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.     

Modification of Sialylation Mediates the Invasive Properties and Chemosensitivity of Human Hepatocellular Carcinoma

Aberrant sialylation is closely associated with malignant phenotypes of tumor cells, including invasiveness and metastasis. This study investigated sialylation with regard to the modification of invasive properties and chemosensitivity in human hepatocellular carcinoma (HCC) cell lines and the association between the sialyltransferase gene family and clinicopathological characteristics in HCC patients. Using mass spectrometry analysis, we found that the composition profiling of sialylated N-glycans differed between MHCC97H and MHCC97L cells with different metastatic potential. The expressional profiles of 20 sialyltransferase genes showed differential expression in two cell lines, transitional and tumor tissues, from the same patients. Two genes, ST6GAL1 and ST8SIA2, were detected as overexpressed in MHCC97H and MHCC97L cells. The altered expression levels of ST6GAL1 and ST8SIA2 corresponded to a changed invasive phenotype and chemosensitivity of MHCC97H and MHCC97L cells both in vitro and in vivo. Further data indicated that manipulation of the expression of the two genes led to altered activity of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway. Targeting the PI3K/Akt pathway by its specific inhibitor wortmannin or by Akt RNA interference resulted in a reduced capacity for invasion and chemoresistance of MHCC97H cells. Our results imply that sialylation may function as an internal factor, regulating the invasion and chemosensitivity of HCC, probably through ST6GAL1 or ST8SIA2 regulation of the activity of the PI3K/Akt pathway.Metastasis of tumor cells and the development of resistance to antitumor therapies are the major causes of death in cancer patients. Specific changes in the glycosylation patterns of cell surface glycoproteins have been shown to enhance the metastatic efficiency of tumor cells, in particular that of terminal sialylation (1). It is well known that alterations in cell surface sialylated antigens affect many cellular properties—for example, cell–cell adhesion, cell–extracellular matrix adhesion, immune defense, cell metastasis, and invasion abilities (2–5). Sialyltransferases catalyze the transfer of sialic acid from cytidine 5′-monophospho-N-acetylneuraminic acid to terminal positions of glycoprotein and glycolipid carbohydrate groups (6).The sialyltransferase (ST)¹ family is a family of anabolic enzymes consisting of 20 members that are divided into three subfamilies (7). α-2,3-sialyltransferases mediate the transfer of sialic acid with an α-2,3-linkage to it with terminal Gal residues (ST3GalI-VI). α-2,6-sialyltransferases mediate the transfer of sialic acid with an α-2,6-linkage to it with terminal Gal (ST6GalI-II) (8, 9) or GalNAc residues (ST6GalNAcI-VI). α-2,8-sialyltransferases mediate the transfer of sialic acid with an α-2,8-linkage (ST8SiaI-VI). Changes in specific sialyltransferase expression in several tumors have been reported. ST3GalIII modulates pancreatic cancer cell motility and adhesion in vitro and enhances its metastatic potential in vivo (10). The high expression of ST3GalIV is associated with the malignant behavior of gastric cancer cells (11). ST6GalI is up-regulated in colon adenocarcinoma, and its expression is positively correlated with tumor cell invasiveness and metastasis (12–14). ST6GalNAcI expression is sufficient to enhance the tumorigenicity of MDA-MB-231 breast cancer cells (15). Overexpression of ST6GalNAcII has been correlated with poor patient survival (16). ST6GalNAcV has recently been reported to mediate brain metastasis of breast cancer cells (17). ST8Sia I is also overexpressed in neuroectoderm-derived malignant tumors such as melanoma, glioblastoma, and neuroblastoma, as well as in estrogen receptor negative breast cancer, where it plays a role in cell proliferation, migration, adhesion, and angiogenesis (18).The phosphoinositide 3 kinase (PI3K)/Akt pathway is involved in many cellular processes, including proliferation, differentiation, apoptosis, cell cycle progression, cell motility, tumorigenesis, tumor growth, and angiogenesis (19, 20). In addition, several reports highlight that the PI3K/Akt pathway is responsible for the proliferation, invasion, metastasis, and drug resistance of hepatocellular carcinoma (HCC), and targeting PI3K/AKT inhibits the proliferation and tumorigenesis of HCC cells (21, 22). MicroRNA-7 plays a substantial role in inhibiting the tumorigenesis and reversing the metastasis of HCC through the PI3K/Akt/mTOR signaling pathway in vitro and in vivo (23). The proliferation and invasion of HCC cells are inhibited by lipocalin 2 through the blockade of PI3K/Akt signaling (24). Activation of the PI3K/Akt pathway mediates rapamycin and sorafenib resistance in HCC cells (25, 26). However, little is known about the ST family and its signaling pathway in relation to malignant phenotypes of human HCC.Therefore, the aims of the present study were to determine sialylated oligosaccharide alteration and expression levels of ST genes among the MHCC97H and MHCC97L cell lines and HCC patient cells by using MS and real-time PCR. In addition, we investigated whether the ST gene family participates in the regulation of tumor invasion and chemosensitivity via the PI3K/Akt pathway and the possible mechanisms.     

