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. 2007 Nov;27(22):7906-17.
doi: 10.1128/MCB.01369-07. Epub 2007 Sep 17.

Regulation of neuron survival through an intersectin-phosphoinositide 3'-kinase C2beta-AKT pathway

Margaret Das  1 , Erica Scappini, Negin P Martin, Katy A Wong, Sara Dunn, Yun-Ju Chen, Stephanie L H Miller, Jan Domin, John P O'Bryan
Affiliations

Regulation of neuron survival through an intersectin-phosphoinositide 3'-kinase C2beta-AKT pathway

Margaret Das et al. Mol Cell Biol. 2007 Nov.

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 C2beta (PI3K-C2beta) as an ITSN binding protein, suggesting that ITSN may regulate a PI3K-C2beta-AKT survival pathway. ITSN associated with PI3K-C2beta on a subset of endomembrane vesicles and enhanced both basal and growth factor-stimulated PI3K-C2beta activity, resulting in AKT activation. The use of pharmacological inhibitors, dominant negatives, and rescue experiments revealed that PI3K-C2beta 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.

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Figures

FIG. 1.
FIG. 1.
Silencing ITSN in N1E-115 neuroblastoma cells. (A) Stable shRNAs were transfected into N1E-115 cells and either clonal (designated with a number) or polyclonal (P) cell lines were selected. ITSN expression was determined by Western blot analysis of cell lysates. The relative ITSN expression was determined by dividing the ITSN signal by the calreticulin signal and normalizing to the ratio from the N1E-115 sample. CALR, calreticulin as a normalization control for loading. (B) N1E-115-derived lines were examined for their differentiation potential by removal of serum. Similar results were obtained by differentiation with DMSO (data not shown). (C) Cell survival was quantified using a CellTiterGlo kit for measuring total ATP levels. Silencing ITSN resulted in dramatically reduced survival following serum withdraw but did not affect the growth of cells in the presence of serum (data not shown). N1E, N1E-115 cells; pSR, N1E-115 cells stably transfected with empty pSuperRetro vector.
FIG. 2.
FIG. 2.
Silencing ITSN decreases survival of primary cortical neurons through an apoptotic pathway. (A) Primary rat cortical neurons were infected with lentivirus expressing green fluorescent protein alone (LVTH) or green fluorescent protein and shRNA to ITSN (M1635) and observed for differences at the indicated days postinfection. (B) Survival of neurons was quantified by CellTiterGlo ATP assay. (C) Presence of apoptotic cells was determined by TUNEL staining. At least 250 cells per condition were scored for TUNEL staining. ITSN expression in the individual samples is shown below the graph. Equal protein amounts were loaded in each lane. L, LVTH-infected cultures; M, M1635-infected cultures; ITSN-L, ITSN long isoform; ITSN-S, ITSN short isoform; Tuj1, neuron-specific tubulin. (D) Silencing ITSN results in decreased pAKT levels. The lysates whose results are shown in panel C were analyzed for the levels of pAKT versus total AKT. The numbers below the panels represent the percent reduction in AKT activation, i.e., pAKT/total AKT, compared to the ratio in LVTH-infected cells for that day. DPI, days postinfection.
FIG. 3.
FIG. 3.
Stable silencing of ITSN does not affect constitutive endocytosis. (A) N1E-115 cells transfected with the indicated pSR construct were incubated with biotinylated transferrin (bTfn). Uptake of transferrin was quantified by Western blot analysis with an HRP-linked streptavidin antibody. Calr, calreticulin as control for loading. (B) N1E-115 cells stably silenced for ITSN (M1635) or vector control cells (pSR) were incubated with HRP and then assayed for uptake as previously described (16).
FIG. 4.
FIG. 4.
ITSN interacts with PI3K-C2β. (A) ITSN expression constructs. At the NH2 terminus are two EH domains, followed by a coiled-coil (CC) region and five SH3 domains, A through E. Each of the indicated proteins possesses an HA epitope tag at the NH2 terminus. (B) Yeast two-hybrid screening identified PI3K-C2β as an ITSN binding protein. Only the coexpression of ITSN and PI3K-C2β was sufficient to activate HIS3 expression, allowing growth on medium lacking histidine. Putative ITSN-binding sequences are highlighted in gray and underlined. Heavy underlining dentoes the putative Ras-binding region. (C) The modular structure of PI3K-C2β is shown at the top of the panel (15). RBD, putative PI3K Ras