Critical Role of a Ferritin-Like Protein in the Control of Listeria monocytogenes Cell Envelope Structure and Stability under β-lactam Pressure

The human pathogen Listeria monocytogenes is susceptible to the β-lactam antibiotics penicillin G and ampicillin, and these are the drugs of choice for the treatment of listerial infections. However, these antibiotics exert only a bacteriostatic effect on this bacterium and consequently, L. monocytogenes is regarded as β-lactam tolerant. It is widely accepted that the phenomenon of bacterial tolerance to β-lactams is due to the lack of adequate autolysin activity, but the mechanisms of L. monocytogenes tolerance to this class of antibiotics are poorly characterized. A ferritin-like protein (Fri) was recently identified as a mediator of β-lactam tolerance in L. monocytogenes, but its function in this process remains unknown. The present study was undertaken to improve our understanding of L. monocytogenes tolerance to β-lactams and to characterize the role of Fri in this phenomenon. A comparative physiological analysis of wild-type L. monocytogenes and a fri deletion mutant provided evidence of a multilevel mechanism controlling autolysin activity in cells grown under β-lactam pressure, which leads to a reduction in the level and/or activity of cell wall-associated autolysins. This is accompanied by increases in the amount of teichoic acids, cell wall thickness and cell envelope integrity of L. monocytogenes grown in the presence of penicillin G, and provides the basis for the innate β-lactam tolerance of this bacterium. Furthermore, this study revealed the inability of the L. monocytogenes Δ fri mutant to deplete autolysins from the cell wall, to adjust the content of teichoic acids and to maintain their D-alanylation at the correct level when treated with penicillin G, thus providing further evidence that Fri is involved in the control of L. monocytogenes cell envelope structure and stability under β-lactam pressure.     

Protein Phosphatase 2A Catalytic Subunit α Plays a MyD88-Dependent,Central Role in the Gene-Specific Regulation of Endotoxin Tolerance

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Highlights? PP2Ac is constitutively activated and targets MyD88 in LPS-tolerized macrophages ? Constitutively active PP2Ac shifts a proinflammatory MyD88 to its prosurvival mode ? Constitutively active PP2Ac reprograms gene-specific chromatin modification landscape ? Constitutively active PP2Ac broadly defines ET at both signaling and epigenetic levels     

Receptor-Associated Protein (RAP) Plays a Central Role in Modulating Aβ Deposition in APP/PS1 Transgenic Mice

Background

Receptor associated protein (RAP) functions in the endoplasmic reticulum (ER) to assist in the maturation of several membrane receptor proteins, including low density lipoprotein receptor-related protein (LRP) and lipoprotein receptor 11 (SorLA/LR11). Previous studies in cell and mouse model systems have demonstrated that these proteins play roles in the metabolism of the amyloid precursor protein (APP), including processes involved in the generation, catabolism and deposition of β-amyloid (Aβ) peptides.

Methodology/Principal Findings

Mice transgenic for mutant APPswe and mutant presenilin 1 (PS1dE9) were mated to mice with homozygous deletion of RAP. Unexpectedly, mice that were homozygous null for RAP and transgenic for APPswe/PS1dE9 showed high post-natal mortality, necessitating a shift in focus to examine the levels of amyloid deposition in APPswe/PS1dE9 that were hemizygous null for RAP. Immunoblot analysis confirmed 50% reductions in the levels of RAP with modest reductions in the levels of proteins dependent upon RAP for maturation [LRP trend towards a 20% reduction ; SorLA/LR11 statistically significant 15% reduction (p<0.05)]. Changes in the levels of these proteins in the brains of [APPswe/PS1dE9](+/−)/RAP(+/−) mice correlated with 30–40% increases in amyloid deposition by 9 months of age.

Conclusions/Significance

Partial reductions in the ER chaperone RAP enhance amyloid deposition in the APPswe/PS1dE9 model of Alzheimer amyloidosis. Partial reductions in RAP also affect the maturation of LRP and SorLA/LR11, which are each involved in several different aspects of APP processing and Aβ catabolism. Together, these findings suggest a central role for RAP in Alzheimer amyloidogenesis.     

