Semaphorin3A Signaling Mediated by Fyn-dependent Tyrosine Phosphorylation of Collapsin Response Mediator Protein 2 at Tyrosine 32

Collapsin response mediator protein 2 (CRMP2) is an intracellular protein that mediates signaling of Semaphorin3A (Sema3A), a repulsive axon guidance molecule. Fyn, a Src-type tyrosine kinase, is involved in the Sema3A signaling. However, the relationship between CRMP2 and Fyn in this signaling pathway is still unknown. In our research, we demonstrated that Fyn phosphorylated CRMP2 at Tyr³² residues in HEK293T cells. Immunohistochemical analysis using a phospho-specific antibody at Tyr³² of CRMP showed that Tyr³²-phosphorylated CRMP was abundant in the nervous system, including dorsal root ganglion neurons, the molecular and Purkinje cell layer of adult cerebellum, and hippocampal fimbria. Overexpression of a nonphosphorylated mutant (Tyr³² to Phe³²) of CRMP2 in dorsal root ganglion neurons interfered with Sema3A-induced growth cone collapse response. These results suggest that Fyn-dependent phosphorylation of CRMP2 at Tyr³² is involved in Sema3A signaling.Collapsin response mediator proteins (CRMPs)⁴ have been identified as intracellular proteins that mediate Semaphorin3A (Sema3A) signaling in the nervous system (1). CRMP2 is one of the five members of the CRMP family. CRMPs also mediate signal transduction of NT3, Ephrin, and Reelin (2–4). CRMPs interact with several intracellular molecules, including tubulin, Numb, kinesin1, and Sra1 (5–8). CRMPs are involved in axon guidance, axonal elongation, cell migration, synapse maturation, and the generation of neuronal polarity (1, 2, 4, 5).CRMP family proteins are known to be the major phosphoproteins in the developing brain (1, 9). CRMP2 is phosphorylated by several Ser/Thr kinases, such as Rho kinase, cyclin-dependent kinase 5 (Cdk5), and glycogen synthase kinase 3β (GSK3β) (2, 10–13). The phosphorylation sites of CRMP2 by these kinases are clustered in the C terminus and have already been identified. Rho kinase phosphorylates CRMP2 at Thr⁵⁵⁵ (10). Cdk5 phosphorylates CRMP2 at Ser⁵²², and this phosphorylation is essential for sequential phosphorylations by GSK3β at Ser⁵¹⁸, Thr⁵¹⁴, and Thr⁵⁰⁹ (2, 11–13). These phosphorylations disrupt the interaction of CRMP2 with tubulin or Numb (2, 3, 13). The sequential phosphorylation of CRMP2 by Cdk5 and GSK3β is an essential step in Sema3A signaling (11, 13). Furthermore, the neurofibrillary tangles in the brains of people with Alzheimer disease contain hyperphosphorylated CRMP2 at Thr⁵⁰⁹, Ser⁵¹⁸, and Ser⁵²² (14, 15).CRMPs are also substrates of several tyrosine kinases. The phosphorylation of CRMP2 by Fes/Fps and Fer has been shown to be involved in Sema3A signaling (16, 17). Phosphorylation of CRMP2 at Tyr⁴⁷⁹ by a Src family tyrosine kinase Yes regulates CXCL12-induced T lymphocyte migration (18). We reported previously that Fyn is involved in Sema3A signaling (19). Fyn associates with PlexinA2, one of the components of the Sema3A receptor complex. Fyn also activates Cdk5 through the phosphorylation at Tyr¹⁵ of Cdk5 (19). In dorsal root ganglion (DRG) neurons from fyn-deficient mice, Sema3A-induced growth cone collapse response is attenuated compared with control mice (19). Furthermore, we recently found that Fyn phosphorylates CRMP1 and that this phosphorylation is involved in Reelin signaling (4). Although it has been shown that CRMP2 is involved in Sema3A signaling (1, 11, 13), the relationship between Fyn and CRMP2 in Sema3A signaling and the tyrosine phosphorylation site(s) of CRMPs remain unknown.Here, we show that Fyn phosphorylates CRMP2 at Tyr³². Using a phospho-specific antibody against Tyr³², we determined that the residue is phosphorylated in vivo. A nonphosphorylated mutant CRMP2Y32F inhibits Sema3A-induced growth cone collapse. These results indicate that tyrosine phosphorylation by Fyn at Tyr³² is involved in Sema3A signaling.     

