Involvement of Grb2 Adaptor Protein in Nucleophosmin-Anaplastic Lymphoma Kinase (NPM-ALK)-mediated Signaling and Anaplastic Large Cell Lymphoma Growth

Most anaplastic large cell lymphomas (ALCL) express oncogenic fusion proteins derived from chromosomal translocations or inversions of the anaplastic lymphoma kinase (ALK) gene. Frequently ALCL carry the t(2;5) translocation, which fuses the ALK gene to the nucleophosmin (NPM1) gene. The transforming activity mediated by NPM-ALK fusion induces different pathways that control proliferation and survival of lymphoma cells. Grb2 is an adaptor protein thought to play an important role in ALK-mediated transformation, but its interaction with NPM-ALK, as well as its function in regulating ALCL signaling pathways and cell growth, has never been elucidated. Here we show that active NPM-ALK, but not a kinase-dead mutant, bound and induced Grb2 phosphorylation in tyrosine 160. An intact SH3 domain at the C terminus of Grb2 was required for Tyr¹⁶⁰ phosphorylation. Furthermore, Grb2 did not bind to a single region but rather to different regions of NPM-ALK, mainly Tyr^152–156, Tyr⁵⁶⁷, and a proline-rich region, Pro^415–417. Finally, shRNA knockdown experiments showed that Grb2 regulates primarily the NPM-ALK-mediated phosphorylation of SHP2 and plays a key role in ALCL cell growth.     

Identification and characterization of a nuclear interacting partner of anaplastic lymphoma kinase (NIPA)

Anaplastic large-cell lymphoma is a subtype of non-Hodgkin lymphomas characterized by the expression of CD30. More than half of these lymphomas carry a chromosomal translocation t(2;5) leading to expression of the oncogenic tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). NPM-ALK is capable of transforming fibroblasts and lymphocytes in vitro and of causing lymphomas in mice. Previously, we and others demonstrated phospholipase C-gamma and phosphatidylinositol 3-kinase as crucial downstream signaling mediators of NPM-ALK-induced oncogenicity. In this study, we used an ALK fusion protein as bait in a yeast two-hybrid screen identifying NIPA (nuclear interacting partner of ALK) as a novel downstream target of NPM-ALK. NIPA encodes a 60-kDa protein that is expressed in a broad range of human tissues and contains a classical nuclear translocation signal in its C terminus, which directs its nuclear localization. NIPA interacts with NPM-ALK and other ALK fusions in a tyrosine kinase-dependent manner and is phosphorylated in NPM-ALK-expressing cells on tyrosine and serine residues with serine 354 as a major phosphorylation site. Overexpression of NIPA in Ba/F3 cells was able to protect from apoptosis induced by IL-3 withdrawal. Mutations of the nuclear translocation signal or the Ser-354 phosphorylation site impaired the antiapoptotic function of NIPA. In NPM-ALK-transformed Ba/F3 cells, apoptosis triggered by wortmannin treatment was enhanced by overexpression of putative dominant-negative NIPA mutants. These results implicate an antiapoptotic role for NIPA in NPM-ALK-mediated signaling events.     

Mechanism of Phosphorylation-induced Activation of Phospholipase C-γ Isozymes

The lipase activity of most phospholipases C (PLCs) is basally repressed by a highly degenerate and mostly disordered X/Y linker inserted within the catalytic domain. Release of this auto-inhibition is driven by electrostatic repulsion between the plasma membrane and the electronegative X/Y linker. In contrast, PLC-γ isozymes (PLC-γ1 and -γ2) are structurally distinct from other PLCs because multiple domains are present in their X/Y linker. Moreover, although many tyrosine kinases directly phosphorylate PLC-γ isozymes to enhance their lipase activity, the underlying molecular mechanism of this activation remains unclear. Here we define the mechanism for the unique regulation of PLC-γ isozymes by their X/Y linker. Specifically, we identify the C-terminal SH2 domain within the X/Y linker as the critical determinant for auto-inhibition. Tyrosine phosphorylation of the X/Y linker mediates high affinity intramolecular interaction with the C-terminal SH2 domain that is coupled to a large conformational rearrangement and release of auto-inhibition. Consequently, PLC-γ isozymes link phosphorylation to phospholipase activation by elaborating upon primordial regulatory mechanisms found in other PLCs.     

PLC-γ1 Signaling Plays a Subtype-Specific Role in Postbinding Cell Entry of Influenza A Virus

Host signaling pathways and cellular proteins play important roles in the influenza viral life cycle and can serve as antiviral targets. In this study, we report the engagement of host phosphoinositide-specific phospholipase γ1 (PLC-γ1) in mediating cell entry of influenza virus H1N1 but not H3N2 subtype. Both PLC-γ1-specific inhibitor and short hairpin RNA (shRNA) strongly suppress the replication of H1N1 but not H3N2 viruses in cell culture, suggesting that PLC-γ1 plays an important subtype-specific role in the influenza viral life cycle. Further analyses demonstrate that PLC-γ1 activation is required for viral postbinding cell entry. In addition, H1N1, but not H3N2, infection leads to the phosphorylation of PLC-γ1 at Ser 1248 immediately after infection and independent of viral replication. We have further shown that H1N1-induced PLC-γ1 activation is downstream of epidermal growth factor receptor (EGFR) signaling. Interestingly, both H1N1 and H3N2 infections activate EGFR, but only H1N1 infection leads to PLC-γ1 activation. Taking our findings together, we have identified for the first time the subtype-specific interplay of host PLC-γ1 signaling and H1N1 virus that is critical for viral uptake early in the infection. Our study provides novel insights into how virus interacts with the cellular signaling network by demonstrating that viral determinants can regulate how the host signaling pathways function in virally infected cells.     

