John P. O'Bryan

The Ubiquitin-Interacting Motifs Target the Endocytic Adaptor Protein Epsin for Ubiquitination

The covalent attachment of ubiquitin to proteins is an evolutionarily conserved signal for rapid protein degradation. However, additional cellular functions for ubiquitination are now emerging, including regulation of protein trafficking and endocytosis. For example, recent genetic studies suggested a role for ubiquitination in regulating epsin, a modular endocytic adaptor protein that functions in the assembly of clathrin-coated vesicles; however, biochemical evidence for this notion has been lacking. Epsin consists of an epsin NH(2)-terminal homology (ENTH) domain that promotes the interaction with phospholipids, several AP2 binding sites, two clathrin binding sequences, and several Eps15 homology (EH) domain binding motifs. Interestingly, epsin also possesses several recently described ubiquitin-interacting motifs (UIMs) that have been postulated to bind ubiquitin. Here, we demonstrate that epsin is predominantly monoubiquitinated and resistant to proteasomal degradation. The UIMs are necessary for epsin ubiquitination but are not the site of ubiquitination. Finally, we demonstrate that the isolated UIMs from both epsin and an unrelated monoubiquitinated protein, Eps15, are sufficient to promote ubiquitination of a chimeric glutathione-S-transferase (GST)-UIM fusion protein. Thus, our data suggest that UIMs may serve as a general signal for ubiquitination.

The Mammalian ShcB and ShcC Phosphotyrosine Docking Proteins Function in the Maturation of Sensory and Sympathetic Neurons

Shc proteins possess SH2 and PTB domains and serve a scaffolding function in signaling by a variety of receptor tyrosine kinases. There are three known mammalian Shc genes, of which ShcB and ShcC are primarily expressed in the nervous system. We have generated null mutations in ShcB and ShcC and have obtained mice lacking either ShcB or ShcC or both gene products. ShcB-deficient animals exhibit a loss of peptidergic and nonpeptidergic nociceptive sensory neurons, which is not enhanced by additional loss of ShcC. Mice lacking both ShcB and ShcC exhibit a significant loss of neurons within the superior cervical ganglia, which is not observed in either mutant alone. The results indicate that these Shc family members possess both unique and overlapping functions in regulating neural development and suggest physiological roles for ShcB/ShcC in TrkA signaling.

Intersectin Can Regulate the Ras/MAP Kinase Pathway Independent of Its Role in Endocytosis

We previously identified intersectin, a multiple EH and SH3 domain-containing protein, as a component of the endocytic machinery. Overexpression of the SH3 domains of intersectin blocks transferrin receptor endocytosis, possibly by disrupting targeting of accessory proteins of clathrin-coated pit formation. More recently, we identified mammalian Sos, a guanine-nucleotide exchange factor for Ras, as an intersectin SH3 domain-binding partner. We now demonstrate that overexpression of intersectins SH3 domains blocks activation of Ras and MAP kinase in various cell lines. Several studies suggest that activation of MAP kinase downstream of multiple receptor types is dependent on endocytosis. Thus, the dominant-negative effect of the SH3 domains on Ras/MAP kinase activation may be indirectly mediated through a block in endocytosis. Consistent with this idea, incubating cells at 4 °C or with phenylarsine oxide, treatments previously established to inhibit EGF receptor endocytosis, blocks EGF-dependent activation of MAP kinase. However, under these conditions, Ras activity is unaffected and overexpression of the SH3 domains of intersectin is still able to block Ras activation. Thus, intersectin SH3 domain overexpression can effect EGF-mediated MAP kinase activation directly through a block in Ras, consistent with a functional role for intersectin in Ras activation.

Rac and Rho Mediate Opposing Hormonal Regulation of the Ether-A-Go-Go-Related Potassium Channel

BACKGROUND Previous studies of ion channel regulation by G proteins have focused on the larger, heterotrimeric GTPases, which are activated by heptahelical membrane receptors. In contrast, studies of the Rho family of smaller, monomeric, Ras-related GTPases, which are activated by cytoplasmic guanine nucleotide exchange factors, have focused on their role in cytoskeletal regulation. RESULTS Here we demonstrate novel functions for the Rho family GTPases Rac and Rho in the opposing hormonal regulation of voltage-activated, ether-a-go-go-related potassium channels (ERG) in a rat pituitary cell line, GH(4)C(1). The hypothalamic neuropeptide, thyrotropin-releasing hormone (TRH) inhibits ERG channel activity through a PKC-independent process that is blocked by RhoA(19N) and the Clostridium botulinum C3 toxin, which inhibit Rho signaling. The constitutively active, GTPase-deficient mutant of RhoA(63L) rapidly inhibits the channels when the protein is dialysed directly into the cell through the patch pipette, and inhibition persists when the protein is overexpressed. In contrast, GTPase-deficient Rac1(61L) stimulates ERG channel activity. The thyroid hormone triiodothyronine (T3), which antagonizes TRH action in the pituitary, also stimulates ERG channel activity through a rapid process that is blocked by Rac1(17N) and wortmannin but not by RhoA(19N). CONCLUSIONS Rho stimulation by G(13)-coupled receptors and Rac stimulation by nuclear hormones through PI3-kinase may be general mechanisms for regulating ion channel activity in many cell types. Disruption of these novel signaling cascades is predicted to contribute to several specific human neurological diseases, including epilepsy and deafness.