Evidence of a Role for Phosphatidylinositol 3-Kinase Activation in the Blocking of Apoptosis by Polyomavirus Middle T Antigen

A polyomavirus mutant (315YF) blocked in binding phosphatidylinositol 3-kinase (PI 3-kinase) has previously been shown to be partially deficient in transformation and to induce fewer tumors and with a significant delay compared to wild-type virus. The role of polyomavirus middle T antigen-activated PI 3-kinase in apoptosis was investigated as a possible cause of this behavior. When grown in medium containing 1d-3-deoxy-3-fluoro-myo-inositol to block formation of 3′-phosphorylated phosphatidylinositols, F111 rat fibroblasts transformed by wild-type polyomavirus (PyF), but not normal F111 cells, showed a marked loss of viability with evidence of apoptosis. Similarly, treatment with wortmannin, an inhibitor of PI 3-kinase, stimulated apoptosis in PyF cells but not in normal cells. Activation of Akt, a serine/threonine kinase whose activity has been correlated with regulation of apoptosis, was roughly twofold higher in F111 cells transformed by either wild-type virus or mutant 250YS blocked in binding Shc compared to cells transformed by mutant 315YF. In the same cells, levels of apoptosis were inversely correlated with Akt activity. Apoptosis induced by serum withdrawal in Rat-1 cells expressing a temperature-sensitive p53 was shown to be at least partially p53 independent. Expression of either wild-type or 250YS middle T antigen inhibited apoptosis in serum-starved Rat-1 cells at both permissive and restrictive temperatures for p53. Mutant 315YF middle T antigen was partially defective for inhibition of apoptosis in these cells. The results indicate that unlike other DNA tumor viruses which block apoptosis by inactivation of p53, polyomavirus achieves protection from apoptotic death through a middle T antigen–PI 3-kinase–Akt pathway that is at least partially p53 independent.Programmed cell death occurs during normal development and under certain pathological conditions. In mammalian cells, apoptosis can be induced by a variety of stimuli, including DNA damage (45), virus infection (54, 57), oncogene activation (25), and serum withdrawal (34, 37). Apoptosis can also be blocked by a number of factors, including adenovirus E1B 55- or 19-kDa proteins (9, 16), baculovirus p35 and iap genes (10), Bcl-2 (36, 61), and survival factors (12, 21). DNA tumor viruses have evolved mechanisms that both trigger and inhibit apoptosis. These frequently involve binding and inactivation of tumor suppressor proteins. E7 in some papillomaviruses (22), E1A in adenovirus (31, 43, 64), and large T antigen in simian virus 40 (SV40) (17) bind Rb and/or p300 and lead to upregulation of p53, which is thought to trigger apoptosis in virus-infected cells. The same viruses also inhibit apoptosis by inactivating p53 by various mechanisms (44, 63, 67). In contrast, the mechanism by which polyomavirus interacts with apoptotic pathways in the cell is not known; no direct interaction with p53 by any of the proteins encoded by this virus has been demonstrated (19, 62).The principal oncoprotein of polyomavirus is the middle T antigen. Neoplastic transformation by polyomavirus middle T antigen has as a central feature its association with and activation of members of the Src family of tyrosine kinases p60^c-src (13) and p62^c-yes (42). The major known consequence of these interactions is phosphorylation of middle T antigen on specific tyrosine residues creating binding sites for other signaling proteins. Phosphorylation at tyrosines 250, 315, and 322 promotes binding to Shc (18), the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) (59), and phospholipase Cγ-1 (58), respectively. Recognition of multiple signaling pathways emanating from middle T antigen has led to a keen interest in identifying their downstream biochemical effects, which collectively lead to the emergence of neoplastic transformation and presumably underlie the dramatic ability of the virus to induce many kinds of tumors in the mouse.Previous work has shown that the binding of PI 3-kinase to middle T antigen is essential for full transformation of rat fibroblasts in culture (8) and for rapid development of a broad spectrum of tumors in mice (30), for translocation of the GLUT1 transporter (68), and activation of p70 S6 kinase (14). While the mutant 315YF (blocked in PI 3-kinase activation) was able to induce some tumors, it did so at reduced frequencies and with an average latency three times longer than that of either the wild-type virus or a mutant, 250YS, blocked in binding Shc (4, 30). Recent studies have indicated a role of PI 3-kinase in blocking apoptosis in nonviral systems. Growth factor receptors acting through protein tyrosine kinases may prevent apoptosis by activating PI 3-kinase in PC12 cells, T lymphocytes, hematopoietic progenitors, and rat fibroblasts (7, 48, 56, 65, 66). The failure of mutant 315YF to induce full transformation of cells in culture and to induce the rapid development of tumors in mice could therefore be related, at least in part, to a failure to block apoptosis. In this study, we focus on the question of whether middle T antigen–PI 3-kinase interaction is involved in blocking apoptosis in cells transformed by polyomavirus.     