Compartmentalized IgE Receptor–mediated Signal Transduction in Living Cells

Several receptor-mediated signal transduction pathways, including EGF and IgE receptor pathways, have been proposed to be spatially restricted to plasma membrane microdomains. However, the experimental evidence for signaling events in these microdomains is largely based on biochemical fractionation and immunocytochemical studies and only little is known about their spatial dynamics in living cells. Here we constructed green fluorescent protein–tagged SH2 domains to investigate where and when IgE receptor (FcεRI)–mediated tyrosine phosphorylation occurs in living tumor mast cells. Strikingly, within minutes after antigen addition, tandem SH2 domains from Syk or PLC-γ1 translocated from a uniform cytosolic distribution to punctuate plasma membrane microdomains. Colocalization experiments showed that the microdomains where tyrosine phosphorylation occurred were indistinguishable from those stained by cholera toxin B, a marker for glycosphingolipids. Competitive binding studies with coelectroporated unlabeled Syk, PLC-γ1, and other SH2 domains selectively suppressed the induction of IgE receptor–mediated calcium signals as well as the binding of the fluorescent SH2 domains. This supports the hypothesis that PLC-γ1 and Syk SH2 domains selectively bind to Syk and IgE receptors, respectively. Unlike the predicted prelocalization of EGF receptors to caveolae microdomains, fluorescently labeled IgE receptors were found to be uniformly distributed in the plasma membrane of unstimulated cells and only transiently translocated to glycosphingolipid rich microdomains after antigen addition. Thus, these in vivo studies support a plasma membrane signaling mechanism by which IgE receptors transiently associate with microdomains and induce the spatially restricted activation of Syk and PLC-γ1.     

Stoichiometric Structure-Function Analysis of the Prolactin Receptor Signaling Domain by Receptor Chimeras

The intracellular domain of the prolactin (PRL) receptor (PRLr) is required for PRL-induced signaling and proliferation. To identify and test the functional stoichiometry of those PRLr motifs required for transduction and growth, chimeras consisting of the extracellular domain of either the α or β subunit of human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GM-CSFr) and the intracellular domain of the rat PRLr were synthesized. Because the high-affinity binding of GM-CSF results from the specific pairing of one α- and one β-GM-CSFr, use of GM-CSFr/PRLr chimera enabled targeted dimerization of the PRLr intracellular domain. To that end, the extracellular domains of the α- and β-GM-CSFr were conjugated to one of the following mutations: (i) PRLr C-terminal truncations, termed α278, α294, α300, α322, or β322; (ii) PRLr tyrosine replacements, termed Y309F, Y382F, or Y309+382F; or, (iii) PRLr wild-type short, intermediate, or long isoforms. These chimeras were cotransfected into the cytokine-responsive Ba/F3 line, and their expression was confirmed by ligand binding and Northern and Western blot analyses. Data from these studies revealed that heterodimeric complexes of the wild type with C-terminal truncation mutants of the PRLr intracellular domain were incapable of ligand-induced signaling or proliferation. Replacement of any single tyrosine residue (Y309F or Y382F) in the dimerized PRLr complex resulted in a moderate reduction of receptor-associated Jak2 activation and proliferation. In contrast, trans replacement of these residues (i.e., αY309F and βY382F) markedly reduced ligand-driven Jak2 activation and proliferation, while cis replacement of both tyrosine residues in a single intracellular domain (i.e., αY309+382F) produced an inactive signaling complex. Analysis of these GM-CSFr–PRLr complexes revealed equivalent levels of Jak2 in association with the mutant receptor chains, suggesting that the tyrosine residues at 309 and 382 do not contribute to Jak association, but instead to its activation. Heterodimeric pairings of the intracellular domains from the known PRLr receptor isoforms (short-intermediate, short-long, and intermediate-long) also yielded inactive receptor complexes. These data demonstrate that the tyrosine residues at 309 and 382, as well as additional residues within the C terminus of the dimerized PRLr complex, contribute to PRL-driven signaling and proliferation. Furthermore, these findings indicate a functional requirement for the pairing of Y309 and Y382 in trans within the dimerized receptor complex.     