binding domain; PI3K C2, outlier PKC-related Ca+2 binding domain specific to PI3Ks; PI3K Acc, PI3K accessory domain; PI3K Cat, PI3K catalytic domain; PX, PhoX homology domain; C2, PKC-related Ca+2 binding domain. Shown below the diagram is an alignment of the PRDs from PI3K-C2β orthologs, with a consensus sequence at the bottom. C. elegans, Caenorhabditis elegans; C. briggsae, Caenorhabditis briggsae. (D) GST, GST-PI3K NT (aa 26 to 356 of PI3K-C2β), or GST-PI3K-C2α C2 was incubated with lysates from 293T cells transfected with either ITSN (+) or empty vector (−). PI3K-C2β (NT) but not the C2 domain of PI3K-C2α (C2) or GST by itself bound ITSN (top panel). Expression of HA-ITSN is shown in the lower panel. (E) PI3K-C2β binds specifically to SH3A of ITSN. GST fusions of the individual SH3 domains were used to precipitate endogenous PI3K-C2β from lysates of 293T cells. SH3A and, to a lesser extent, SH3C bound PI3K, while SH3B, -D, and -E and GST alone did not (top panel). In contrast, Cbl bound SH3A, -C, and -E (middle panel). The purified SH3 domains are shown in the bottom panel. GST-SH3B of ITSN runs anomalously on gels. The gels shown are representative of identical data from three separate experiments. Molecular masses (kDa) are shown on the left.
FIG. 5.
FIG. 5.
ITSN associates with PI3K-C2β in vivo. (A) Immunocytochemical staining of endogenous ITSN (green) and endogenous PI3K-C2β (red) from LAN-1 human neuroblastoma cells. ITSN staining is shown in panels a and d. PI3K-C2β staining is shown in panels b and e. Overlap of ITSN and PI3K-C2β is shown in panels c and f. Panels g and h show LAN-1 cells stained with anti-rabbit or anti-mouse secondary antibody, respectively, as negative controls. Scale bars, 20 μm. (B) HA epitope-tagged ITSN proteins were expressed in 293T cells, immunoprecipitated with anti-HA, and then examined for association with endogenous PI3K-C2β (top panel). The bottom panel shows the expression levels for the HA-tagged ITSN proteins. Arrows indicate the positions of PI3K-C2β and the ITSN long (ITSN-L) and ITSN short (ITSN-S) isoforms. In similar experiments, antibodies to PI3K-C2α did not interact with ITSN (data not shown). (C) HA epitope-tagged ITSN truncation mutants were expressed in cells, immunoprecipitated as described for panel B, and then examined for association with endogenous PI3K-C2β. The position of PI3K-C2β is indicated by the arrow. The band above this represents a nonspecific contaminating band. The data shown are representative of the results of three independent experiments. Molecular masses (kDa) are shown on the left. (D) Mutation of the PRD of PI3K-C2β inhibits the interaction with ITSN. Full-length fragments of PI3K or PI3K NT fused to CFP were coexpressed with HA-ITSN. ITSN was immunopurified using HA antibodies (top and middle panels), and the association of PI3K was determined by Western blot analysis with green fluorescent protein antibodies (top panel). The middle panel represents a Western blot of the HA immunoprecipitates with HA antibody, demonstrating the level of HA-ITSN in each sample. The PA mutants of PI3K and PI3K NT did not interact with ITSN. The expression of each of the proteins in the cell lysates is shown in the lower panels. Molecular masses (kDa) are shown on the left. α, anti; IP, immunoprecipation; IB, immunoblot.
FIG. 6.
FIG. 6.
ITSN colocalizes with PI3K-C2β in vivo. (A) BiFC analysis. VN-ITSN was coexpressed with either VC-tagged PI3K-C2β (wild type or PRD mutant) or VC fused to a nonspecific peptide (VC-pep). Mutation of the PRD (PA mutant, middle panels) of PI3K dramatically reduced interaction with ITSN as measured by BiFC. Confocal settings were identical for each condition. Cells were transfected in the early evening and analyzed the following morning to minimize the expression of the transfected constructs. CFP was cotransfected with the BiFC constructs to mark transfected cells. The scale bar represents 20 μm. (B) BiFC-tagged proteins are expressed at near endogenous levels. The first lane corresponds to lysates from untransfected cells. The remaining lanes correspond to the samples shown in panel A. Endogenous ITSN is visible as a doublet in COS cell lysates. Calr, calreticulin as a loading control for the Western blots. The VC-nonspecific peptide fusion protein (VC-pep) was visible as a peptide of ∼16 kDa on the lower portion of the HA blot (not shown). WT, wild type; α, anti.
FIG. 7.
FIG. 7.
