The protein histidine phosphatase LHPP is a tumour suppressor.

Histidine phosphorylation, the so-called hidden phosphoproteome, is a poorly characterized post-translational modification of proteins. Here we describe a role of histidine phosphorylation in tumorigenesis. Proteomic analysis of 12 tumours from an mTOR-driven hepatocellular carcinoma mouse model revealed that NME1 and NME2, the only known mammalian histidine kinases, were upregulated. Conversely, expression of the putative histidine phosphatase LHPP was downregulated specifically in the tumours. We demonstrate that LHPP is indeed a protein histidine phosphatase. Consistent with these observations, global histidine phosphorylation was significantly upregulated in the liver tumours. Sustained, hepatic expression of LHPP in the hepatocellular carcinoma mouse model reduced tumour burden and prevented the loss of liver function. Finally, in patients with hepatocellular carcinoma, low expression of LHPP correlated with increased tumour severity and reduced overall survival. Thus, LHPP is a protein histidine phosphatase and tumour suppressor, suggesting that deregulated histidine phosphorylation is oncogenic.

The human Descemet's membrane and lens capsule: Protein composition and biomechanical properties.

The Descemet's membrane (DM) and the lens capsule (LC) are two ocular basement membranes (BMs) that are essential in maintaining stability and structure of the cornea and lens. In this study, we investigated the proteomes and biomechanical properties of these two materials to uncover common and unique properties. We also screened for possible protein changes during diabetes. LC-MS/MS was used to determine the proteomes of both BMs. Biomechanical measurements were conducted by atomic force microscopy (AFM) in force spectroscopy mode, and complemented with immunofluorescence microscopy. Proteome analysis showed that all six existing collagen IV chains represent 70% of all LC-protein, and are thus the dominant components of the LC. The DM on the other hand is predominantly composed of a single protein, TGF-induced protein, which accounted for around 50% of all DM-protein. Four collagen IV-family members in DM accounted for only 10% of the DM protein. Unlike the retinal vascular BMs, the LC and DM do not undergo significant changes in their protein compositions during diabetes. Nanomechanical measurements showed that the endothelial/epithelial sides of both BMs are stiffer than their respective stromal/anterior-chamber sides, and both endothelial and stromal sides of the DM were stiffer than the epithelial and anterior-chamber sides of the LC. Long-term diabetes did not change the stiffness of the DM and LC. In summary, our analyses show that the protein composition and biomechanical properties of the DM and LC are different, i.e., the LC is softer than DM despite a significantly higher concentration of collagen IV family members. This finding is unexpected, as collagen IV members are presumed to be responsible for BM stiffness. Diabetes had no significant effect on the protein composition and the biomechanical properties of both the DM and LC.

Quantitative proteomics and phosphoproteomics on serial tumor biopsies from a sorafenib-treated HCC patient.

Compensatory signaling pathways in tumors confer resistance to targeted therapy, but the pathways and their mechanisms of activation remain largely unknown. We describe a procedure for quantitative proteomics and phosphoproteomics on snap-frozen biopsies of hepatocellular carcinoma (HCC) and matched nontumor liver tissue. We applied this procedure to monitor signaling pathways in serial biopsies taken from an HCC patient before and during treatment with the multikinase inhibitor sorafenib. At diagnosis, the patient had an advanced HCC. At the time of the second biopsy, abdominal imaging revealed progressive disease despite sorafenib treatment. Sorafenib was confirmed to inhibit MAPK signaling in the tumor, as measured by reduced ribosomal protein S6 kinase phosphorylation. Hierarchical clustering and enrichment analysis revealed pathways broadly implicated in tumor progression and resistance, such as epithelial-to-mesenchymal transition and cell adhesion pathways. Thus, we describe a protocol for quantitative analysis of oncogenic pathways in HCC biopsies and obtained first insights into the effect of sorafenib in vivo. This protocol will allow elucidation of mechanisms of resistance and enable precision medicine.