Differential 14-3-3 Affinity Capture Reveals New Downstream Targets of Phosphatidylinositol 3-Kinase Signaling

We devised a strategy of 14-3-3 affinity capture and release, isotope differential (d₀/d₄) dimethyl labeling of tryptic digests, and phosphopeptide characterization to identify novel targets of insulin/IGF1/phosphatidylinositol 3-kinase signaling. Notably four known insulin-regulated proteins (PFK-2, PRAS40, AS160, and MYO1C) had high d₀/d₄ values meaning that they were more highly represented among 14-3-3-binding proteins from insulin-stimulated than unstimulated cells. Among novel candidates, insulin receptor substrate 2, the proapoptotic CCDC6, E3 ubiquitin ligase ZNRF2, and signaling adapter SASH1 were confirmed to bind to 14-3-3s in response to IGF1/phosphatidylinositol 3-kinase signaling. Insulin receptor substrate 2, ZNRF2, and SASH1 were also regulated by phorbol ester via p90RSK, whereas CCDC6 and PRAS40 were not. In contrast, the actin-associated protein vasodilator-stimulated phosphoprotein and lipolysis-stimulated lipoprotein receptor, which had low d₀/d₄ scores, bound 14-3-3s irrespective of IGF1 and phorbol ester. Phosphorylated Ser¹⁹ of ZNRF2 (RTRAYpS¹⁹GS), phospho-Ser⁹⁰ of SASH1 (RKRRVpS⁹⁰QD), and phospho- Ser⁴⁹³ of lipolysis-stimulated lipoprotein receptor (RPRARpS⁴⁹³LD) provide one of the 14-3-3-binding sites on each of these proteins. Differential 14-3-3 capture provides a powerful approach to defining downstream regulatory mechanisms for specific signaling pathways.Activated tyrosine kinase receptors generally drive cells to assimilate nutrients; regulate partitioning of the assimilate to make storage polymers and biosynthetic precursors and for energy production; and promote cellular survival, growth, division, movement, and differentiation. From this spectrum, each cell displays a specific subset of responses depending on the hormone, specific receptors, cross-talk from other signaling pathways, metabolic conditions, and cellular complement of effector proteins. For example, insulin stimulates glucose uptake and glycogen synthesis in skeletal muscle, whereas IGF1¹ promotes survival, growth, and proliferation of many cell types (1, 2).Many of these cellular responses are mediated via PI 3-kinase, which generates phosphatidylinositol 3,4,5-trisphosphate, promoting the activation of AGC protein kinases such as PKB/Akt and other signaling components (1, 3). PI 3-kinase is activated by binding to tyrosine-phosphorylated receptors such as the platelet-derived growth factor receptor or via adaptor molecules such as insulin receptor substrates, which are phosphorylated by the activated insulin receptor. Deregulated PI 3-kinase and downstream signaling has been linked to problems with wound healing, immune responses, neurodegeneration, and cardiovascular disease; decreased PI 3-kinase signaling may underlie insulin resistance and type II diabetes; and this pathway is often activated in human tumors (4, 5). To help pinpoint drug targets for these diseases we must define the mechanisms linking PI 3-kinase and other signaling pathways to downstream effectors and understand specificity with respect to different hormone/cell type combinations.Many missing substrates of PI 3-kinase/AGC kinases must be found to explain all the cellular responses to insulin and growth factors (3). Several targets of PI 3-kinase/PKB signaling, including TSC2 (6), PRAS40 (7), AS160 (8), and FYVE domain-containing phosphatidylinositol 3-phosphate 5-kinase (9) were identified using the anti-PAS antibody, which loosely recognizes the minimal phosphorylated consensus for PKB, which is RXRXX(pS/pT) where pS is phosphoserine and pT is phosphothreonine. Another helpful feature for identifying new downstream targets is that phosphorylation by PKB sometimes creates binding sites for 14-3-3s, which are dimeric proteins that bind to specific phosphorylated sites on target proteins. Thus PKB promotes the binding of 14-3-3s to proteins including PFKFB2 cardiac PFK-2 (10, 11), BimEL (12), β-catenin (13), p27(Kip1) (14), PRAS40 (7), FOXO1 (15), Miz1 (16), TBC1D4 (AS160 (17, 18), and TBC1D1 (19). Functionally 14-3-3s can trigger changes in the conformations of their targets and alter how targets interact with other proteins. Consistent with 14-3-3/target interactions being important in cellular responses to growth factors and insulin, reagents that compete with targets for binding to 14-3-3s inhibit the IGF1-stimulated increase in the glycolytic stimulator fructose-2,6-bisphosphate (10) and PKB-dependent cell survival (20).Some 14-3-3-binding sites on the above named proteins can also be phosphorylated by other basophilic protein kinases (21). For example, AS160 and TBC1D1 are two related RabGAPs (GTPase-activating protein for Rabs) regulated by multisite phosphorylation that regulate trafficking of GluT4 transporter to the plasma membrane for uptake of glucose. The two 14-3-3-binding sites on AS160 can be phosphorylated by PKB, p90RSK, serum- and glucocorticoid-inducible kinase, and other kinases, whereas