Cdc42 and the Phosphatidylinositol 3-Kinase-Akt Pathway Are Essential for PspC-mediated Internalization of Pneumococci by Respiratory Epithelial Cells

The pneumococcal surface protein C (PspC) is a major adhesin of Streptococcus pneumoniae, the cause of lobar pneumonia and invasive diseases. PspC interacts in a human-specific manner with the ectodomain of the human polymeric immunoglobulin receptor (pIgR) produced by respiratory epithelial cells. By adopting the retrograde machinery of human pIgR, this protein-protein interaction promotes colonization and transcytosis across the epithelial layer. Here, we explored the role of Rho family guanosine triphosphatases (GTPases), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) for ingestion of pneumococci via the human pIgR. Inhibition experiments suggested that the host-cell actin microfilaments and microtubules are essential for this pneumococcal uptake mechanism. By using specific GTPase-modifying toxins, inhibitors, and GTPase expression constructs we demonstrate that Cdc42, but not Rac1 and RhoA are involved in PspC-mediated invasion of pneumococci into host cells. Accordingly, Cdc42 is time-dependently activated during ingestion of pneumococci. In addition, PI3K and Akt are essential for ingestion of pneumococci by respiratory epithelial cells via the PspC-pIgR interaction. The subunit p85α of PI3K and Akt was activated during the infection process. Moreover, Akt activation upon pneumococcal invasion depends on PI3K. In conclusion, our results illustrate for the first time key signaling molecules of host cells that are required for PspC-pIgR-mediated invasion of pneumococci into epithelial cells. This unique and specific bacterial entry process is dependent on the cooperation and activation of Rho family GTPase Cdc42, PI3K, and Akt.Streptococcus pneumoniae (pneumococci) is (are) the etiologic agent of community-acquired pneumonia and life-threatening invasive diseases such as septicemia and bacterial meningitis (1). Pneumococci use several strategies to colonize the respiratory tract, which is considered to be the initial and essential step prior to their transmigration into the lungs and bloodstream. Adherence of pneumococci to host cells is facilitated by serum or matrix proteins such as Factor H, thrombospondin-1, and vitronectin (2–4). More importantly, pneumococci produce adhesins, which interact directly with cellular receptors and, consequently, these interactions promote bacterial adherence to and invasion into host cells (5). The pneumococcal surface protein C (PspC),³ also referred to as CbpA or SpsA, is a multifunctional choline-binding protein and a major adhesin of pneumococci residing on mucosal respiratory surfaces. PspC interacts directly and in a human-specific manner with the ectodomain of the polymeric immunoglobulin receptor (pIgR), which is also known as the secretory component (SC) (6). The PspC-hpIgR interaction has been characterized in detail on the molecular level and also on the structural level with regard to the PspC protein. A hexameric peptide within the N-terminal repeat domains (termed R1 or R2) of PspC recognizes human-specific amino acids in ectodomains D3 and D4 of pIgR (6–9). After binding to pIgR, pneumococci are ingested and transcytosed across epithelial cells by adopting the pIgR retrograde transcytosis machinery (7, 10). Additionally, the N terminus of PspC interacts in a human-specific manner with the innate immune regulator Factor H, and this interaction mediates immune evasion and adherence to host cells (2, 11–13).The pIgR, which is broadly expressed by epithelial cells of the respiratory tract, mediates the transport of polymeric IgA (dIgA) or pIgM across the mucosal epithelial barriers from the basolateral to apical surface (14). Although unloaded pIgR undergoes constitutive transcytosis, binding of dIgA stimulates the receptor transcytosis in in vitro and in vivo situations (15, 16). The model of pIgR-dIgA transcytosis from the basolateral to the apical cell surface is based largely on studies using Madin-Darby canine kidney (MDCK) cells expressing exogenous rabbit or rat pIgR (15–17). The studies provided important insights into receptor sorting, intracellular compartments involved in transcytosis, and regulation of the endocytic pathways (14). After endocytosis in clathrin-coated vesicles at the basolateral surface, pIgR is delivered in an actin- and microtubule-dependent manner to the common recycling endosomes. At the apical surface unloaded receptor can be recycled and transported in retrograde. The dIgA-stimulated pIgR transcytosis is regulated by Rho family GTPases, phosphatidylinositol-3-kinase (PI3K), and requires the production of secondary messengers, including inositol 1,4,5-triphosphate and free intracellular calcium (17–23). In addition, the activation of these signaling molecules depends on the Src family protein tyrosine kinase p62^yes and may stimulate a network of downstream pathways (24). Although it has become clear that pneumococci can adopt the pIgR-transcytosis machinery for invasion, the induced signal transduction cascades have not yet been explored. The goal of this study was, therefore, to assess the induced intracellular signaling pathways during PspC-hpIgR-mediated pneumococcal invasion into host cells. We asked whether this process depends on the dynamics of the actin cytoskeleton as suggested by earlier observations by electron microscopy (5) and which member(s) of the Rho family of small GTPases are the key players in this uptake mechanism. In addition, we have analyzed the role of the PI3K and of protein kinase B (Akt; also known as PKB). Akt is phosphorylated during activation, and phosphorylation at Ser-473 depends on PI3K activity (25, 26). By using GTPase-modifying toxins, pharmacological inhibitors, GTPase constructs, and GTPase activation assays we demonstrate for the first time that pneumococcal invasion via the PspC-hpIgR interaction requires the small GTPase member Cdc42, PI3K, and Akt activity.     

