARD syndrome, cardio-facio-cutaneous syndrome, Costello syndrome, NF1 and the Legius syndrome, share characteristic overlapping features, including predisposition to develop multiple types of cancer. Individuals with NF1 and Noonan syndrome have a higher risk of haematological malignancies, including acute leukemia and the rare disorder juvenile myelomonocytic leukemia. Although rare, inherited predispositions to myeloid leukemia have uncovered a critical role of hyperactive RAS-MAPK signaling in normal myeloid growth and leukemogenesis. We previously reported the observation of an 11-month-old boy with a SPRED1 constitutional mutation, who developed an AML [6]. RAS mutations account for less than 10–15 % of pediatric AML and FLT3-ITD mutations do not exceed 5–8 %, which is in contrast with the higher frequency of MAPK/PI3K up-regulation observed in these leukemia. It is reasonable to think that negative-feedback regulators for RAS signaling are involved with AML transformation at genetic or epigenetic level [7]. Future studies are needed to integrate both somatic and germline variants in RAS-MAPK regulators, to provide comprehensive characterization of genetic risk factors for AML and shed light on the functional consequences of these alterations. This latter point is of utmost importance to develop more rational therapies aimed to inhibit the complex network of RAS-MAPK aberrant activation.