NMDA receptor-dependent GABAB receptor internalization via CaMKII phosphorylation of serine 867 in GABAB1.

GABAB receptors are the G-protein-coupled receptors for GABA, the main inhibitory neurotransmitter in the brain. GABAB receptors are abundant on dendritic spines, where they dampen postsynaptic excitability and inhibit Ca2+ influx through NMDA receptors when activated by spillover of GABA from neighboring GABAergic terminals. Here, we show that an excitatory signaling cascade enables spines to counteract this GABAB-mediated inhibition. We found that NMDA application to cultured hippocampal neurons promotes dynamin-dependent endocytosis of GABAB receptors. NMDA-dependent internalization of GABAB receptors requires activation of Ca2+/Calmodulin-dependent protein kinase II (CaMKII), which associates with GABAB receptors in vivo and phosphorylates serine 867 (S867) in the intracellular C terminus of the GABAB1 subunit. Blockade of either CaMKII or phosphorylation of S867 renders GABAB receptors refractory to NMDA-mediated internalization. Time-lapse two-photon imaging of organotypic hippocampal slices reveals that activation of NMDA receptors removes GABAB receptors within minutes from the surface of dendritic spines and shafts. NMDA-dependent S867 phosphorylation and internalization is predominantly detectable with the GABAB1b subunit isoform, which is the isoform that clusters with inhibitory effector K+ channels in the spines. Consistent with this, NMDA receptor activation in neurons impairs the ability of GABAB receptors to activate K+ channels. Thus, our data support that NMDA receptor activity endocytoses postsynaptic GABAB receptors through CaMKII-mediated phosphorylation of S867. This provides a means to spare NMDA receptors at individual glutamatergic synapses from reciprocal inhibition through GABAB receptors.

A major role for the Plasmodium falciparum ApiAP2 protein PfSIP2 in chromosome end biology.

The heterochromatic environment and physical clustering of chromosome ends at the nuclear periphery provide a functional and structural framework for antigenic variation and evolution of subtelomeric virulence gene families in the malaria parasite Plasmodium falciparum. While recent studies assigned important roles for reversible histone modifications, silent information regulator 2 and heterochromatin protein 1 (PfHP1) in epigenetic control of variegated expression, factors involved in the recruitment and organization of subtelomeric heterochromatin remain unknown. Here, we describe the purification and characterization of PfSIP2, a member of the ApiAP2 family of putative transcription factors, as the unknown nuclear factor interacting specifically with cis-acting SPE2 motif arrays in subtelomeric domains. Interestingly, SPE2 is not bound by the full-length protein but rather by a 60kDa N-terminal domain, PfSIP2-N, which is released during schizogony. Our experimental re-definition of the SPE2/PfSIP2-N interaction highlights the strict requirement of both adjacent AP2 domains and a conserved bipartite SPE2 consensus motif for high-affinity binding. Genome-wide in silico mapping identified 777 putative binding sites, 94% of which cluster in heterochromatic domains upstream of subtelomeric var genes and in telomere-associated repeat elements. Immunofluorescence and chromatin immunoprecipitation (ChIP) assays revealed co-localization of PfSIP2-N with PfHP1 at chromosome ends. Genome-wide ChIP demonstrated the exclusive binding of PfSIP2-N to subtelomeric SPE2 landmarks in vivo but not to single chromosome-internal sites. Consistent with this specialized distribution pattern, PfSIP2-N over-expression has no effect on global gene transcription. Hence, contrary to the previously proposed role for this factor in gene activation, our results provide strong evidence for the first time for the involvement of an ApiAP2 factor in heterochromatin formation and genome integrity. These findings are highly relevant for our understanding of chromosome end biology and variegated expression in P. falciparum and other eukaryotes, and for the future analysis of the role of ApiAP2-DNA interactions in parasite biology.