Requirement for Phospholipase C-γ1 Enzymatic Activity in Growth Factor-Induced Mitogenesis

The cytoplasmic regions of the receptors for epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) bind and activate phospholipase C-γ1 (PLC-γ1) and other signaling proteins in response to ligand binding outside the cell. Receptor binding by PLC-γ1 is a function of its SH2 domains and is required for growth factor-induced cell cycle progression into the S phase. Microinjection into MDCK epithelial cells and NIH 3T3 fibroblasts of a polypeptide corresponding to the noncatalytic SH2-SH2-SH3 domains of PLC-γ1 (PLC-γ1 SH2-SH2-SH3) blocked growth factor-induced S-phase entry. Treatment of cells with diacylglycerol (DAG) or DAG and microinjected inositol-1,4,5-triphosphate (IP₃), the products of activated PLC-γ1, did not stimulate cellular DNA synthesis by themselves but did suppress the inhibitory effects of the PLC-γ1 SH2-SH2-SH3 polypeptide but not the cell cycle block imposed by inhibition of the adapter protein Grb2 or p21 Ras. Two c-fos serum response element (SRE)-chloramphenicol acetyltransferase (CAT) reporter plasmids, a wild-type version, wtSRE-CAT, and a mutant, pm18, were used to investigate the function of PLC-γ1 in EGF- and PDGF-induced mitogenesis. wtSRE-CAT responds to both protein kinase C (PKC)-dependent and -independent signals, while the mutant, pm18, responds only to PKC-independent signals. Microinjection of the dominant-negative PLC-γ1 SH2-SH2-SH3 polypeptide greatly reduced the responses of wtSRE-CAT to EGF stimulation in MDCK cells and to PDGF stimulation in NIH 3T3 cells but had no effect on the responses of mutant pm18. These results indicate that in addition to Grb2-mediated activation of Ras, PLC-γ1-mediated DAG production is required for EGF- and PDGF-induced S-phase entry and gene expression, possibly through activation of PKC.     

The Tyrosine 343 Residue of Nucleophosmin (NPM)-Anaplastic Lymphoma Kinase (ALK) Is Important for Its Interaction with SHP1, a Cytoplasmic Tyrosine Phosphatase with Tumor Suppressor Functions

The cytoplasmic tyrosine phosphatase SHP1 has been shown to inhibit the oncogenic fusion protein nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK), and loss of SHP1 contributes to NPM-ALK-mediated tumorigenesis. In this study, we aimed to further understand how SHP1 interacts and regulates NPM-ALK. We employed an in vitro model in which GP293 cells were transfected with various combinations of NPM-ALK (or mutants) and SHP1 (or mutants) expression vectors. We found that SHP1 co-immunoprecipitated with NPM-ALK, but not the enzymatically inactive NPM-ALK^K210R mutant, or the mutant in which all three functionally important tyrosine residues (namely, Tyr³³⁸, Tyr³⁴², and Tyr³⁴³) in the kinase activation loop (KAL) of ALK were mutated. Interestingly, whereas mutation of Tyr³³⁸ or Tyr³⁴² did not result in any substantial change in the NPM-ALK/SHP1 binding (assessed by co-immunoprecipitation), mutation of Tyr³⁴³ abrogated this interaction. Furthermore, the NPM-ALK/SHP1 binding was readily detectable when each of the remaining 8 tyrosine residues known to be phosphorylated were mutated. Although the expression of SHP1 effectively reduced the level of tyrosine phosphorylation of NPM-ALK, it did not affect that of the NPM-ALK^Y343F mutant. In soft agar clonogenic assay, SHP1 expression significantly reduced the tumorigenicity of NPM-ALK but not that of NPM-ALK^Y343F. In conclusion, we identified Tyr³⁴³ of NPM-ALK as the crucial site for mediating the NPM-ALK/SHP1 interaction. Our results also support the notion that the tumor suppressor effects of SHP1 on NPM-ALK are dependent on its ability to bind to this oncogenic protein.     

A kinetic model of phospholipase C-γ1 linking structure-based insights to dynamics of enzyme autoinhibition and activation

Phospholipase C-γ1 (PLC-γ1) is a receptor-proximal enzyme that promotes signal transduction through PKC in mammalian cells. Because of the complexity of PLC-γ1 regulation, a two-state (inactive/active) model does not account for the intricacy of activation and inactivation steps at the plasma membrane. Here, we introduce a structure-based kinetic model of PLC-γ1, considering interactions of its regulatory Src homology 2 (SH2) domains and perturbation of those dynamics upon phosphorylation of Tyr783, a hallmark of activation. For PLC-γ1 phosphorylation to dramatically enhance enzyme activation as observed, we found that high intramolecular affinity of the C-terminal SH2 (cSH2) domain–pTyr783 interaction is critical, but this affinity need not outcompete the autoinhibitory interaction of the cSH2 domain. Under conditions for which steady-state PLC-γ1 activity is sensitive to the rate of Tyr783 phosphorylation, maintenance of the active state is surprisingly insensitive to the phosphorylation rate, since pTyr783 is well protected by the cSH2 domain while the enzyme is active. In contrast, maintenance of enzyme activity is sensitive to the rate of PLC-γ1 membrane (re)binding. Accordingly, we found that hypothetical PLC-γ1 mutations that either weaken autoinhibition or strengthen membrane binding influence the activation kinetics differently, which could inform the characterization of oncogenic variants. Finally, we used this newly informed kinetic scheme to refine a spatial model of PLC/PKC polarization during chemotaxis. The refined model showed improved stability of the polarized pattern while corroborating previous qualitative predictions. As demonstrated here for PLC-γ1, this approach may be adapted to model the dynamics of other receptor- and membrane-proximal enzymes.     