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.     

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.     

Phosphorylation of RACK1 on Tyrosine 52 by c-Abl Is Required for Insulin-like Growth Factor I-mediated Regulation of Focal Adhesion Kinase

Focal Adhesion Kinase (FAK) activity is controlled by growth factors and adhesion signals in tumor cells. The scaffolding protein RACK1 (receptor for activated C kinases) integrates insulin-like growth factor I (IGF-I) and integrin signaling, but whether RACK1 is required for FAK function is unknown. Here we show that association of FAK with RACK1 is required for both FAK phos pho ryl a tion and dephos pho ryl a tion in response to IGF-I. Suppression of RACK1 by small interfering RNA ablates FAK phos pho ryl a tion and reduces cell adhesion, cell spreading, and clonogenic growth. Peptide array and mutagenesis studies localize the FAK binding interface to blades I-III of the RACK1 β-propeller and specifically identify a set of basic and hydrophobic amino acids (Arg-47, Tyr-52, Arg-57, Arg-60, Phe-65, Lys-127, and Lys-130) as key determinants for association with FAK. Mutation of tyrosine 52 alone is sufficient to disrupt interaction of RACK1 with FAK in cells where endogenous RACK1 is suppressed by small interfering RNA. Cells expressing a Y52F mutant RACK1 are impaired in adhesion, growth, and foci formation. Comparative analyses of homology models and crystal structures for RACK1 orthologues suggest a role for Tyr-52 as a site for phos pho ryl a tion that induces conformational change in RACK1, switching the protein into a FAK binding state. Tyrosine 52 is further shown to be phos pho ryl a ted by c-Abl kinase, and the c-Abl inhibitor STI571 disrupts FAK interaction with RACK1. We conclude that FAK association with RACK1 is regulated by phos pho ryl a tion of Tyr-52. Our data reveal a novel mechanism whereby IGF-I and c-Abl control RACK1 association with FAK to facilitate adhesion signaling.RACK1² is a tryptophan-aspartate (WD) repeat containing protein that acts as a scaffolding protein in a wide array of signaling events (1, 2). It has been reported to both regulate and promote cell migration in different cell types (3–5). RACK1 scaffolds proteins at focal adhesions and is capable of mediating both focal adhesion assembly and disassembly (4, 6, 7). RACK1 also scaffolds core kinases of the ERK pathway in response to adhesion signals and modulates the phosphorylation of focal adhesion proteins including focal adhesion kinase (FAK) and paxillin (8, 9). In transformed cells RACK1 integrates signaling from the IGF-I receptor (IGF-IR) and β1 integrin by forming a scaffolding complex that includes these receptors as well as signaling molecules that promote cell migration (5, 10, 11). Cooperation between IGF-IR and β1 integrin signaling is essential for growth of certain tumors (12), and we propose that RACK1 has an important role in this.The interaction of RACK1 with the IGF-IR requires integrins to be ligated and also requires a domain in the C terminus of the IGF-IR that is essential for IGF-IR function in anchorage-independent growth, cell survival, and cell migration (13, 14). Ligand-mediated activation of the IGF-IR leads to recruitment of certain proteins to RACK1 such as IRS-1, β1 integrin, and dissociation of other proteins from RACK1 such as PP2A and Src. Competitive binding to RACK1 occurs for some of these proteins. For example, IGF-I-mediated dissociation of PP2A from RACK1 is required for recruitment of β1 integrin, and both PP2A and β1 integrin compete for binding to tyrosine 302 in RACK1 (5, 15).RACK1 is located in areas of cell protrusion that are rich in paxillin (4, 7) and can increase the phosphorylation of FAK (7). FAK is a well characterized kinase in mediating integrin signaling and is associated with the enhanced migratory potential of several cancer cell types (16–18). FAK is phosphorylated on tyrosine 397 in response to the clustering of integrins (for review, see Ref. 19) or by activation of the EGF and platelet-derived growth factor receptors (20–23). This results in recruitment of Src and subsequent phosphorylation of target proteins that are associated with focal adhesion formation and activation of mitogen-activated protein kinase pathways. FAK becomes rapidly dephosphorylated when cells are detached, and this is thought to be essential for focal adhesion dissolution and cell migration. FAK dephosphorylation can be stimulated by IGF-I (5, 24–27). Interestingly, we have observed that IGF-I-mediated dephosphorylation of FAK is enhanced in cells overexpressing RACK1, which also have enhanced migratory potential and increased activation of mitogen-activated protein kinase pathways (28). However, it is not known how the phosphorylation and subsequent dephosphorylation of FAK are coordinated. In particular, the role of RACK1 in regulation of FAK phosphorylation remains undefined. Here we investigated this in the context of IGF-I and adhesion signaling by determining the role of RACK1 in FAK function.     

Functional Proteomics Identifies Acinus L as a Direct Insulin- and Amino Acid-Dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Substrate

