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 C2␤ (PI3K-C2␤) as an ITSN binding protein, suggesting that ITSN may regulate a PI3K-C2␤-AKT survival pathway. ITSN associated with PI3K-C2␤ on a subset of endomembrane vesicles and enhanced both basal and growth factor-stimulated PI3K-C2␤ activity, resulting in AKT activation. The use of pharmacological inhibitors, dominant negatives, and rescue experiments revealed that PI3K-C2␤ 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 (ITSN) is a modular scaffold with multiple protein interaction domains that is conserved among metazoa. At the amino terminus are two Eps15 homology (EH) domains that bind NPF motifs on proteins such as epsin (36). The EH domains are followed by a coiled-coil domain that enables ITSN to homo-and heterodimerize with proteins such as Eps15 (24). The carboxy terminus consists of five Src homology 3 (SH3) domains that interact with Pro-rich motifs on a variety of proteins, several of which are involved in regulating endocytosis. Indeed, a subset of ITSNЈs SH3 domains are potent inhibitors of clathrin-coated pit formation (26). Recent studies on the Drosophila melanogaster ortholog of ITSN, Dap160, indicate that this scaffold functions as a stabilizing or recruitment factor for components of the clathrin-coated pit (14, 17). The loss of Dap160 function results in fewer coated vesicles, as well as enlarged vesicles, indicating that ITSN functions in both the formation and maturation of endocytic vesicles. Consistent with this role in Drosophila, silencing ITSN expression in mammalian cells results in defects in epidermal growth factor receptor (EGFR) internalization (18).Although ITSN is clearly linked with endocytosis, increasing evidence suggests that ITSNЈs role within the cell is not limited to this process (1,10,18,19,(27)(28)(29)32). The cloning of mammalian ITSN revealed a longer, spliced product containing a guanine nucleotide exchange factor domain specific for Cdc42 (10, 27). Through the EH domains, ITSN activates a Jun Nterminal protein kinase-dependent pathway that cooper...

Receptor Tyrosine Kinase Ubiquitylation Involves the Dynamic Regulation of Cbl-Spry2 by Intersectin 1 and the Shp2 Tyrosine Phosphatase

“…On the following day, cells were transfected with 0.5 g of plasmids encoding proteins fused to pHA-VC155N and pFLAG-VN173N. ITSN1-S, Cbl, and Spry2 BiFC constructs have been previous described (3,25). The VC-Shp2 construct encoding wild-type Shp2 was kindly provided by G. Carnegie (University of Illinois at Chicago) and has been previously described (26).…”

Classes of phosphoinositide 3-kinases at a glance

“…The PI3KC2a Nterminal region appears to inhibit kinase activity, which can be released by clathrin binding (Gaidarov et al, 2001), and PI3KC2a has been implicated in clathrin-mediated endocytosis (Posor et al, 2013). The PI3KC2b N-terminus also binds the scaffold protein intersectin, which promotes increased PtdIns(3)P synthesis (Das et al, 2007). Unlike the PI3KC2a and -b isoforms, PI3KC2c protein interactions have not been tested.…”

Modulation of Intersectin-1s Lung Expression Induces Obliterative Remodeling and Severe Plexiform Arteriopathy in the Murine Pulmonary Vascular Bed

“…47,48 Recently, we have shown that chronic ITSN deficiency up-regulated extracellular signal regulated kinase (Erk) 1/2 MAPK activity, suppressed the proapoptotic Bad, and altered Smads signaling downstream of the transforming growth factor-b receptor-1, resulting in a proliferative apoptotic-resistant EC phenotype. 12 In a PAH environment, ITSN deficiency and concurrent expression of granzyme B cleavage products of ITSN, able to regulate the p38 to Erk1/2 activity ratio, appear to be critical for initiation of the aberrant EC proliferation.…”