Membrane-induced Allosteric Control of Phospholipase C-β Isozymes

All peripheral membrane proteins must negotiate unique constraints intrinsic to the biological interface of lipid bilayers and the cytosol. Phospholipase C-β (PLC-β) isozymes hydrolyze the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP₂) to propagate diverse intracellular responses that underlie the physiological action of many hormones, neurotransmitters, and growth factors. PLC-β isozymes are autoinhibited, and several proteins, including Gα_q, Gβγ, and Rac1, directly engage distinct regions of these phospholipases to release autoinhibition. To understand this process, we used a novel, soluble analog of PIP₂ that increases in fluorescence upon cleavage to monitor phospholipase activity in real time in the absence of membranes or detergents. High concentrations of Gα_q or Gβ₁γ₂ did not activate purified PLC-β3 under these conditions despite their robust capacity to activate PLC-β3 at membranes. In addition, mutants of PLC-β3 with crippled autoinhibition dramatically accelerated the hydrolysis of PIP₂ in membranes without an equivalent acceleration in the hydrolysis of the soluble analog. Our results illustrate that membranes are integral for the activation of PLC-β isozymes by diverse modulators, and we propose a model describing membrane-mediated allosterism within PLC-β isozymes.     

Structural basis for the recognition of nucleophosmin-anaplastic lymphoma kinase oncoprotein by the phosphotyrosine binding domain of Suc1-associated neurotrophic factor-induced tyrosine-phosphorylated target-2

The nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion oncoprotein, formed by the t(2;5) chromosomal translocation in anaplastic large-cell lymphomas, has constitutive tyrosine kinase activity and interacts with a number of signaling molecules. One of the interacting partners of NPM-ALK is the adaptor protein, Suc1-associated neurotrophic factor-induced tyrosine-phosphorylated target (SNT), and mutations that deprive NPM-ALK of all three of the SNT-binding sites significantly reduced the transforming activity. In this study, the interactions of the three binding sites in NPM-ALK with the phosphotyrosine binding (PTB) domain of SNT-2 were analyzed. First, by isothermal titration calorimetry, we found that the phosphorylation-independent binding site in NPM-ALK interacts with the SNT-2 PTB domain more tightly than the phosphorylation-dependent binding sites. Second, the solution structure of the SNT-2 PTB domain in complex with the nonphosphorylated NPM-ALK peptide was determined by nuclear magnetic resonance spectroscopy. The NPM-ALK peptide interacts with the hydrophobic surface of the PTB domain and intermolecularly extends the PTB β-sheet. This interaction mode is much broader and more extensive than those of the phosphorylation-dependent binding sites. Our results indicate that the higher binding activity of the phosphorylation-independent binding site is caused by additional hydrophobic interactions.     

Studies of Phosphoproteomic Changes Induced by Nucleophosmin-Anaplastic Lymphoma Kinase (ALK) Highlight Deregulation of Tumor Necrosis Factor (TNF)/Fas/TNF-related Apoptosis-induced Ligand Signaling Pathway in ALK-positive Anaplastic Large Cell Lymphoma