The serine/threonine kinase mammalian target of rapamycin (mTOR) governs growth, metabolism, and aging in response to insulin and amino acids (aa), and is often activated in metabolic disorders and cancer. Much is known about the regulatory signaling network that encompasses mTOR, but surprisingly few direct mTOR substrates have been established to date. To tackle this gap in our knowledge, we took advantage of a combined quantitative phosphoproteomic and interactomic strategy. We analyzed the insulin- and aa-responsive phosphoproteome upon inhibition of the mTOR complex 1 (mTORC1) component raptor, and investigated in parallel the interactome of endogenous mTOR. By overlaying these two datasets, we identified acinus L as a potential novel mTORC1 target. We confirmed acinus L as a direct mTORC1 substrate by co-immunoprecipitation and MS-enhanced kinase assays. Our study delineates a triple proteomics strategy of combined phosphoproteomics, interactomics, and MS-enhanced kinase assays for the de novo-identification of mTOR network components, and provides a rich source of potential novel mTOR interactors and targets for future investigation.The serine/threonine kinase mammalian target of rapamycin (mTOR)¹ is conserved in all eukaryotes from yeast to mammals (1). mTOR is a central controller of cellular growth, whole body metabolism, and aging, and is frequently deregulated in metabolic diseases and cancer (2). Consequently, mTOR as well as its upstream and downstream cues are prime candidates for targeted drug development to alleviate the causes and symptoms of age-related diseases (3, 4). The identification of novel mTOR regulators and effectors thus remains a major goal in biomedical research. A vast body of literature describes a complex signaling network around mTOR. However, our current comparatively detailed knowledge of mTOR''s upstream cues contrasts with a rather limited set of known direct mTOR substrates.mTOR exists in two structurally and functionally distinct multiprotein complexes, termed mTORC1 and mTORC2. Both complexes contain mTOR kinase as well as the proteins mLST8 (mammalian lethal with SEC thirteen 8) (5–7), and deptor (DEP domain-containing mTOR-interacting protein) (8). mTORC1 contains the specific scaffold protein raptor (regulatory-associated protein of mTOR) (9, 10), whereas mTORC2 contains the specific binding partners rictor (rapamycin-insensitive companion of mTOR) (5–7), mSIN1 (TORC2 subunit MAPKAP1) (11–13), and PRR5/L (proline rich protein 5/-like) (14–16). The small macrolide rapamycin acutely inhibits mTORC1, but can also have long-term effects on mTORC2 (17, 18). More recently, ATP-analogs (19) that block both mTOR complexes, such as Torin 1 (20), have been developed. As rapamycin has already been available for several decades, our knowledge of signaling events associated with mTORC1 as well as its metabolic inputs and outputs is much broader as compared with mTORC2. mTORC1 responds to growth factors (insulin), nutrients (amino acids, aa) and energy (ATP). In response, mTORC1 activates anabolic processes (protein, lipid, nucleotide synthesis) and blocks catabolic processes (autophagy) to ultimately allow cellular growth (21). The insulin signal is transduced to mTORC1 via the insulin receptor (IR), and the insulin receptor substrate (IRS), which associates with class I phosphoinositide 3-kinases (PI3Ks). Subsequent phosphatidylinositol 3,4,5 trisphosphate (PIP3) binding leads to relocalization of the AGC kinases phosphoinositide-dependent