Protein kinase CK2 accumulation in ��oncophilic�� cells: causes and effects

At variance with protein kinases expressed by oncogenes, CK2 is endowed with constitutive activity under normal conditions, and no CK2 gain-of-function mutants are known. Its amount, however, is abnormally high in malignant cells where it appears to be implicated in many of the cell biology phenomena associated with cancer. These observations can be reconciled assuming that tumor cells develop an overdue reliance ("non-oncogene addiction") on abnormally high CK2 level. While the potential of this latter to generate an environment favorable to neoplasia is consistent with the global antiapoptotic and prosurvival role played by CK2, it is not clear what is determining accumulation of CK2 in cells "predisposed" to become malignant. Exploiting the apoptosis sensitive (S) or resistant (R) CEM cell model, characterized by sharply different CK2 levels, we have now correlated the level and degradation rate of CK2 to those of the chaperone proteins Hsp90 and Cdc37. We show in particular that persistence of high CK2 level in R-CEM, as opposed to S-CEM, is accompanied by the presence of an immunospecific form of Cdc37 not detectable in S-CEM and refractory to staurosporine-induced degradation.     

The enhancement of stability of p53 in MTBP induced p53–MDM2 regulatory network

We have modeled an MTBP-MDM2–p53 regulatory network by integrating p53–MDM2 autoregulatory model (Proctor and Gray, 2008) with the effect of a cellular protein MTBP (MDM2 binding protein) which is allowed to bind with MDM2 (Brady et al., 2005). We study this model to investigate the activation of p53 and MDM2 steady state levels induced by MTBP protein under different stress conditions. Our simulation results in three approaches namely deterministic, Chemical Langevin equation and stochastic simulation of Master equation show a clear transition from damped limit cycle oscillation to fixed point oscillation during a certain time period with constant stress condition in the cell. This transition is the signature of transition of p53 and MDM2 levels from activated state to stabilized steady state levels. We present various phase diagrams to show the transition between unstable and stable states of p53 and MDM2 concentration levels and also their possible relations among critical value of the parameters at which the respective protein level reach stable steady states. In the stochastic approach, the dynamics of the proteins become noise induced process depending on the system size. We found that this noise enhances the stability of the p53 steady state level.     

Role of plant-specific N-terminal domain of maize CK2β1 subunit in CK2β functions and holoenzyme regulation

Protein kinase CK2 is a highly pleiotropic Ser/Thr kinase ubiquituous in eukaryotic organisms. CK2 is organized as a heterotetrameric enzyme composed of two types of subunits: the catalytic (CK2α) and the regulatory (CK2β). The CK2β subunits enhance the stability, activity and specificity of the holoenzyme, but they can also perform functions independently of the CK2 tetramer. CK2β regulatory subunits in plants differ from their animal or yeast counterparts, since they present an additional specific N-terminal extension of about 90 aminoacids that shares no homology with any previously characterized functional domain. Sequence analysis of the N-terminal domain of land plant CK2β subunit sequences reveals its arrangement through short, conserved motifs, some of them including CK2 autophosphorylation sites. By using maize CK2β1 and a deleted version (ΔNCK2β1) lacking the N-terminal domain, we have demonstrated that CK2β1 is autophosphorylated within the N-terminal domain. Moreover, the holoenzyme composed with CK2α1/ΔNCK2β1 is able to phosphorylate different substrates more efficiently than CK2α1/CK2β1 or CK2α alone. Transient overexpression of CK2β1 and ΔNCK2β1 fused to GFP in different plant systems show that the presence of N-terminal domain enhances aggregation in nuclear speckles and stabilizes the protein against proteasome degradation. Finally, bimolecular fluorescence complementation (BiFC) assays show the nuclear and cytoplasmic location of the plant CK2 holoenzyme, in contrast to the individual CK2α/β subunits mainly observed in the nucleus. All together, our results support the hypothesis that the plant-specific N-terminal domain of CK2β subunits is involved in the down-regulation of the CK2 holoenzyme activity and in the stabilization of CK2β1 protein. In summary, the whole amount of data shown in this work suggests that this domain was acquired by plants for regulatory purposes.     

