Mutual regulation of Src family kinases and the neurotrophin receptor TrkB.

The neurotrophin receptor tyrosine kinase TrkB is critical to diverse biological processes. We investigated the interplay of Src family kinases (SFKs) and TrkB to better understand mechanisms of TrkB signaling in physiological and pathological conditions. We compared and contrasted the role of SFKs in TrkB signaling following activation of TrkB by two mechanisms, its transactivation by zinc, and its activation by its prototypic neurotrophin ligand, brain-derived neurotrophic factor (BDNF). Using biochemical, pharmacological, and chemical genetic studies of cultured rodent neurons, we found that zinc promotes preferential phosphorylation of Tyr-705/Tyr-706 of TrkB by a SFK-dependent but TrkB kinase-independent mechanism, a signaling event critical for transactivation of TrkB by zinc. By contrast, SFK activity is not essential for BDNF-mediated activation of TrkB, yet SFK activity is increased as a consequence of TrkB activation by BDNF. Moreover, BDNF-induced phosphorylation of Tyr-705/Tyr-706 of TrkB was inhibited by SFK inhibitors, implicating a regulatory role of SFKs in TrkB activation by BDNF. In sum, SFKs are activated by TrkB and, in turn, SFKs can promote TrkB activation. We propose models depicting the mutual regulation of SFKs and TrkB following activation of TrkB by zinc and BDNF.

Zinc-mediated transactivation of TrkB potentiates the hippocampal mossy fiber-CA3 pyramid synapse.

The receptor tyrosine kinase, TrkB, is critical to diverse functions of the mammalian nervous system in health and disease. Evidence of TrkB activation during epileptogenesis in vivo despite genetic deletion of its prototypic neurotrophin ligands led us to hypothesize that a non-neurotrophin, the divalent cation zinc, can transactivate TrkB. We found that zinc activates TrkB through increasing Src family kinase activity by an activity-regulated mechanism independent of neurotrophins. One subcellular locale at which zinc activates TrkB is the postsynaptic density of excitatory synapses. Exogenous zinc potentiates the efficacy of the hippocampal mossy fiber (mf)-CA3 pyramid synapse by a TrkB-requiring mechanism. Long-term potentiation of this synapse is impaired by deletion of TrkB, inhibition of TrkB kinase activity, and by CaEDTA, a selective chelator of zinc. The activity-dependent activation of synaptic TrkB in a neurotrophin-independent manner provides a mechanism by which this receptor can regulate synaptic plasticity.

Neuregulin-1 enhances depolarization-induced GABA release.

Neuregulin-1 (NRG1), a regulator of neural development, has been shown to regulate neurotransmission at excitatory synapses. Although ErbB4, a key NRG1 receptor, is expressed in glutamic acid decarboxylase (GAD)-positive neurons, little is known about its role in GABAergic transmission. We show that ErbB4 is localized at GABAergic terminals of the prefrontal cortex. Our data indicate a role of NRG1, both endogenous and exogenous, in regulation of GABAergic transmission. This effect was blocked by inhibition or mutation of ErbB4, suggesting the involvement of ErbB4. Together, these results indicate that NRG1 regulates GABAergic transmission via presynaptic ErbB4 receptors, identifying a novel function of NRG1. Because both NRG1 and ErbB4 have emerged as susceptibility genes of schizophrenia, these observations may suggest a mechanism for abnormal GABAergic neurotransmission in this disorder.

Neuregulin-induced expression of the acetylcholine receptor requires endocytosis of ErbB receptors.

Neuregulin-induced expression of the acetylcholine receptor (AChR) contributes to high concentration of the receptor at the neuromuscular junction (NMJ). Neuregulin-1 activates ErbB tyrosine kinases and subsequently intracellular kinases including Erk that is required for induced AChR expression. Recent studies demonstrate that ligand-induced internalization may regulate signaling of various receptor tyrosine kinases. However, the role of induced ErbB endocytosis in regulating AChR expression was unclear. Here we provide evidence that ErbB tyrosine kinases became rapidly internalized in response to neuregulin. The internalization required the kinase activity of ErbB proteins and involved a clathrin-dependent endocytic pathway. Moreover, neuregulin-induced Erk activation and AChR expression were attenuated when ErbB endocytosis was blocked. These results indicate that ErbB proteins undergo endocytosis in response to neuregulin, and this process is required for neuregulin signaling and induced AChR expression.

Human homologue of Drosophila CNK interacts with Ras effector proteins Raf and Rlf.

Connector enhancer of KSR (CNK) is a multidomain protein that participates in Ras signaling in Drosophila eye development. In this report we identify the human homologue of CNK, termed CNK2A, and a truncated alternatively spliced variant, CNK2B. We characterize CNK2 phosphorylation, membrane localization, and interaction with Ras effector molecules. Our results show that MAPK signaling appears to play a role in the phosphorylation of CNK2 in vivo. CNK2 is found in both membrane and cytoplasmic fractions of the cell. In MDCK cells, full-length CNK2 is localized to the lateral plasma membrane. Consistent with previous reports, we show CNK2 interacts with Raf. CNK2 interaction was mapped to the regulatory and kinase domains of Raf, as well as to the carboxyl-terminal half of CNK2. CNK2 also interacts with the Ral signaling components, Ral GTPase, and the RalGDS family member Rlf. CNK2 interaction was mapped to the GEF domain of Rlf. The ability of CNK2 to interact with both Ras effector proteins Raf and Rlf suggests that CNK2 may integrate signals between MAPK and Ral pathways through a complex interplay of components.