The Ews/Fli-1 Fusion Gene Switches the Differentiation Program of Neuroblastomas to Ewing Sarcoma/Peripheral Primitive Neuroectodermal Tumors

Neuroblastoma (NB) and the Ewing sarcoma (ES)/peripheral primitive neuroectodermal tumor (PNET) family are pediatric cancers derived from neural crest cells. Although NBs display features of the sympathetic nervous system, ES/PNETs express markers consistent with parasympathetic differentiation. To examine the control of these differentiation markers, we generated NB × ES/PNET somatic cell hybrids. NB-specific markers were suppressed in the hybrids, whereas ES/PNET-specific markers were unaffected. These results suggested that the Ews/Fli-1 fusion gene, resulting from a translocation unique to ES/PNETs, might account for the loss of NB-specific markers. To test this hypothesis, we generated two different NB cell lines that stably expressed the Ews/Fli-1 gene. We observed that heterologous expression of the Ews/Fli-1 protein led to the suppression of NB-specific markers and de novo expression of ES/PNET markers. To determine the extent of changes in differentiation, we used the Affymetrix GeneChip Array system to observe global transcriptional changes of genes. This analysis revealed that the gene expression pattern of the Ews/Fli-1-expressing NB cells resembled that observed in pooled ES/PNET cell lines and differed significantly from the NB parental cells. Therefore, we propose that Ews/Fli-1 contributes to the etiology of ES/PNET by subverting the differentiation program of its neural crest precursor cell to a less differentiated and more proliferative state.

Regulation of Neuron Survival through an Intersectin-Phosphoinositide 3′-Kinase C2β-AKT Pathway

ABSTRACT While endocytosis attenuates signals from plasma membrane receptors, recent studies suggest that endocytosis also serves as a platform for the compartmentalized activation of cellular signaling pathways. Intersectin (ITSN) is a multidomain scaffolding protein that regulates endocytosis and has the potential to regulate various biochemical pathways through its multiple, modular domains. To address the biological importance of ITSN in regulating cellular signaling pathways versus in endocytosis, we have stably silenced ITSN expression in neuronal cells by using short hairpin RNAs. Decreasing ITSN expression dramatically increased apoptosis in both neuroblastoma cells and primary cortical neurons. Surprisingly, the loss of ITSN did not lead to major defects in the endocytic pathway. Yeast two-hybrid analysis identified class II phosphoinositide 3′-kinase C2β (PI3K-C2β) as an ITSN binding protein, suggesting that ITSN may regulate a PI3K-C2β-AKT survival pathway. ITSN associated with PI3K-C2β on a subset of endomembrane vesicles and enhanced both basal and growth factor-stimulated PI3K-C2β activity, resulting in AKT activation. The use of pharmacological inhibitors, dominant negatives, and rescue experiments revealed that PI3K-C2β and AKT were epistatic to ITSN. This study represents the first demonstration that ITSN, independent of its role in endocytosis, regulates a critical cellular signaling pathway necessary for cell survival.

INTERSECTIN REGULATES EPIDERMAL GROWTH FACTOR RECEPTOR ENDOCYTOSIS, UBIQUITYLATION, AND SIGNALING

Receptor tyrosine kinases (RTKs) are critical for normal cell growth, differentiation, and development, but they contribute to various pathological conditions when disrupted. Activation of RTKs stimulates a plethora of pathways, including the ubiquitylation and endocytosis of the receptor itself. Although endocytosis terminates RTK signaling, it has emerged as a requisite step in RTK activation of signaling pathways. We have discovered that the endocytic scaffolding protein intersectin (ITSN) cooperated with epidermal growth factor receptor (EGFR) in the regulation of cell growth and signaling. However, a biochemical link between ITSN and EGFR was not defined. In this study, we demonstrate that ITSN is a scaffold for the E3 ubiquitin ligase Cbl. ITSN forms a complex with Cbl in vivo mediated by the Src homology (SH) 3 domains binding to the Pro-rich COOH terminus of Cbl. This interaction stimulates the ubiquitylation and degradation of the activated EGFR. Furthermore, silencing ITSN by RNA interference attenuated EGFR internalization as well as activation of the extracellular signal-regulated kinasemitogen-activated protein kinase pathway, thereby demonstrating the importance of ITSN in EGFR function. Given the cooperativity between ITSN and additional RTKs, these results point to an important evolutionarily conserved, regulatory role for ITSN in RTK function that is necessary for both signaling from receptors as well as the ultimate termination of receptor signaling.