protein kinase 1 (PDK1) and Akt (also termed protein kinase B, PKB) to the plasma membrane, where PDK1 phosphorylates Akt at T308 (22, 23). In response, Akt phosphorylates and inhibits the heterocomplex formed by the tuberous sclerosis complex proteins 1 and 2 (TSC1-TSC2) (24, 25). TSC1-TSC2 is the inhibitory, GTPase-activating protein for the mTORC1-inducing GTPase Ras homolog enriched in brain (rheb) (26–30), which activates mTORC1 at the lysosome. mTORC1 localization depends on the presence of aa, which in a rag GTPase-dependent manner induce mTORC1 relocalization to lysosomes (31, 32). Low energy levels are sensed by the AMP-dependent kinase (AMPK), which in turn phosphorylates the TSC1-TSC2 complex (33) and raptor (34), thereby inhibiting mTORC1.mTORC1 phosphorylates its well-described downstream substrate S6-kinase (S6K) at T389, the proline-rich Akt substrate of 40 kDa (PRAS40) at S183, and the translational repressor 4E-binding protein (4E-BP) at T37/46 (35–41). Unphosphorylated 4E-BP binds and inhibits the translation initiation factor 4G (eIF4G), which within the eIF4F complex mediates the scanning process of the ribosome to reach the start codon. Phosphorylation by mTORC1 inhibits 4E-BP''s interaction with eIF4E, thus allowing for assembly of eIF4F, and translation initiation (42, 43). More recently, also the IR-activating growth factor receptor-bound protein 10 (Grb10) (44, 45), the autophagy-initiating Unc-51-like kinase ULK1 (46), and the trifunctional enzymatic complex CAD composed of carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase (47, 48), which is required for nucleotide synthesis, have been described as direct mTORC1 substrates.mTORC2 activation is mostly described to be mediated by insulin, and this is mediated by a PI3K variant that is distinct from the PI3K upstream of mTORC1 (49, 50). Furthermore, mTORC2 responds to aa (5, 51). In response, mTORC2 phosphorylates the AGC kinases Akt at S473 (52–55), and serum and glucocorticoid kinase SGK (56) and protein kinase C alpha (PKCalpha) (7) within their hydrophobic motifs (57, 58), to control cellular motility (5–7), hepatic glycolysis, and lipogenesis (59). In addition, mTOR autophosphorylation at S2481 has been established as an mTORC2 readout in several cell lines including HeLa cells (49).Given the multiplicity of effects via which mTOR controls cellular and organismal growth and metabolism, it is surprising that only relatively few direct mTOR substrates have been established to date. Proteomic studies are widely used to identify novel interactors and substrates of protein kinases. Two studies have recently shed light on the interaction of rapamycin and ATP-analog mTOR inhibitors with TSC2 inhibition in mammalian cells (44, 45), and one study has analyzed the effects of raptor and rictor knockouts in non-stimulated cells (48).In this work, we report a functional proteomics approach to study mTORC1 substrates. We used an inducible raptor knockdown to inhibit mTORC1 in HeLa cells, and analyzed the effect in combination with insulin and aa induction by quantitative phosphoproteomics using stable isotope labeling by amino acids in cell culture (SILAC) (60). In parallel, we purified endogenous mTOR complexes and studied the interactome of mTOR by SILAC-MS. Through comparative data evaluation, we identified acinus L as a potential novel aa/insulin-sensitive mTOR substrate. We further validated acinus L by co-immunoprecipitation and MS-enhanced kinase assays as a new direct mTORC1 substrate.     