Retinal Degeneration Slow (RDS) Glycosylation Plays a Role in Cone Function and in the Regulation of RDS·ROM-1 Protein Complex Formation

The photoreceptor-specific glycoprotein retinal degeneration slow (RDS, also called PRPH2) is necessary for the formation of rod and cone outer segments. Mutations in RDS cause rod and cone-dominant retinal disease, and it is well established that both cell types have different requirements for RDS. However, the molecular mechanisms for this difference remain unclear. Although RDS glycosylation is highly conserved, previous studies have revealed no apparent function for the glycan in rods. In light of the highly conserved nature of RDS glycosylation, we hypothesized that it is important for RDS function in cones and could underlie part of the differential requirement for RDS in the two photoreceptor subtypes. We generated a knockin mouse expressing RDS without the N-glycosylation site (N229S). Normal levels of RDS and the unglycosylated RDS binding partner rod outer segment membrane protein 1 (ROM-1) were found in N229S retinas. However, cone electroretinogram responses were decreased by 40% at 6 months of age. Because cones make up only 3–5% of photoreceptors in the wild-type background, N229S mice were crossed into the nrl^−/− background (in which all rods are converted to cone-like cells) for biochemical analysis. In N229S/nrl^−/− retinas, RDS and ROM-1 levels were decreased by ∼60% each. These data suggest that glycosylation of RDS is required for RDS function or stability in cones, a difference that may be due to extracellular versus intradiscal localization of the RDS glycan in cones versus rods.     

Plasticity of the ��-Trefoil Protein Fold in the Recognition and Control of Invertebrate Predators and Parasites by a Fungal Defence System

Discrimination between self and non-self is a prerequisite for any defence mechanism; in innate defence, this discrimination is often mediated by lectins recognizing non-self carbohydrate structures and so relies on an arsenal of host lectins with different specificities towards target organism carbohydrate structures. Recently, cytoplasmic lectins isolated from fungal fruiting bodies have been shown to play a role in the defence of multicellular fungi against predators and parasites. Here, we present a novel fruiting body lectin, CCL2, from the ink cap mushroom Coprinopsis cinerea. We demonstrate the toxicity of the lectin towards Caenorhabditis elegans and Drosophila melanogaster and present its NMR solution structure in complex with the trisaccharide, GlcNAcβ1,4[Fucα1,3]GlcNAc, to which it binds with high specificity and affinity in vitro. The structure reveals that the monomeric CCL2 adopts a β-trefoil fold and recognizes the trisaccharide by a single, topologically novel carbohydrate-binding site. Site-directed mutagenesis of CCL2 and identification of C. elegans mutants resistant to this lectin show that its nematotoxicity is mediated by binding to α1,3-fucosylated N-glycan core structures of nematode glycoproteins; feeding with fluorescently labeled CCL2 demonstrates that these target glycoproteins localize to the C. elegans intestine. Since the identified glycoepitope is characteristic for invertebrates but absent from fungi, our data show that the defence function of fruiting body lectins is based on the specific recognition of non-self carbohydrate structures. The trisaccharide specifically recognized by CCL2 is a key carbohydrate determinant of pollen and insect venom allergens implying this particular glycoepitope is targeted by both fungal defence and mammalian immune systems. In summary, our results demonstrate how the plasticity of a common protein fold can contribute to the recognition and control of antagonists by an innate defence mechanism, whereby the monovalency of the lectin for its ligand implies a novel mechanism of lectin-mediated toxicity.     

Role of protein kinases CK1α and CK2 in multiple myeloma: regulation of pivotal survival and stress-managing pathways

Multiple myeloma (MM) is a malignant tumor of transformed plasma cells. MM pathogenesis is a multistep process. This cancer can occur de novo (rarely) or it can develop from monoclonal gammopathy of undetermined significance (most of the cases). MM can be asymptomatic (smoldering myeloma) or clinically active. Malignant plasma cells exploit intrinsic and extrinsic bone marrow microenvironment-derived growth signals. Upregulation of stress-coping pathways is also instrumental to maintain MM cell growth. The phylogenetically related Ser/Thr kinases CSNK1A1 (CK1α) and CSNK2 (CK2) have recently gained a growing importance in hematologic malignancies arising both from precursors and from mature blood cells. In multiple myeloma, CK1α or CK2 sustain oncogenic cascades, such as the PI3K/AKT, JAK/STAT, and NF-κB, as well as propel stress-related signaling that help in coping with different noxae. Data also suggest that these kinases modulate the delivery of growth factors and cytokines from the bone marrow stroma. The “non-oncogene addiction” phenotype generated by the increased activity of CK1α and CK2 in multiple myeloma contributes to malignant plasma cell proliferation and survival and represents an Achilles’ heel for the activity of small ATP competitive CK1α or CK2 inhibitors.     