Mitogenesis and endocytosis: What's at the INTERSECTIoN?

Endocytosis is a regulated physiological process by which cell surface proteins are internalized along with extracellular factors such as nutrients, pathogens, peptides, toxins, etc. The process begins with the invagination of small regions of the plasma membrane which ultimately form intracellullar vesicles. These internalized vesicles may shuttle back to the plasma membrane to recycle the membrane components or they may be targeted for degradation. One role for endocytosis is in the attenuation of receptor signaling. For example, desensitization of activated membrane bound receptors such as G-protein coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs) occurs, in part, through endocytosis of the activated receptor. However, accumulating evidence suggests that endocytosis also mediates intracellular signaling. In this review, we discuss the experimental data that implicate endocytosis as a critical component in cellular signal transduction, both in the initiation of a signal as well as in the termination of a signal. Furthermore, we focus our attention on a recently described adaptor protein, intersectin (ITSN), which provides a link to both the endocytic and the mitogenic machinery of a cell. Thus, ITSN functions at a crossroad in the biochemical regulation of cell function.

Inhibition of RAS function through targeting an allosteric regulatory site

RAS GTPases are important mediators of oncogenesis in humans. However, pharmacological inhibition of RAS has proved challenging. Here we describe a functionally critical region, located outside the effector lobe of RAS, that can be targeted for inhibition. We developed NS1, a synthetic binding protein (monobody) that bound with high affinity to both GTP- and GDP-bound states of H-RAS and K-RAS but not N-RAS. NS1 potently inhibited growth factor signaling and oncogenic H-RAS- and K-RAS-mediated signaling and transformation but did not block oncogenic N-RAS, BRAF or MEK1. NS1 bound the α4-β6-α5 region of RAS, which disrupted RAS dimerization and nanoclustering and led to blocking of CRAF-BRAF heterodimerization and activation. These results establish the importance of the α4-β6-α5 interface in RAS-mediated signaling and define a previously unrecognized site in RAS for inhibiting RAS function.

INTERSECTINg Pathways in Cell Biology

The intersectin family of scaffolding proteins link signaling and endocytic pathways. A challenge faced by multicellular organisms is cellular communication, which is critical for the regulation of cell growth, differentiation, development, and apoptosis. Cells must communicate with one another to respond to changes in the environment. In one cellular communication strategy, binding of extracellular ligands to transmembrane receptors activates a cascade of signaling events in cells, leading to rearrangement of the cytoskeleton, changes in enzyme activity, or generation of second messengers. Inactivation of these signaling events is also necessary to prevent chronic stimulation or activation of cells, such as that seen in cancer. One mechanism for decreasing receptor signaling is the removal of activated receptors from the plasma membrane through endocytosis, a regulated process of membrane internalization. However, endocytosis is also involved in the positive flow of signals in cells, and endocytic vesicles have emerged as platforms that enable compartmentalized signaling to provide temporal and spatial control of signal transduction. Thus, a key area of research in biology lies in defining the molecular links between cellular signaling pathways and the endocytic pathway. The intersectin family of scaffold proteins can interact with components of signaling pathways as well as those in the endocytic pathway and, thus, may link these two sets of pathways. This review, which includes three figures and 95 references, discusses the role of intersectins in regulating endocytosis and their link to various signal transduction pathways, describes the involvement of intersectins in human diseases, and highlights future directions of investigation. The endocytic pathway is involved in activation and inhibition of cellular signaling. Thus, defining the regulatory mechanisms that link endocytosis and cellular signaling is of interest. An emerging link between these processes is a family of proteins called intersectins (ITSNs). These multidomain proteins serve as scaffolds in the assembly of endocytic vesicles and also regulate components of various signaling pathways, including kinases, guanosine triphosphatases, and ubiquitin ligases. This review summarizes research on the role of ITSNs in regulating both endocytic and signal transduction pathways, discusses the link between ITSNs and human disease, and highlights future directions in the study of ITSNs.