The oncogenic fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), found exclusively in a subset of ALK-positive anaplastic large cell lymphoma, promotes tumorigenesis by exerting its constitutively active tyrosine kinase activity. Thus, characterization of the NPM-ALK-induced changes in the phosphoproteome will likely provide insights into the biology of this oncoprotein. To achieve this goal, we used a strategy of combining sequential affinity purification of phosphopeptides and LC/MS. GP293 cells transfected with either NPM-ALK or an NPM-ALK mutant with decreased tyrosine kinase activity (negative control) were used. We identified 506 phosphoproteins detectable in NPM-ALK-expressing cells but not in the negative control. Bioinformatics analysis revealed that these phosphoproteins carry a wide diversity of biological functions, some of which have not been described in association with NPM-ALK, such as the tumor necrosis factor (TNF)/Fas/tumor necrosis factor-related apoptosis-induced ligand (TRAIL) signaling pathway and the ubiquitin proteasome degradation pathway. In particular, modulations of the TNF/Fas/TRAIL pathway by NPM-ALK were supported by our antibody microarray data. Further validation of the TNF/Fas/TRAIL pathway was performed in ALK⁺ anaplastic large cell lymphoma (ALCL) cell lines with knockdown of NPM-ALK using short interference RNA, resulting in the loss of the tyrosine phosphorylation of tumor necrosis factor receptor-associated protein 1 (TRAP1) and receptor-interacting protein 1, two crucial TNF signaling molecules. Functional analyses revealed that knockdown of TRAP1 facilitated cell death induced by TRAIL or doxorubicin in ALK⁺ ALCL cells. This suggests that down-regulation of TRAP1 in combination with TRAIL or doxorubicin might be a potential novel therapeutic strategy for ALK⁺ ALCL. These findings demonstrated that our strategy allowed the identification of novel proteins downstream of NPM-ALK that contribute to the maintenance of neoplastic phenotype and holds great potential for future studies of cellular tyrosine kinases in normal states and diseases.Anaplastic lymphoma kinase (ALK)¹-positive anaplastic large cell lymphoma (ALK⁺ ALCL) is a subtype of T/null cell non-Hodgkin lymphoma that is characterized by its consistent expression of CD30 and anaplastic cytology (1). ALK⁺ ALCL is relatively uncommon among adults, accounting for ∼3% of adult non-Hodgkin lymphoma, whereas it is frequent in children, representing 10–20% of all lymphoma (2). Approximately 80% of ALK⁺ ALCL tumors carry the t(2;5)(p23;q35) chromosomal translocation that fuses the tyrosine kinase domain of ALK with the oligomerization domain of the nucleophosmin (NPM) protein. This chromosomal translocation results in the formation of the chimeric protein NPM-ALK (3–7). NPM-ALK has been shown to have propensity to dimerize, and this process triggers autophosphorylation of NPM-ALK and results in constitutive activation of the ALK tyrosine kinase. Once activated, NPM-ALK binds, phosphorylates, and constitutively activates a host of proteins involved in various cellular signaling pathways, including those of PI3K/RAC-α serine/threonine-protein kinase (AKT) (8, 9), JAK/STAT (10), mTOR (11–13), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)/ERK (14), and Ras/MAPK (15).As a tyrosine kinase, major attention was drawn to NPM-ALK in identifying the tyrosine phosphorylation, whereas other aspects of this oncogenic protein were not fully explored. Two LC/MS studies were performed previously (16, 17). Using quantitative LC/MS, Boccalatte et al. (16) reported 47 phosphoproteins in a panel of ALK⁺ ALCL cell lines that showed significant changes in their phosphorylation status upon ALK inhibition by short hairpin RNA or a tyrosine kinase inhibitor. Using LC/MS, Rush et al. (17) recently found ∼70 phosphoproteins from each of the two ALK⁺ ALCL cell lines, although whether the phosphorylation of these proteins is related to NPM-ALK was not determined. In both studies, only phosphotyrosine-containing proteins were captured by using a phosphotyrosine antibody immobilized to a solid phase prior to LC/MS analysis.However, mounting evidence has shown that NPM-ALK-induced signaling can culminate in the activation of oncogenic pathways via regulating serine/threonine phosphorylation (e.g. mTOR, c-Jun, nuclear interacting partner of ALK (NIPA), and AKT) (13, 18, 19). It is highly possible that NPM-ALK induces serine/threonine phosphorylation of downstream targets through recruitment and activation of various serine/threonine kinases like previously reported JNK and ERK (12, 18). In this regard, we have reported that NPM-ALK physically interacts with multiple serine/threonine kinases, including ribosomal protein S6 kinase α-3, serine/threonine/tyrosine kinase 1 (STYK1), serine/threonine-protein kinase PRP4, and serine/threonine-protein kinase PCTAIRE-3 (20). Therefore, comprehensive characterization of phosphoproteomic changes (i.e. serine, threonine, and tyrosine phosphorylation) induced by NPM-ALK will likely further our understanding of the pathobiology of NPM-ALK.Although comprehensive characterization of phosphoproteomic changes induced by oncogenic tyrosine kinases (such as NPM-ALK) is highly useful, this approach is met with multiple technical challenges. It is intrinsically difficult to detect phosphoproteins because the phosphopeptides produced from the phosphoproteins are generally present at a much lower amount compared with their native counterparts. In an attempt to overcome this difficulty, several enrichment approaches that are designed to isolate phosphorylated amino acid residues have been described, and they include IMAC through metal complexation with the phosphate group (21–23), acid-based interaction with titanium oxide (24–28), and solution charge-based enrichment by strong cation exchange chromatography (23, 29). Specific capture of phosphopeptides is possible by elimination of the phosphate group and subsequent introduction of an affinity tag (30) or conversion to methyl ester derivatives (21, 31, 32). In addition, several groups have combined various ion exchange approaches with IMAC to further enrich phosphopeptides prior to LC/MS/MS analysis (33–35). These works and others reported were mainly focused on improving the enrichment of phosphopeptides to facilitate the detection of as many phosphopeptides as possible. However, because tyrosine residues are present at much lower concentrations than serine or threonine (i.e. serine/threonine/tyrosine ratio is 1800:200:1), important proteins carrying phosphotyrosine residues often escape detection and are under-represented in the final results. To address this issue, several research groups recently reported the utilization of anti-phosphotyrosine antibodies to specifically enrich the phosphotyrosine-containing peptides within the digest (16, 17, 36) or intact protein prior to digestion (32). The use of anti-phosphotyrosine antibody alone, as used in the two previous studies of NPM-ALK (16, 17), is prone to nonspecific proteins binding to the anti-phosphotyrosine antibodies, resulting in a high background that may prevent the detection of the relatively weak signals of phosphotyrosine-containing peptides. Specific capture of phosphotyrosine-containing peptides has been enhanced by phosphotyrosine protein enrichment followed by IMAC (37, 38). Recently, Heck and co-workers (39) have reported an optimized enrichment method for phosphotyrosine peptides based on immunoaffinity purification. This method allowed a high level of enrichment; they were able to identify 1112 unique phosphotyrosine peptides derived from 4 mg of starting materials. However, biologically important proteins carrying phosphoserine and phosphothreonine residues were excluded with this approach. To identify both phosphotyrosine-containing proteins and phosphoserine-/phosphothreonine-containing proteins, parallel experiments involving in IMAC enrichment mainly for phosphoserine/phosphothreonine protein identification and anti-phosphotyrosine enrichment mainly for phosphotyrosine protein identification have been reported (23). However, this work required a doubled amount of the starting materials.To characterize the phosphoproteomic changes induced by NPM-ALK in a comprehensive and efficient manner, we developed a strategy and applied it to enrich phosphopeptides prior to the LC/MS analysis. The design of this enrichment strategy is based on three theoretical advantages. 1) It enables comprehensive identification of all three types of phosphorylated peptides. 2) It facilitates the detection of the phosphotyrosine-containing peptides despite their relatively low abundance compared with phosphoserine- and phosphothreonine-containing peptides. 3) It does not require parallel experiments; i.e. the same starting materials were used for profiling both phosphoserine-/phosphothreonine-containing proteins and phosphotyrosine-containing proteins. In the first step, all the phosphopeptides were enriched by using IMAC. In the second step, using an anti-phosphotyrosine specific antibody, we separated the phosphotyrosine pool from that of phosphoserine and phosphothreonine. Because of the prior enrichment by IMAC, the nonspecific protein binding to the phosphotyrosine antibody will be minimized. The phosphoserine/phosphothreonine pool was fractionated by strong cation exchange chromatography to facilitate their detection by LC/MS. Our results suggest that this strategy is highly useful in the comprehensive characterization of the phosphoproteome, particularly in enhancing our ability to detect phosphotyrosine peptides.One of the key objectives of this study was to comprehensively assess the phosphoproteomic changes induced by oncogenic kinase NPM-ALK that can serve as a proof of principle for future related biological studies. To achieve this goal, our experimental approach was designed to compare the qualitative phosphoproteomic changes between unaltered NPM-ALK- and NPM-ALK^FFF mutant-transfected cells, focusing on similar absolute changes in phosphorylation statuses, by using our strategy. We did not use a quantitative proteomics approach to analyze the phosphoproteomic changes induced by NPM-ALK because of the special property of this oncoprotein. Most of the previous reports (40–42) and our previous publication (43) have shown that phosphorylation of proteins downstream of NPM-ALK were only present in the presence of NPM-ALK; in the absence of NPM-ALK, the phosphorylation statuses of proteins downstream of NPM-ALK were lost. Using quantitative proteomics approaches, such as stable isotope labeling with amino acid in cell culture or isobaric tags for relative and absolute quantitation, will sacrifice the sensitivity of identification. Therefore, in this study, we used a qualitative phosphoproteomics approach instead of using a quantitative phosphoproteomics approach.In this study, we applied this protocol to compare the phosphorylation profile changes between the unaltered NPM-ALK- and NPM-ALK^FFF mutant-transfected cells and identify a set of phosphorylated proteins likely associated with NPM-ALK activity. It was found that the phosphoproteins identified regulate a diversity of key cellular pathways, some of which have never been reported to be associated with NPM-ALK. To document the biological relevance of these identified phosphoproteins, validation experiments were performed using NPM-ALK- and NPM-ALK^FFF mutant-transfected cells as well as ALK⁺ ALCL cells. The TNF/Fas/tumor necrosis factor-related apoptosis-induced ligand (TRAIL) signaling pathway was validated to be modulated by NPM-ALK in ALK⁺ ALCL. Functional analysis showed that knockdown of tumor necrosis factor receptor-associated protein 1 (TRAP1), one of the crucial TNF/Fas/TRAIL signaling molecules, enhanced TRAIL- or chemotherapeutic drug doxorubicin-induced cell death. These findings provide a potential therapeutic strategy for ALK⁺ ALCL and novel insight into the NPM-ALK-mediated lymphomagenesis.     