ITSN association with PI3K-C2β is enhanced upon growth factor stimulation and results in AKT activation. (A) A431 cells were stably transfected with either empty vector (−) or ITSN (+). Following EGF stimulation (100 ng/ml) for the indicated times, PI3K-C2β was immunoprecipitated and examined for association with ITSN by Western blot analysis. Molecular masses (kDa) are shown on the left. (B) Lipid kinase activity in PI3K-C2β immunoprecipitates from ITSN-expressing cells was enhanced following EGF stimulation. A431 cells transfected with either empty vector or HA-tagged ITSN were stimulated with EGF for the indicated times. PI3K-C2β was immunoprecipitated, and the Ca+2-dependent lipid kinase activity was measured. (C) EGF enhanced ITSN-associated PI3K-C2β activity. HA immunoprecipitates from A431 cells whose results are shown in panel B were stimulated without or with EGF (100 ng/ml, 10 min) and then subjected to a PI3K assay as described in Materials and Methods. Experiments were performed in triplicate, and the data represent the means ± standard errors of the means (SEM) of the results. (D) ITSN overexpression enhances AKT activation. COS cells were transfected with the indicated expression construct along with HA-tagged AKT. Cells were serum-starved and then stimulated without (−) or with (+) EGF (100 ng/ml, 10 min), lysed, and AKT immunoprecipitated with an anti-HA antibody. The immunoprecipitates were analyzed for activated AKT using a phosphospecific antibody (pSer473) and total AKT (anti-HA). The relative AKT activation was determined by dividing the level of pAKT by total AKT and normalizing to the level in the unstimulated vector control sample (YFP alone). The graph represents the means ± SEM of the results of three independent experiments. (E) ITSN activation of AKT is PI3K-dependent. AKT activation was determined as described for panel D, except that cells were treated overnight with LY294002 or control vehicle (DMSO). Results are the means ± SEM of the results of three independent experiments.
FIG. 8.
FIG. 8.
PI3K-C2β and AKT are epistatic to ITSN. (A) Wild-type N1E-115 cells were differentiated in the presence of PI3K inhibitor (LY294002) or control vehicle (DMSO), and cell survival was quantified using an ATP assay. (B) N1E-115 cells were stably transfected with either empty vector or a kinase-dead AKT construct (AKT-DN). Cells were grown in the presence of serum (top panels) or differentiated by removal of serum (bottom panels). (C) Parallel cultures from the samples shown in panel B were incubated with CellTiterGlo reagent to quantify cell survival. Expression of kinase-dead AKT (AKT DN) resulted in increased cell death. (D) ITSN-silenced cells (M1635) were stably transfected with either CFP or CFP-PI3K-C2β. Cells were then grown in the presence of serum or differentiated by removal of serum. (E) ITSN-silenced cells (M1635) were stably transfected with either empty vector or HA-tagged wild-type (AKT-WT), activated (Myr-AKT), or kinase-dead (AKT-DN) AKT. Cells were then grown in the presence of serum (top panels) or differentiated by removal of serum (bottom panels). (F) Survival of cells shown in panel E was determined using CellTiterGlo reagent as described for panel C. (G) Expression of various HA-tagged AKT proteins was determined by Western blot analysis of cell lysates with anti-HA antibodies (top panel). These same lysates were also analyzed for expression of ITSN. pSR, N1E-115 cells stably transfected with empty pSuperRetro vector; M1635, N1E-115 cells stably transfected with the pSR-M1635 shRNA construct that silences ITSN expression. The M1635 cells were also stably transfected with AKT expression constructs (wild type [WT], kinase dead [DN], or activated [Myr]) or empty vector (vector). α, anti.
FIG. 9.
FIG. 9.
Endocytosis in ITSN-silenced cells. Internalization of Alexa 488-transferrin was measured in the various N1E-115 cells. (A) Confocal image analysis revealed no difference in the uptake of transferrin in the various N1E-115 clones. pcDNA, vector alone; WT, wild type. (B) Transferrin (Tf) internalization was quantified for each of the cell lines. The results shown are the means ± SEM of the results of two independent experiments in which relative levels of transferrin internalization were determined for the panel of cell lines. The differences between samples were not statistically significant.
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