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.     

Quantitative Proteomics with siRNA Screening Identifies Novel Mechanisms of Trastuzumab Resistance in HER2 Amplified Breast Cancers

HER2 is a receptor tyrosine kinase that is overexpressed in 20% to 30% of human breast cancers and which affects patient prognosis and survival. Treatment of HER2-positive breast cancer with the monoclonal antibody trastuzumab (Herceptin) has improved patient survival, but the development of trastuzumab resistance is a major medical problem. Many of the known mechanisms of trastuzumab resistance cause changes in protein phosphorylation patterns, and therefore quantitative proteomics was used to examine phosphotyrosine signaling networks in trastuzumab-resistant cells. The model system used in this study was two pairs of trastuzumab-sensitive and -resistant breast cancer cell lines. Using stable isotope labeling, phosphotyrosine immunoprecipitations, and online TiO₂ chromatography utilizing a dual trap configuration, ∼1700 proteins were quantified. Comparing quantified proteins between the two cell line pairs showed only a small number of common protein ratio changes, demonstrating heterogeneity in phosphotyrosine signaling networks across different trastuzumab-resistant cancers. Proteins showing significant increases in resistant versus sensitive cells were subjected to a focused siRNA screen to evaluate their functional relevance to trastuzumab resistance. The screen revealed proteins related to the Src kinase pathway, such as CDCP1/Trask, embryonal Fyn substrate, and Paxillin. We also identify several novel proteins that increased trastuzumab sensitivity in resistant cells when targeted by siRNAs, including FAM83A and MAPK1. These proteins may present targets for the development of clinical diagnostics or therapeutic strategies to guide the treatment of HER2+ breast cancer patients who develop trastuzumab resistance.HER2 is a member of the epidermal growth factor receptor (EGFR)/ErbB family of receptor tyrosine kinases. Under normal physiologic conditions, HER2 tyrosine kinase signaling is tightly regulated spatially and temporally by the requirement for it to heterodimerize with a ligand bound family member, such as EGFR, HER3/ErbB3, or HER4/ErbB4 (1). However, in 20% to 30% of human breast cancer cases, HER2 gene amplification is present, resulting in a high level of HER2 protein overexpression and unregulated, constitutive HER2 tyrosine kinase signaling (2, 3). HER2 gene amplified breast cancer, also termed HER2-positive breast cancer, carries a poor prognosis, but the development of the HER2 targeted monoclonal antibody trastuzumab (Herceptin) has significantly improved patient survival (2). Despite the clinical effectiveness of trastuzumab, the development of drug resistance significantly increases the risk of patient death. This poses a major medical problem, as most metastatic HER2-positive breast cancer patients develop trastuzumab resistance over the course of their cancer treatment (4). The treatment approach for HER2+ breast cancer patients after trastuzumab resistance develops is mostly a trial-and-error process that subjects the patient to increased toxicity. Therefore, there is a substantial medical need for strategies to overcome trastuzumab resistance.Multiple trastuzumab-resistance mechanisms have been identified, and they alter signaling networks and protein