one of the 14-3-3-binding sites on TBC1D1 is also a substrate of the energy-sensing kinase AMP-activated protein kinase (17–19). Thus, the relative sensitivity of glucose trafficking to insulin and AMP-activated protein kinase activators in different tissues may depend in part on the distribution of AS160 and TBC1D1. Other insulin-regulated 14-3-3 targets, such as myosin 1C (22), are also convergence points for phosphorylation by more than one AGC and/or Ca²⁺/calmodulin-dependent protein kinase.Here many more proteins than those already identified were found to display 14-3-3 and/or PAS binding signals when the PI 3-kinase pathway was activated in cells against a “background” of other proteins whose 14-3-3 and PAS binding status was unaffected by PI 3-kinase signaling. We aimed to pick out the PI 3-kinase-regulated proteins, which was challenging given the hundreds of 14-3-3 binding partners in mammalian cells (10, 23–27). We used 14-3-3 affinity capture and release, identified phosphopeptides, and devised a quantitative proteomics approach in which 14-3-3-binding proteins from insulin-stimulated versus unstimulated cells were labeled with formaldehyde containing light or heavy isotopes, respectively. Biochemical checking of candidates from these screens, which included proteins with links to diabetes, cancers, and neurodegenerative disorders, confirmed the identification of novel downstream targets of PI 3-kinase, some of which are also convergence points for regulation by MAPK/p90RSK signaling.     

Regulation of Phosphatidylinositol Kinases and Metabolism by Wnt3a and Dvl

Wnt signaling plays important roles in various physiological and pathophysiological processes. The pathway that leads to β-catenin stabilization is initiated by Wnt binding to its cell surface receptors, which induces the formation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) via activation of phosphatidylinositol 4-phosphate 5-kinase (PIP5K) type I. Here, we show that Wnt also stimulated the production of phosphatidylinositol 4-phosphate (PtdIns(4)P), which depended on Frizzled (Fz), Dishevelled (Dvl), and phosphatidylinositol 4-kinase (PI4K) type IIα in HEK293T cells. Dvl directly interacted with and activated PI4KIIα by increasing its V_max for ATP and PtdIns. In addition, Dvl regulated PI4KIIα and PIP5KI via different domains. Moreover, Dvl, PI4KIIα, and PIP5KI appeared to form a ternary complex upon Wnt3a stimulation. This complex may allow efficient production of PtdIns(4,5)P₂ from PtdIns, which is far more abundant than PtdIns(4)P in cells. Therefore, this study provides new insights into the mechanism by which Wnt3a regulates the production of PtdIns(4,5)P₂.The Wnt family of secretory glycoproteins plays important roles in regulation of embryonic development and tumorigenesis. They also regulate many other physiological and pathophysiological processes, including bone development, neuronogenesis, adipogenesis, myogenesis, organogenesis, and lipid and glucose metabolism (1–5). Studies using Drosophila and Xenopus embryos as well as mammalian cells have established a canonical Wnt signaling pathway that leads to stabilization of β-catenin. In the absence of Wnt, a number of proteins, including Axin, adenomatous polyposis coli (APC), casein kinase 1 (CK1), glycogen synthase kinase-3β (GSK3β),³ form a complex that facilitates β-catenin phosphorylation by CK1 and GSK3β. This phosphorylation targets β-catenin for ubiquitination and proteasome-mediated proteolytic degradation (3, 6). Some of the Wnt proteins bind to two cell surface receptors Fz and low density lipoprotein receptor-related protein (LRP) 5/6 and initiate a signaling cascade that eventually leads to the suppression of β-catenin phosphorylation by GSK3β and stabilization of β-catenin.Because the finding that the canonical Wnt proteins transduce signals by inducing the interaction between LRP5/6 and Axin (7), more has been learned about the mechanisms by which this interaction is regulated by Wnt proteins. Studies have indicated that two phosphorylation events at the C-terminal intracellular domain of LRP5/6, the phosphorylation of Thr¹⁴⁷⁹ by CKIγ (8, 9) and of Ser¹⁴⁹⁰ by GSK3 (10, 11), were required for the interaction. We recently showed that Wnt3a stimulated the production of PtdIns (4,5)P₂, which in turn regulated the phosphorylation of LRP5/6 at Thr¹⁴⁷⁹ and Ser¹⁴⁹⁰ (12). We also showed that Wnt3a regulated phosphatidylinositol 4-phosphate 5-kinase type I (PIP5KI) activity by inducing the interaction between Dvl and PIP5KI (12). Moreover, Dvl could directly stimulate the lipid kinase activity of PIP5KI (12).PtdIns(4,5)P₂ plays important roles in various cellular functions, including membrane trafficking, cytoskeletal reorganization, migration, ion channel activation, and signal transduction (13). It, however, represents less than 1% of plasma membrane phospholipids and is primarily synthesized in most cells by sequential phosphorylation of PtdIns on the D4 and D5 positions of the inositol ring by two PtdIns kinases, PI4K and PIP5KI, respectively (14, 15). While PtdIns(4)P, the substrate for PIP5KI, is also accounted for around 1% of plasma membrane phospholipids, PtdIns, the substrate for PI4K, is very abundant. Thus, Wnt3a may have to stimulate PI4K activity to provide enough substrate for PIP5KI in PtdIns(4,5)P₂ production.Two types of PI4K (PI4KI and PI4KII) have been characterized in mammalian cells. There are two isoforms of PI4KII (PI4KIIα and PI4KIIβ) and two isoforms of PI4KI (PI4KIα and PI4KIβ) (16). In our previous study, we demonstrated the involvement of PI4KIIα in Wnt signaling. siRNA-mediated knockdown in mammalian cells and morpholino-mediated suppression in Xenopus embryos of PI4KIIα inhibited LRP6 phosphorylation and Wnt signaling. In this report, we examined whether Wnt3a regulates the lipid kinase activity of PI4KIIα and found that Wnt3a could induce an increase in the level of PtdIns(4)P in a Dvl- and Fz-dependent manner. In addition, the Dvl protein was found to directly interact with and activate PI4KIIα. Moreover, different domains of Dvl appeared to be involved in the regulation of PI4KIIα and PIP5KI, and Wnt3a induced the formation of a complex of Dvl, PI4KIIα, and PIP5KI possibly for more efficient production of PtdIns (4,5)P₂ in cells.     

RGS16 Inhibits Breast Cancer Cell Growth by Mitigating Phosphatidylinositol 3-Kinase Signaling

Aberrant activity of the phosphatidylinositol 3-kinase (PI3K) pathway supports growth of many tumors including those of breast, lung, and prostate. Resistance of breast cancer cells to targeted chemotherapies including tyrosine kinase inhibitors (TKI) has been linked to persistent PI3K activity, which may in part be due to increased membrane expression of epidermal growth factor (EGF) receptors (HER2 and HER3). Recently we found that proteins of the RGS (regulator of G protein signaling) family suppress PI3K activity downstream of the receptor by sequestering its p85α subunit from signaling complexes. Because a substantial percentage of breast tumors have RGS16 mutations and reduced RGS16 protein expression, we investigated the link between regulation of PI3K activity by RGS16 and breast cancer cell growth. RGS16 overexpression in MCF7 breast cancer cells inhibited EGF-induced proliferation and Akt phosphorylation, whereas shRNA-mediated extinction of RGS16 augmented cell growth and resistance to TKI treatment. Exposure to TKI also reduced RGS16 expression in MCF7 and BT474 cell lines. RGS16 bound the amino-terminal SH2 and inter-SH2 domains of p85α and inhibited its interaction with the EGF receptor-associated adapter protein Gab1. These results suggest that the loss of RGS16 in some breast tumors enhances PI3K signaling elicited by growth factors and thereby promotes proliferation and TKI evasion downstream of HER activation.The role of the PI3K³ pathway in cell proliferation and survival, adhesion, metabolism, migration, drug resistance, and cytoskeletal rearrangement is well documented (1–3). Mutations in PI3K and dysregulation of the PI3K pathway have been implicated in many human cancers including lymphoma, multiple myeloma, and melanoma (4–8). Because the PI3K signal is a gatekeeper for tumor growth, an understanding of its regulation is critical for the therapeutic intervention of cancer.PI3K, which catalyzes the production of phosphatidylinositol 3,4,5-trisphosphate from phosphatidylinositol 3,4-bisphosphate (9, 10), is activated by extracellular receptor tyrosine kinases including the EGF receptor (EGFR or HER) family, platelet-derived growth factor receptor, and the insulin growth factor receptor. HER stimulation activates Class IA PI3Ks consisting of dimers of p85α or β and either p110α, β, and δ catalytic subunits (11). Tyrosine phosphorylation of the adapter protein Grb2-associated binder 1 (Gab1) recruits p85 to the EGFR complex through a Src homology 2 (SH2) domain in p85 (12), which co-localizes the catalytic p110 subunit and membrane phospholipid substrates at the plasma membrane. Phosphatidylinositol 3,4,5-trisphosphate generated by PI3K activity recruits phosphoinositide-dependent kinase 1 through its pleckstrin homology domain, which in turn phosphorylates the mitogenic and antiapoptotic kinase Akt. Substrates of Akt include mTOR, BAD, IKK, FOXO, p27, MDM2, and GSK3β, all of which are signaling molecules with vital functions in cell cycle regulation and growth (3). Overexpression of Akt has been shown in several tumors such as ovarian and breast carcinoma and may lead directly to transformation of malignant melanoma (5).Proteins of the RGS (regulator of G protein signaling) family mediate cellular desensitization to G protein-coupled receptor stimulation. RGS proteins act as GTPase-accelerating proteins to reduce the life span of activated (GTP-bound) Gα subunits of the G protein-coupled receptor signal-transducing heterotrimeric G protein (13). The R4 subfamily of RGSs (RGS1, 2, 4, 5, 8, 13, 16, 18, and 21) are the smallest members of the family, containing few residues outside of the ∼120-amino acid RGS domain that mediates binding to Gα