Ligand-dependent recruitment of the ErbB4 signaling complex into neuronal lipid rafts.

Neuregulin (NRG) regulates synapse formation and synaptic plasticity, but little is known about the regulation of NRG signaling at synapses. Here we show that the NRG receptor ErbB4 was localized in anatomically defined postsynaptic densities in the brain. In cultured cortical neurons, ErbB4 was recruited to the neuronal lipid raft fraction after stimulation by NRG. Along with ErbB4, adaptor proteins Grb2 and Shc were translocated to lipid rafts by NRG stimulation. In transfected human embryonic kidney 293 cells, the partitioning of ErbB4 into a detergent-insoluble fraction that includes lipid rafts was increased by PSD-95 (postsynaptic density-95), through interaction of the ErbB4 C terminus with the PDZ [PSD-95/Discs large/zona occludens-1] domains of PSD-95. Disruption of lipid rafts inhibited NRG-induced activation of Erk and prevented NRG-induced blockade of induction of long-term potentiation at hippocampal CA1 synapses. Thus, our results indicate that NRG stimulation causes translocation of ErbB4 into lipid rafts and that lipid rafts are necessary for signaling by ErbB4.

Erbin suppresses the MAP kinase pathway.

We present evidence here that Erbin is a negative regulator of the Ras-Raf-Erk signaling pathway. Expression of Erbin decreases transcription of the AChR epsilon-subunit gene, an event that is mediated by Erk activation. Although it interacts with the ErbB2 C terminus through the PDZ domain, Erbin has no effect on ErbB2 tyrosine phosphorylation or binding to the adaptor proteins Shc and Grb2. In contrast, expression of Erbin greatly impairs activation of Erk, but not Akt, by ligands that activate receptor tyrosine kinases. Moreover, Erbin inhibits the Erk activation by active Ras, while it fails to do so in the presence of active Raf-1. Erbin associates with active Ras, but not inactive Ras nor Raf. Consistently, Erbin interferes with the interaction between Ras and Raf both in vivo and in vitro. Finally, overexpression of Erbin leads to inhibition of NGF-induced neuronal differentiation of PC12 cells, whereas down-regulation of endogenous Erbin by specific siRNA exhibits an opposite effect. Collectively, our study has identified Erbin as a novel suppressor of the Ras signaling by disrupting the Ras-Raf interaction.

Compartmentalized NRG signaling and PDZ domain-containing proteins in synapse structure and function.

The synapse-specific synthesis of the acetylcholine receptor (AChR) is mediated by multiple mechanisms including compartmentalized signaling induced by neuregulin (NRG). This paper presents evidence that NRG receptors--ErbB receptor tyrosine kinases interact with distinct PDZ domain-containing proteins that are localized at the neuromuscular junction (NMJ). ErbB4 associates with the PSD-95 (also known as SAP90)-family members including PSD-95, SAP97, and SAP102 whereas ErbB2 interacts with Erbin and PICK1. Although, ErbB kinases are concentrated at the NMJ, they are not colocalized with the AChR in cultured muscle cells even in the presence of agrin. Co-expression of PSD-95 causes ErbB4 to form clusters in COS cells. We propose that PDZ domain-containing proteins play a role in anchoring ErbB proteins at the neuromuscular junction, and/or mediating downstream signaling pathways. Such mechanisms could be important for the maintenance and function of the synapse.

Presenilin-dependent gamma-secretase-like intramembrane cleavage of ErbB4.

An unusual protease gamma-secretase requires functional presenilins and cleaves substrates (e.g. amyloid beta-protein precursor and Notch) with very loose amino acid sequence specificity within the transmembrane region. Here we report that ErbB4, a tyrosine kinase receptor for neuregulins, is a substrate for presenilin-dependent gamma-secretase. Our studies show that constitutive ectodomain shedding of full-length ErbB4 yields the approximately 80-kDa membrane-associated C-terminal fragment (B4-CTF). Subsequent intramembrane cleavage of the B4-CTF was inhibited in the cells devoid of functional presenilins or by treatment of cells with a gamma-secretase inhibitor, leading to enhanced accumulation of B4-CTF. Furthermore, an in vitro gamma-secretase assay demonstrated that the intracellular domain of ErbB4 (B4-ICD) was produced and subsequently released into the soluble fraction in a presenilin-dependent manner. We have also shown that ectopically expressed B4-ICD is localized to the nucleus, suggesting that the presenilin-dependent cleavage of ErbB4 generates the soluble B4-ICD that functions in the nucleus presumably at transcriptional level. Our study indicates that ErbB4 represents a first receptor tyrosine kinase that undergoes intramembrane proteolysis and may mediate a novel signaling function independent of its canonical role as a receptor tyrosine kinase. Our studies also support the idea that presenilins play a generic role in intramembrane cleavage of selected type I membrane proteins.