Phospholipase C-β1 and β4 Contribute to Non-Genetic Cell-to-Cell Variability in Histamine-Induced Calcium Signals in HeLa Cells

A uniform extracellular stimulus triggers cell-specific patterns of Ca²⁺ signals, even in genetically identical cell populations. However, the underlying mechanism that generates the cell-to-cell variability remains unknown. We monitored cytosolic inositol 1,4,5-trisphosphate (IP₃) concentration changes using a fluorescent IP₃ sensor in single HeLa cells showing different patterns of histamine-induced Ca²⁺ oscillations in terms of the time constant of Ca²⁺ spike amplitude decay and the Ca²⁺ oscillation frequency. HeLa cells stimulated with histamine exhibited a considerable variation in the temporal pattern of Ca²⁺ signals and we found that there were cell-specific IP₃ dynamics depending on the patterns of Ca²⁺ signals. RT-PCR and western blot analyses showed that phospholipase C (PLC)-β1, -β3, -β4, -γ1, -δ3 and -ε were expressed at relatively high levels in HeLa cells. Small interfering RNA-mediated silencing of PLC isozymes revealed that PLC-β1 and PLC-β4 were specifically involved in the histamine-induced IP₃ increases in HeLa cells. Modulation of IP₃ dynamics by knockdown or overexpression of the isozymes PLC-β1 and PLC-β4 resulted in specific changes in the characteristics of Ca²⁺ oscillations, such as the time constant of the temporal changes in the Ca²⁺ spike amplitude and the Ca²⁺ oscillation frequency, within the range of the cell-to-cell variability found in wild-type cell populations. These findings indicate that the heterogeneity in the process of IP₃ production, rather than IP₃-induced Ca²⁺ release, can cause cell-to-cell variability in the patterns of Ca²⁺ signals and that PLC-β1 and PLC-β4 contribute to generate cell-specific Ca²⁺ signals evoked by G protein-coupled receptor stimulation.     