Topoisomerase IIα mediates E2F-1-induced chemosensitivity and is a target for p53-mediated transcriptional repression

Mutations of the retinoblastoma tumor suppressor, pRb, or its cyclin-cyclin-dependent kinase (CDK) regulatory kinases or CDK inhibitors, allows unrestrained E2F activity, leading to unregulated cell cycle progression. However, overexpression of E2F-1 also sensitizes cells to apoptosis, suggesting that targeting this pathway may be of therapeutic benefit. Enforced expression of E2F-1 in interleukin-3-dependent myeloid cells led to preferential sensitivity to the topoisomerase II inhibitor, etoposide, which was independent of p53 accumulation. Pretreatment of the E2F-1-expressing cells with ICRF-193, a second topoisomerase II inhibitor that does not cause DNA damage, protected these cells against etoposide-induced apoptosis. However, ICRF-193 cooperated with other DNA-damaging agents to induce apoptosis. Enforced expression of E2F-1 led to accumulation of p53 protein. An E2F-1 mutant that is defective in inducing cell cycle progression also induced p53, suggesting that p53 was responding directly to E2F, and not to secondary events caused by inappropriate cell cycle progression (i.e., DNA damage). Thus, topoisomerase II inhibition and DNA damage cooperate to selectively induce apoptosis in cells that have mutations in the pRb pathway.     

Role of Molecular Chaperones in G Protein ��5/Regulator of G Protein Signaling Dimer Assembly and G Protein �¦� Dimer Specificity