proteins and GTPase-accelerating protein activity. We found recently that several R4 RGS proteins interacted with the phosphorylated p85α subunit of PI3K (14). In mast cells, RGS13 inhibited PI3K activation induced by high affinity IgE receptor (FcϵRI) cross-linking by antigen. FcϵRI stimulates PI3K by recruiting its catalytic p110δ subunit through p85 binding to a multi-protein complex that includes Gab2 and Grb2 at the plasma membrane (15). PI3K has an essential function in allergic responses (16). As a result of increased PI3K activation, mice deficient in RGS13 had more IgE-mediated mast cell degranulation and anaphylaxis (14).RGS16, an R4 RGS protein homologous to RGS13, was identified originally as a p53 target gene in breast and colon cancer cells (17, 18). Recent analysis of 222 primary breast cancers found a high rate (50%) of genomic instability at the RGS16 locus (19). Because RGS16 associates with both EGFR (20) and p85α (14), we investigated how it affected the growth and survival of breast cancer cells. We found that RGS16 directly bound the amino-terminal SH2 and inter-SH2 domains of phosphorylated p85α, which mediate p110 and adapter binding and membrane localization (21). RGS16 overexpression in MCF7 breast cancer cells suppressed proliferation and EGF-induced Akt phosphorylation, whereas extinction of RGS16 expression increased cell growth and resistance to TKI treatment. Thus, through regulation of PI3K activity, RGS16 may limit proliferation of mammary cells and render cancer cells more susceptible to TKIs or other therapeutic compounds.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Cross-species Proteomics Reveals Specific Modulation of Signaling in Cancer and Stromal Cells by Phosphoinositide 3-kinase (PI3K) Inhibitors

The tumor microenvironment plays key roles in cancer biology, but its impact on the regulation of signaling pathway activity in cancer cells has not been systemically investigated. We designed an analytical strategy that allows differential analysis of signaling between cancer and stromal cells present in tumor xenografts. We used this approach to investigate how in vivo growth conditions and PI3K inhibitors regulate pathway activities in both cancer and stromal cell populations. We found that, despite inducing more modest changes in protein expression, in vivo growing conditions extensively rewired protein kinase networks in cancer cells. As a result, different sets of phosphorylation sites were modulated by PI3K inhibitors in cancer cells growing in tumors relative to when these cells were in culture. The p110δ PI3K-selective compound CAL-101 (Idelalisib) did not inhibit markers of PI3K activity in cancer or stromal cells; however, unexpectedly, it induced phosphorylation on SQ motifs in both subpopulations of tumor cells in vivo but not in vitro. Thus, the interaction between cancer cells and the stroma modulated the ability of PI3K inhibitors to induce the activation of apoptosis in solid tumors. Our study provides proof-of-principle of a proteomics workflow for measuring signaling specifically in cancer and stromal cells and for investigating how cancer biochemistry is modulated in vivo.Solid tumors contain a heterogeneous population of cells. Transformed epithelial cells recruit different types of somatic cells to the tumor microenvironment where they influence varying aspects of cancer biology. The role of heterotypic communication between normal stromal cells and transformed cancer cells is well established (1, 2). Different somatic cell types, including fibroblasts, epithelial cells, and cells of the immune system—all of which are found in tumors—promote cancer cell development by means of gap-junction intercellular communication, direct cell-to-cell contacts, and by the release of growth factors, enzymes, and cytokines that act on neighboring malignant cells (3–6).The tumor microenvironment determines the ability of cancer cells to survive in specific organs and their ability to proliferate and metastasize (7–9). Growth factors released from tumor-associated stromal cells also influence how cancer cells respond to drug administration (10). Therefore, the advancement of targeted cancer therapies requires an understanding of how the tumor microenvironment modulates the biochemistry of transformed cancer cells. In addition, targeting the tumor stroma is emerging as an intriguing concept for the development of anti-cancer therapies (11). It is therefore important to investigate specific effects of compounds in clinical development on stromal cells in addition to those exerted toward malignant cancer cells (12).Here we investigated the effects that changes in growing conditions from a two-dimensional cell culture to an in vivo three-dimensional tumor environment had in modulating protein and phosphoprotein expression in human cancer cells. For this, we used mass spectrometry (MS) to specifically measure cancer and stromal proteomes and phosphoproteomes within mouse tumor xenografts.We also investigated the effects that the pharmacological