Soluble and Membrane-Bound TGF-β-Mediated Regulation of Intratumoral T Cell Differentiation and Function in B-Cell Non-Hodgkin Lymphoma

While the effect of TGF-β on malignant B cells in non-Hodgkin lymphoma (NHL) has been previously evaluated, studies to specifically define the role of TGF-β in tumor immunity in B-cell NHL are limited. We found that soluble TGF-β, secreted by both lymphoma cells and intratumoral T cells, is present in the serum of patients with B-cell NHL. Soluble TGF-β promoted regulatory T (T_reg) cells by enhancing expression of Foxp3 in CD4⁺ T cells and suppressed effector helper T (T_H) cells by inhibiting expression of IFN-γ and IL-17. Blockade of the IL-2 signaling pathway diminished the effect of soluble TGF-β on T cell differentiation. Furthermore, we found that membrane-bound TGF-β is expressed specifically on the surface of malignant B cells in B-cell NHL. TGF-β was able to bind to the surface of lymphoma B cells through an interaction with heparan sulfate (HS) but not through the TGF-β receptor. We showed that pretreatment of lymphoma B cells with TGF-β significantly inhibits the proliferation and cytokine production of intratumoral T cells. Taken together, these results suggest that tumor-associated soluble and membrane-bound TGF-β are involved in the regulation of intratumoral T cell differentiation and function in B-cell NHL.     

Phospholipase C-β4 Is Essential for the Progression of the Normal Sleep Sequence and Ultradian Body Temperature Rhythms in Mice

Background

The sleep sequence: i) non-REM sleep, ii) REM sleep, and iii) wakefulness, is stable and widely preserved in mammals, but the underlying mechanisms are unknown. It has been shown that this sequence is disrupted by sudden REM sleep onset during active wakefulness (i.e., narcolepsy) in orexin-deficient mutant animals. Phospholipase C (PLC) mediates the signaling of numerous metabotropic receptors, including orexin receptors. Among the several PLC subtypes, the β4 subtype is uniquely localized in the geniculate nucleus of thalamus which is hypothesized to have a critical role in the transition and maintenance of sleep stages. In fact, we have reported irregular theta wave frequency during REM sleep in PLC-β4-deficient mutant (PLC-β4−/−) mice. Daily behavioral phenotypes and metabotropic receptors involved have not been analyzed in detail in PLC-β4−/− mice, however.

Methodology/Principal Findings

Therefore, we analyzed 24-h sleep electroencephalogram in PLC-β4−/− mice. PLC-β4−/− mice exhibited normal non-REM sleep both during the day and nighttime. PLC-β4−/− mice, however, exhibited increased REM sleep during the night, their active period. Also, their sleep was fragmented with unusual wake-to-REM sleep transitions, both during the day and nighttime. In addition, PLC-β4−/− mice reduced ultradian body temperature rhythms and elevated body temperatures during the daytime, but had normal homeothermal response to acute shifts in ambient temperatures (22°C–4°C). Within the most likely brain areas to produce these behavioral phenotypes, we found that, not orexin, but group-1 metabotropic glutamate receptor (mGluR)-mediated Ca²⁺ mobilization was significantly reduced in the dorsal lateral geniculate nucleus (LGNd) of PLC-β4−/− mice. Voltage clamp recordings revealed that group-1 mGluR-mediated currents in LGNd relay neurons (inward in wild-type mice) were outward in PLC-β4−/− mice.

Conclusions/Significance

These lines of evidence indicate that impaired LGNd relay, possibly mediated via group-1 mGluR, may underlie irregular sleep sequences and ultradian body temperature rhythms in PLC-β4−/− mice.     

Functional Characterization of the Kinase Activation Loop in Nucleophosmin (NPM)-Anaplastic Lymphoma Kinase (ALK) Using Tandem Affinity Purification and Liquid Chromatography-Mass Spectrometry

Previous studies have shown that the kinase activation loop (KAL) of the oncogenic fusion protein NPM-ALK regulates its overall tyrosine phosphorylation status and tumorigenicity. Using tandem affinity purification-mass spectrometry, we assessed how the KAL of NPM-ALK regulates the phosphorylation status of its individual tyrosines. Using the lysates of GP293 cells transfected with NPM-ALK, our highly reproducible results showed evidence of phosphorylation in all 3 tyrosines in KAL and 8 tyrosines outside KAL. We created 7 KAL mutants, each of which carried a Tyr-to-Phe mutation of ≥1 of the 3 tyrosines in KAL. A complete loss of the 8 phosphotyrosines outside KAL was found in 3 KAL mutants, and their oncogenicity (assessed by cell viability, colony formation, and the ability to phosphorylate effector proteins) was abrogated. A partial loss of the 8 phosphotyrosines was found in 4 KAL mutants, but their oncogenicity did not show simple correlation with the number of residual phosphotyrosines. Tyr-to-Phe mutations of each of the 8 phosphotyrosines outside KAL did not result in a significant decrease in the oncogenicity. In conclusion, we have provided details of how the KAL in NPM-ALK regulates its tyrosine phosphorylation pattern. Our results challenge some of the current concepts regarding the relationship between the tyrosine phosphorylation and oncogenicity of NPM-ALK.     