phosphorylation patterns in either a direct or an indirect manner. These mechanisms can be grouped into three categories. The first category is the activation of a parallel signaling network by other tyrosine kinases. These kinases include the receptor tyrosine kinases, EGFR, IGF1R, Her3, Met, EphA2, and Axl, as well as the erythropoietin-receptor-mediated activation of the cytoplasmic tyrosine kinases Jak2 and Src (5–11). The second category is the activation of downstream signaling proteins. Multiple studies have demonstrated activation of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway in trastuzumab resistance, which occurs either via deletion of the PTEN lipid phosphatase or mutation of the PI3K genes (12, 13). Activation of Src family kinases or overexpression of cyclin E, which increases the cyclin E–cyclin-dependent kinase 2 signaling pathway, has also been reported (14). The third category includes mechanisms that maintain HER2 signaling even in the presence of trastuzumab. The production of a truncated isoform of HER2, p95HER2, which lacks the trastuzumab binding site, causes constitutive HER2 signaling (15, 16). Overexpression of the MUC4 sialomucin complex inhibits trastuzumab binding to HER2 and thereby maintains HER2 signaling (17, 18).Given that multiple trastuzumab-resistance mechanisms alter signaling networks and protein phosphorylation patterns, we reasoned that mapping phosphotyrosine signaling networks using quantitative proteomics would be a highly useful strategy for analyzing known mechanisms and identifying novel mechanisms of trastuzumab resistance. Quantitative proteomics and phosphotyrosine enrichment approaches have been extensively used to study the EGFR signal networks (19–23). We and others have used these approaches to map the HER2 signaling network (22, 24, 25). Multiple other tyrosine kinase signaling networks were analyzed using quantitative proteomics, including Ephrin receptor, EphB2 (26–28), platelet-derived growth factor receptor (PDGFR) (21), insulin receptor (29, 30), and the receptor for hepatocyte growth factor, c-MET (31).The goal of this study is to identify, quantify, and functionally screen proteins that might be involved in trastuzumab resistance. We used two pairs of HER2 gene amplified trastuzumab-sensitive (parental, SkBr3 and BT474) and -resistant (SkBr3^R and BT474^R) human breast cancer cell lines as models for trastuzumab resistance. These cell lines and their trastuzumab-resistant derivatives have been extensively characterized and highly cited in the breast cancer literature (32, 33). Using stable isotope labeling of amino acids in cell culture (SILAC),¹ phosphotyrosine immunoprecipitations, and online TiO₂ chromatography with dual trap configuration, we quantified the changes in phosphotyrosine containing proteins and interactors between trastuzumab-sensitive and -resistant cells. Several of the known trastuzumab-resistance mechanisms were identified, which serves as a positive control and validation of our approach, and large protein ratio changes were measured in proteins that had not been previously connected with trastuzumab resistance. We then performed a focused siRNA screen targeting the proteins with significantly increased protein ratios. This screen functionally tested the role of the identified proteins and identifies which proteins might have the largest effect on reversing trastuzumab resistance.     