inhibitors of PI3K, namely GDC-0941 or CAL-101, would have on the phosphoproteomes of stromal cells relative to cancer cells in solid tumors. GDC-0941 is an inhibitor with specificity for class I PI3Ks, whereas CAL-101 specificity is restricted to the p110δ isoform of PI3K (13, 14), which in untransformed tissues is mainly found in leukocytes (15). The PI3K signaling pathway is often deregulated in different cancer types (16), including colorectal cancer (17), and both compounds used in this study are in different stages of clinical development (18–20). PI3K signaling has also been implicated in mediating the effects that the microenvironment has on cancer cells (21).We found that in vivo growth conditions had profound effects on phosphoprotein expression, which was reflected on the phosphorylation sites modulated by PI3K inhibitors in vivo relative to in vitro and in their ability to induce apoptotic markers across these two cell culture conditions.     

Adenovirus Endocytosis via αv Integrins Requires Phosphoinositide-3-OH Kinase

Integrins mediate cell adhesion and motility on the extracellular matrix, yet they also promote viral attachment and/or entry. Evidence is presented that adenovirus internalization by α_v integrins requires activation of phosphoinositide-3-OH kinase (PI3K), whereas α_v integrin-mediated cell motility depends on the ERK1/ERK2 mitogen-activated protein kinase pathway. Interaction of adenovirus with α_v integrins induced activation of PI3K. Pharmacologic or genetic disruption of endogenous PI3K activity blocked adenovirus internalization and virus-mediated gene delivery yet had no effect on integrin-mediated cell adhesion or motility. Therefore, integrin ligation engages distinct signaling pathways that promote viral endocytosis or cell movement.Adenovirus entry into host cells depends on α_v integrin binding to the penton base viral coat protein (2, 20, 48). A highly mobile protrusion on the adenovirus penton base contains the arginine-glycine-aspartic acid (RGD) sequence which mediates α_v integrin binding (42). Integrins are more noted for their ability to mediate cell surface recognition of the extracellular matrix, thereby facilitating adhesion, migration (24), and cell growth and differentiation (28). These interactions have been associated with cell differentiation and tissue development, angiogenesis, wound repair, cancer, and inflammation (22).A number of cell signaling molecules that are associated with integrin-mediated cellular processes, including adhesion, survival, and motility, have recently been identified (18, 32, 34). For example, the signaling molecule pp125^FAK focal adhesion kinase (FAK) (35) is localized to clustered integrins following ligation by extracellular matrix proteins. Engagement (clustering) of integrins by its ligands increases tyrosine phosphorylation and activation of FAK (29). Potential downstream substrates of FAK are the ERK1/ERK2 mitogen-activated protein (MAP) kinases (8, 40) and phosphoinositide-3-OH kinase (PI3K) (7, 17).Recent studies have demonstrated that ligation of α_v and β1 integrins by the extracellular matrix leads to engagement of the ERK1/ERK2 MAP kinase pathway (24). Integrin-mediated regulation of the ERK1/ERK2 MAP kinase pathway results in the activation of myosin light chain kinase and subsequently to phosphorylation of myosin light chains. These molecular events culminate in enhanced cell motility. Cell motility, but not cell adhesion or spreading, can be blocked by ERK antisense oligonucleotides or by the compound PD98059, a specific inhibitor of MEK MAP kinase (24), indicating that the ERK1/ERK2 MAP kinase pathway plays a specific role in cell movement.PI3K (44) is another downstream effector of FAK. PI3K is a member of a family of lipid kinases comprised of a p85 regulatory subunit and a p110 catalytic subunit. The p85 subunit of PI3K binds directly to phosphorylated FAK (6). The products of PI3K activation, phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate (PIP₃), are increased in the plasma membrane of activated but not quiescent cells and have been proposed to act as second messengers for a number of cell functions (5), including cell cycle progression (9) and cytoskeletal changes underlying the cell plasma membrane (47). PI3K activation also modulates intracellular protein trafficking (41), although a direct role of PI3K in receptor-mediated endocytosis has not been established.While integrins play an important role in adenovirus entry and in cell migration, the precise mechanisms by which these receptors promote these distinct biological functions are not known. In the studies reported here, we demonstrate that a specific signaling event is involved in the cell entry of a human viral pathogen. Evidence is provided that PI3K is activated upon adenovirus interaction with α_v integrins and that this event is required for adenovirus internalization. Surprisingly, activation of ERK1/ERK2 following integrin ligation was necessary for cell migration but not for internalization of adenovirus.     