Sustained Platelet-Derived Growth Factor Receptor α Signaling in Osteoblasts Results in Craniosynostosis by Overactivating the Phospholipase C-γ Pathway

The development and growth of the skull is controlled by cranial sutures, which serve as growth centers for osteogenesis by providing a pool of osteoprogenitors. These osteoprogenitors undergo intramembranous ossification by direct differentiation into osteoblasts, which synthesize the components of the extracellular bone matrix. A dysregulation of osteoblast differentiation can lead to premature fusion of sutures, resulting in an abnormal skull shape, a disease called craniosynostosis. Although several genes could be linked to craniosynostosis, the mechanisms regulating cranial suture development remain largely elusive. We have established transgenic mice conditionally expressing an autoactivated platelet-derived growth factor receptor α (PDGFRα) in neural crest cells (NCCs) and their derivatives. In these mice, premature fusion of NCC-derived sutures occurred at early postnatal stages. In vivo and in vitro experiments demonstrated enhanced proliferation of osteoprogenitors and accelerated ossification of osteoblasts. Furthermore, in osteoblasts expressing the autoactivated receptor, we detected an upregulation of the phospholipase C-γ (PLC-γ) pathway. Treatment of differentiating osteoblasts with a PLC-γ-specific inhibitor prevented the mineralization of synthesized bone matrix. Thus, we show for the first time that PDGFRα signaling stimulates osteogenesis of NCC-derived osteoblasts by activating the PLC-γ pathway, suggesting an involvement of this pathway in the etiology of human craniosynostosis.     

Phospholipase C-ε Regulates Epidermal Morphogenesis in Caenorhabditis elegans

Migration of cells within epithelial sheets is an important feature of embryogenesis and other biological processes. Previous work has demonstrated a role for inositol 1,4,5-trisphosphate (IP₃)-mediated calcium signalling in the rearrangement of epidermal cells (also known as hypodermal cells) during embryonic morphogenesis in Caenorhabditis elegans. However the mechanism by which IP₃ production is stimulated is unknown. IP₃ is produced by the action of phospholipase C (PLC). We therefore surveyed the PLC family of C. elegans using RNAi and mutant strains, and found that depletion of PLC-1/PLC-ε produced substantial embryonic lethality. We used the epithelial cell marker ajm-1::gfp to follow the behaviour of epidermal cells and found that 96% of the arrested embryos have morphogenetic defects. These defects include defective ventral enclosure and aberrant dorsal intercalation. Using time-lapse confocal microscopy we show that the migration of the ventral epidermal cells, especially of the leading cells, is slower and often fails in plc-1(tm753) embryos. As a consequence plc-1 loss of function results in ruptured embryos with a Gex phenotype (gut on exterior) and lumpy larvae. Thus PLC-1 is involved in the regulation of morphogenesis. Genetic studies using gain- and loss-of-function alleles of itr-1, the gene encoding the IP₃ receptor in C. elegans, demonstrate that PLC-1 acts through ITR-1. Using RNAi and double mutants to deplete the other PLCs in a plc-1 background, we show that PLC-3/PLC-γ and EGL-8/PLC-β can compensate for reduced PLC-1 activity. Our work places PLC-ε into a pathway controlling epidermal cell migration, thus establishing a novel role for PLC-ε.     

Induction of Apoptosis after Expression of PYK2, a Tyrosine Kinase Structurally Related to Focal Adhesion Kinase

Many cells (e.g., epithelial cells) require attachment to the extracellular matrix (ECM) to survive, a phenomenon known as anchorage-dependent cell survival. Disruption of the cell–ECM interactions mediated by the integrin receptors results in apoptosis. Focal adhesion kinase (FAK), a 125-kD protein tyrosine kinase activated by integrin engagement, appears to be involved in mediating cell attachment and survival. Proline-rich tyrosine kinase 2 (PYK2), also known as cellular adhesion kinase β (CAKβ) and related adhesion focal tyrosine kinase, is a second member of the FAK subfamily and is activated by an increase in intracellular calcium levels, or treatment with TNFα and UV light. However, the function of PYK2 remains largely unknown. In this study, we show that over-expression of PYK2, but not FAK, in rat and mouse fibroblasts leads to apoptotic cell death. Using a series of deletion mutants and chimeric fusion proteins of PYK2/FAK, we determined that the NH₂-terminal domain and tyrosine kinase activity of PYK2 were required for the efficient induction of apoptosis. Furthermore, the apoptosis mediated by PYK2 could be suppressed by over-expressing catalytically active v-Src, c-Src, phosphatidylinositol-3-kinase, or Akt/protein kinase B. In addition, it could also be suppressed by overexpressing an ICE or ICE-like proteinase inhibitor, crmA, but not Bcl2. Collectively, our results suggest that PYK2 and FAK, albeit highly homologous in primary structure, appear to have different functions; FAK is required for cell survival, whereas PYK2 induces apoptosis in fibroblasts.