Potentiation of Growth Factor Signaling by Insulin-like Growth Factor-binding Protein-3 in Breast Epithelial Cells Requires Sphingosine Kinase Activity

We have investigated the mechanism underlying potentiation of epidermal growth factor receptor (EGFR) and type 1 insulin-like growth factor receptor (IGFR1) signaling by IGF-binding protein-3 (IGFBP-3) in MCF-10A breast epithelial cells, focusing on a possible involvement of the sphingosine kinase (SphK) system. IGFBP-3 potentiated EGF-stimulated EGF receptor activation and DNA synthesis, and this was blocked by inhibitors of SphK activity or small interference RNA-mediated silencing of SphK1, but not SphK2, expression. Similarly, IGFR1 phosphorylation and DNA synthesis stimulated by LR3-IGF-I (an IGF-I analog not bound by IGFBP-3), were enhanced by IGFBP-3, and this was blocked by SphK1 silencing. SphK1 expression and activity were stimulated by IGFBP-3 ∼2-fold over 24 h. Silencing of sphingosine 1-phosphate receptor 1 (S1P₁) or S1P₃, but not S1P₂, abolished the effect of IGFBP-3 on EGF-stimulated EGFR activation. The effects of IGFBP-3 could be reproduced with exogenous S1P or medium conditioned by cells treated with IGFBP-3, and this was also blocked by inhibition of S1P₁ and S1P₃. These data indicate that potentiation of growth factor signaling by IGFBP-3 in MCF-10A cells requires SphK1 activity and S1P₁/S1P₃, suggesting that S1P, the product of SphK activity and ligand for S1P₁ and S1P₃, is the “missing link” mediating IGF and EGFR transactivation and cell growth stimulation by IGFBP-3.Insulin-like growth factor-binding protein-3 (IGFBP-3)² is one of the family of six IGFBPs that bind the peptide growth factors IGF-I and IGF-II with high affinity and regulate their bioactivity (1). As the predominant carrier of IGFs in the endocrine system, IGFBP-3 regulates the movement of these growth factors from the circulation to target tissues and inhibits their proliferative and antiapoptotic cellular effects by blocking their activation of the type 1 IGF receptor (IGFR1) at the cell surface. In vitro studies in a variety of cell types have revealed that IGFBP-3 may also impact on cell growth and survival independently of modulating IGF bioactivity, inducing cell cycle arrest and apoptosis by regulation of apoptotic effector proteins (2–4) and interaction with nuclear receptors (5–7).There is, however, also evidence of an association between IGFBP-3 and enhanced cell proliferation. Some clinical studies in breast, prostate, pancreatic, renal cell, and non-small cell lung cancers have shown that a high level of tissue expression of IGFBP-3 correlates with increased tumor growth or malignancy (8–13). Although the mechanism linking IGFBP-3 with growth stimulation in vivo remains unclear, we and others have shown that, in vitro, IGFBP-3 can enhance the effects of stimulatory growth factors. Human and bovine skin fibroblasts exposed to low concentrations of exogenous IGFBP-3 exhibit enhanced IGF-stimulated DNA synthesis (14, 15), and similarly, exogenous and endogenous IGFBP-3 enhanced the growth response to IGF-I in the MCF-7 breast cancer cell line (16). We have also shown previously that IGFBP-3 is inhibitory to DNA synthesis in MCF-10A breast epithelial cells in the absence of exogenous growth factors or serum (17), but is growth stimulatory in the presence of EGF in the same cell line (18). There is no evidence that potentiation of EGF or IGF bioactivity by IGFBP-3 requires direct interaction between IGFBP-3 and the growth factor receptors (15, 18), but the mechanism underlying the effects of IGFBP-3 on growth factor signaling has not been elucidated.Recently it was suggested that, in human umbilical vein endothelial cells, an antiapoptotic effect of IGFBP-3 is associated with increased expression and activity of sphingosine kinase 1 (SphK1), and formation of the bioactive sphingolipid sphingosine 1-phosphate (S1P) (19, 20). SphK1 has been shown to have a role in oncogenesis (21), and S1P, acting both as an intracellular second messenger and extracellularly through activation of specific S1P receptors, stimulates cell proliferation and survival (22). In addition to transducing S1P signaling, the G-protein-coupled S1P receptors have been implicated in signal amplification of a variety of growth factors receptors, including the EGF and platelet-derived growth factor receptors, via receptor transactivation (23, 24). In this study we investigated whether the sphingosine kinase system is involved in modulation of growth factor receptor signaling pathways by IGFBP-3 and demonstrate that SphK1 expression is stimulated by IGFBP-3 in MCF-10A cells, and its activity is required for potentiation of EGF and IGF-I signaling by IGFBP-3 in these cells.