Matricellular Protein CCN3 (NOV) Regulates Actin Cytoskeleton Reorganization

CCN3 (NOV), a putative ligand for integrin receptors, is tightly associated with the extracellular matrix and mediates diverse cellular functions, including cell adhesion and proliferation. CCN3 has been shown to negatively regulate growth although it promotes migration in a cell type-specific manner. In this study, overexpression of CCN3 reduces growth and increases intercellular adhesion of breast cancer cells. Interestingly, CCN3 overexpression also led to the formation of multiple pseudopodia that are enriched in actin, CCN3, and vinculin. Breast cancer cells preincubated with exogenous CCN3 protein also induced the same phenotype, indicating that secreted CCN3 is sufficient to induce changes in cell morphology. Surprisingly, extracellular CCN3 is internalized to the early endosomes but not to the membrane protrusions, suggesting pseudopodia-enriched CCN3 may derive from a different source. The presence of an intracellular variant of CCN3 will be consistent with our finding that the cytoplasmic tail of the gap junction protein connexin43 (Cx43) associates with CCN3. Cx43 is a channel protein permitting intercellular communication to occur. However, neither the channel properties nor the protein levels of Cx43 are affected by the CCN3 protein. In contrast, CCN3 proteins are down-regulated in the absence of Cx43. Finally, we showed that overexpression of CCN3 increases the activity of the small GTPase Rac1, thereby revealing a pathway that links Cx43 directly to actin reorganization.The CCN (CYR61/Connective Tissue Growth Factor/Nephroblastoma Overexpressed) family of multimodular proteins mediates diverse cellular functions, including cell adhesion, migration, and proliferation (1–3). Overexpression of CCN3, one of the founding members of the family, inhibits proliferation in most types of tumors such as glioblastoma and Ewing sarcoma (4, 5). Similarly, down-regulation of CCN3 has been suggested to promote melanoma progression (6). On the other hand, CCN3 can also promote migration in sarcoma and glioblastoma (4, 7), although a separate study shows that it decreases the invasion of melanoma (6). Therefore, in contrast to its role in growth suppression, the role of CCN3 signaling in cell motility is less clear.Most evidence suggests CCN3 mediates its effects by binding to the integrin proteins, such as the αVβ3 receptors (8, 9), and that CCN3 alters cell adhesion in an integrin-dependent fashion (4, 10). In melanocytes, the discoidin domain receptor 1 mediates CCN3-dependent adhesion (11). CCN3 has also been observed to associate with Notch1 (12), fibulin 1C (13), S100A4 (14), and the gap junction protein Cx43³ (15, 16), suggesting that CCN3 may also modulate cell growth via non-integrin signaling pathways.Gap junction proteins are best known for forming channels between cells, contributing to intercellular communication by allowing the exchange of small ions and molecules (17, 18). Consequently, attenuated intercellular communication has been implicated in promoting carcinogenesis (19, 20). Recent evidence has indicated that connexins can mediate channel-independent growth control through interaction of their C-terminal cytoplasmic tail with various intracellular signaling molecules (21–23). In addition, many Cx43-interacting proteins, including ZO-1 (zonula occludens-1) (24), Drebrin (25), and N-cadherin (26) associate with F-actin, thus placing Cx43 in close proximity to the actin cytoskeleton.In this study, we show for the first time that CCN3 reorganizes the actin cytoskeleton of the breast cancer cells MDA-MB-231 with the formation of multiple cell protrusions, possibly by activating the small GTPase Rac1. Our results also suggest an alternative route by which Cx43 may be functionally linked to actin cytoskeletal signaling via CCN3.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.