The G protein βγ subunit dimer (Gβγ) and the Gβ5/regulator of G protein signaling (RGS) dimer play fundamental roles in propagating and regulating G protein pathways, respectively. How these complexes form dimers when the individual subunits are unstable is a question that has remained unaddressed for many years. In the case of Gβγ, recent studies have shown that phosducin-like protein 1 (PhLP1) works as a co-chaperone with the cytosolic chaperonin complex (CCT) to fold Gβ and mediate its interaction with Gγ. However, it is not known what fraction of the many Gβγ combinations is assembled this way or whether chaperones influence the specificity of Gβγ dimer formation. Moreover, the mechanism of Gβ5-RGS assembly has yet to be assessed experimentally. The current study was undertaken to directly address these issues. The data show that PhLP1 plays a vital role in the assembly of Gγ2 with all four Gβ1–4 subunits and in the assembly of Gβ2 with all twelve Gγ subunits, without affecting the specificity of the Gβγ interactions. The results also show that Gβ5-RGS7 assembly is dependent on CCT and PhLP1, but the apparent mechanism is different from that of Gβγ. PhLP1 seems to stabilize the interaction of Gβ5 with CCT until Gβ5 is folded, after which it is released to allow Gβ5 to interact with RGS7. These findings point to a general role for PhLP1 in the assembly of all Gβγ combinations and suggest a CCT-dependent mechanism for Gβ5-RGS7 assembly that utilizes the co-chaperone activity of PhLP1 in a unique way.Eukaryotic cells utilize receptors coupled to heterotrimeric GTP-binding proteins (G proteins)³ to mediate a vast array of responses ranging from nutrient-induced migration of single-celled organisms to neurotransmitter-regulated neuronal activity in the human brain (1). Ligand binding to a G protein-coupled receptor (GPCR) initiates GTP exchange on the G protein heterotrimer (composed of Gα, Gβ, and Gγ subunits), which in turn causes the release of Gα-GTP from the Gβγ dimer (2–4). Both Gα-GTP and Gβγ propagate and amplify the signal by interacting with effector enzymes and ion channels (1, 5). The duration and amplitude of the signal is dictated by receptor phosphorylation coupled with arrestin binding and internalization (6) and by regulators of G protein signaling (RGS) proteins, which serve as GTPase-activating proteins for the GTP-bound Gα subunit (7, 8). The G protein signaling cycle is reset as the inactive Gα-GDP reassembles with the Gβγ dimer and Gαβγ re-associates with the GPCR (5).To fulfill its essential role in signaling, the G protein heterotrimer must be assembled post-translationally from its nascent polypeptides. Significant progress has been made recently regarding the mechanism by which this process occurs. It has been clear for some time that the Gβγ dimer must assemble first, followed by subsequent association of Gα with Gβγ (9). What has not been clear was how Gβγ assembly would occur given the fact that neither Gβ nor Gγ is structurally stable without the other. An important breakthrough was the finding that phosducin-like protein 1 (PhLP1) functions as a co-chaperone with the chaperonin containing tailless complex polypeptide 1 (CCT) in the folding of nascent Gβ and its association with Gγ (10–15). CCT is an important chaperone that assists in the folding of actin and tubulin and many other cytosolic proteins, including many β propeller proteins like Gβ (16). PhLP1 has been known for some time to interact with Gβγ and was initially believed to inhibit Gβγ function (17). However, several recent studies have demonstrated that PhLP1 and CCT work together in a highly orchestrated manner to form the Gβγ dimer (10–15).Studies on the mechanism of PhLP1-mediated Gβγ assembly have focused on the most common dimer Gβ1γ2 (10, 13, 14), leaving open questions about the role of PhLP1 in the assembly of the other Gβγ combinations. These are important considerations given that humans possess 5 Gβ genes and 12 Gγ genes with some important splice variants (18, 19), resulting in more than 60 possible combinations of Gβγ dimers. Gβ1–4 share between 80 and 90% sequence identity and are broadly expressed (18, 19). Gβ5, the more atypical isoform, shares only ∼53% identity with Gβ1, carries a longer N-terminal domain, and is only expressed in the central nervous system and retina (20). The Gγ protein family is more heterogeneous than the Gβ family. The sequence identity of the 12 Gγ isoforms extends from 10 to 70% (21), and the Gγ family can be separated into 5 subfamilies (21–23). All Gγ proteins carry C-terminal isoprenyl modifications, which contribute to their association with the cell membrane, GPCRs, Gαs, and effectors (9). Subfamily I Gγ isoforms are post-translationally farnesylated, whereas all others are geranylgeranylated (22, 24).There is some inherent selectivity in the assembly of different Gβγ combinations, but in general Gβ1–4 can form dimers with most Gγ subunits (25). The physiological purpose of this large number of Gβγ combinations has intrigued researchers in the field for many years, and a large body of research indicates that GPCRs and effectors couple to a preferred subset of Gβγ combinations based somewhat on specific sequence complementarity, but even more so on cellular expression patterns, subcellular localization, and post-translational modifications (18).In contrast to Gβ1–4, Gβ5 does not interact with Gγ subunits in vivo, but it instead forms irreversible dimers with RGS proteins of the R7 family, which includes RGS proteins 6, 7, 9, and 11 (26). All R7 family proteins contain an N-terminal DEP (disheveled, Egl-10, pleckstrin) domain, a central Gγ-like (GGL) domain, and a C-terminal RGS domain (8, 26). The DEP domain interacts with the membrane anchoring/nuclear shuttling R7-binding protein, and the GGL domain binds to Gβ5 in a manner similar to other Gβγ associations (27, 28). Like Gβγs, Gβ5 and R7 RGS proteins form obligate dimers required for their mutual stability (26). Without their partner, Gβ5 and R7 RGS proteins are rapidly degraded in cells (26, 29). Gβ5-R7 RGS complexes act as important GTPase-accelerating proteins for G_i/oα and G_qα subunits in neuronal cells and some immune cells (26).It has been recently shown that all Gβ isoforms are able to interact with the CCT complex, but to varying degrees (15). Gβ4 and Gβ1 bind CCT better than Gβ2 and Gβ3, whereas Gβ5 binds CCT poorly (15). These results suggest that Gβ1 and Gβ4 might be more dependent on PhLP1 than the other Gβs, given the co-chaperone role of PhLP1 with CCT in Gβ1γ2 assembly. However, another report has indicated that Gγ2 assembly with Gβ1 and Gβ2 is more PhLP1-dependent than with Gβ3 and Gβ4 (30). Thus, it is not clear from current information whether PhLP1 and CCT participate in assembly of all Gβγ combinations or whether they contribute to the specificity of Gβγ dimer formation, nor is it clear whether they or other chaperones are involved in Gβ5-R7 RGS dimer formation. This report was designed to address these issues.     

Phospholipase Cγ2 Plays a Role in TCR Signal Transduction and T Cell Selection

One of the important signaling events following TCR engagement is activation of phospholipase Cγ (PLCγ). PLCγ has two isoforms, PLCγ1 and PLCγ2. It is known that PLCγ1 is important for TCR signaling and TCR-mediated T cell selection and functions, whereas PLCγ2 is critical for BCR signal transduction and BCR-mediated B cell maturation and functions. In this study, we report that PLCγ2 was expressed in primary T cells, and became associated with linker for activated T cells and Src homology 2-domain containing leukocyte protein of 76 kDa and activated upon TCR stimulation. PLCγ1/PLCγ2 double-deficient T cells displayed further block from CD4 and CD8 double-positive to single-positive transition compared with PLCγ1 single-deficient T cells. TCR-mediated proliferation was further impaired in PLCγ1/PLCγ2 double-deficient T cells compared with PLCγ1 single-deficient T cells. TCR-mediated signal transduction, including Ca(2+) mobilization and Erk activation, was further impaired in PLCγ1/PLCγ2 double-deficient relative to PLCγ1 single-deficient T cells. In addition, in HY TCR transgenic mouse model, thymic positive and negative selections were reduced in PLCγ1 heterozygous- and PLCγ2 homozygous-deficient (PLCγ1(+/-)PLCγ2(-/-)) relative to wild-type, PLCγ2 single-deficient (PLCγ2(-/-)), or PLCγ1 heterozygous-deficient (PLCγ1(+/-)) mice. Taken together, these data demonstrate that PLCγ2 participates in TCR signal transduction and plays a role in T cell selection.     

Role of STN1 and DNA Polymerase α in Telomere Stability and Genome-Wide Replication in Arabidopsis

The CST (Cdc13/CTC1-STN1-TEN1) complex was proposed to have evolved kingdom specific roles in telomere capping and replication. To shed light on its evolutionary conserved function, we examined the effect of STN1 dysfunction on telomere structure in plants. STN1 inactivation in Arabidopsis leads to a progressive loss of telomeric DNA and the onset of telomeric defects depends on the initial telomere size. While EXO1 aggravates defects associated with STN1 dysfunction, it does not contribute to the formation of long G-overhangs. Instead, these G-overhangs arise, at least partially, from telomerase-mediated telomere extension indicating a deficiency in C-strand fill-in synthesis. Analysis of hypomorphic DNA polymerase α mutants revealed that the impaired function of a general replication factor mimics the telomeric defects associated with CST dysfunction. Furthermore, we show that STN1-deficiency hinders re-replication of heterochromatic regions to a similar extent as polymerase α mutations. This comparative analysis of stn1 and pol α mutants suggests that STN1 plays a genome-wide role in DNA replication and that chromosome-end deprotection in stn1 mutants may represent a manifestation of aberrant replication through telomeres.     

Tetra-substituted imidazoles as a new class of inhibitors of the p53–MDM2 interaction

Capitalizing on crystal structure information obtained from a previous effort in the search for non peptide inhibitors of the p53–MDM2 interaction, we have discovered another new class of compounds able to disrupt this protein–protein interaction, an important target in oncology drug research. The new inhibitors, based on a tetra-substituted imidazole scaffold, have been optimized to low nanomolar potency in a biochemical assay following a structure-guided approach. An appropriate strategy has allowed us to translate the high biochemical potency in significant anti-proliferative activity on a p53-dependent MDM2 amplified cell line.     

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.