Tear Proteomic Predictive Biomarker Model for Ocular Graft Versus Host Disease Classification.

Purpose: Diagnosis of ocular graft-versus-host disease (oGVHD) is hampered by a lack of clinically-validated biomarkers. This study aims to predict disease severity on the basis of tear protein expression in mild oGVHD. Methods: Forty-nine patients with and without chronic oGVHD after AHCT were recruited to a cross-sectional observational study. Patients were stratified using NIH guidelines for oGVHD severity: NIH 0 (none; n = 14), NIH 1 (mild; n = 9), NIH 2 (moderate; n = 16), and NIH 3 (severe; n = 10). The proteomic profile of tears was analyzed using liquid chromatography-tandem mass spectrometry. Random forest and penalized logistic regression were used to generate classification and prediction models to stratify patients according to disease severity. Results: Mass spectrometry detected 785 proteins across all samples. A random forest model used to classify patients by disease grade achieved F1-measure values for correct classification of 0.95 (NIH 0), 0.8 (NIH 1), 0.74 (NIH 2), and 0.83 (NIH 3). A penalized logistic regression model was generated by comparing patients without oGVHD and those with mild oGVHD and applied to identify potential biomarkers present early in disease. A panel of 13 discriminant markers achieved significant diagnostic accuracy in identifying patients with moderate-to-severe disease. Conclusions: Our work demonstrates the utility of tear protein biomarkers in classifying oGVHD severity and adds further evidence indicating ocular surface inflammation as a main driver of oGVHD clinical phenotype. Translational Relevance: Expression levels of a 13-marker tear protein panel in AHCT patients with mild oGVHD may predict development of more severe oGVHD clinical phenotypes.

Exploring the dynamics of the yeast proteome by means of 2-DE.

In this study we combined pulse chase experiments and 2-DE in order to investigate how newly synthesized proteins are processed or modified to yield a functional yeast proteome. This approach allowed us to follow the fate of 560 native yeast proteins from the time they were synthesized up to several hours later. Among these, 81 were observed to vary during the chase, either increasing or decreasing. In addition, 60 were found to be modified immediately after their synthesis. Taking advantage of protein identifications, we characterized a wide variety of post-translational events responsible for these changes, such as protein turnover, protein maturation and different types of PTMs. These events operate over very different time scales, ranging from the brief period required for co-translational modifications to one generation time or more. In light of these results, the functional proteome of exponentially growing cells appears to be the product of a permanent remodelling process that modifies native proteins far beyond the time they have been synthesized. This study also allowed us to obtain information on the half-lives of 260 abundant yeast proteins.

Yeast proteome map (last update).

The identification of proteins separated on 2-D gels is essential to exploit the full potential of 2-D gel electrophoresis for proteomic investigations. For this purpose we have undertaken the systematic identification of Saccharomyces cerevisiae proteins separated on 2-D gels. We report here the identification by mass spectrometry of 100 novel yeast protein spots that have so far not been tackled due to their scarcity on our standard 2-D gels. These identifications extend the number of protein spots identified on our yeast 2-D proteome map to 716. They correspond to 485 unique proteins. Among these, 154 were resolved into several isoforms. The present data set can now be expanded to report for the first time a map of 363 protein isoforms that significantly deepens our knowledge of the yeast proteome. The reference map and a list of all identified proteins can be accessed on the Yeast Protein Map server (www.ibgc.u-bordeaux2.fr/YPM).

A modified KESTREL search reveals a basophilic substrate consensus for the Saccharomyces cerevisiae Npr1 protein kinase.

The Saccharomyces cerevisiae nitrogen permease reactivator Npr1 is a hyperphosphorylated protein that belongs to a family of Ser/Thr protein kinases dedicated to the regulation of plasma membrane transporters. Its activity is regulated by the Tor (target of rapamycin) signaling pathway. Inhibition of the Tor proteins by treating yeast cells with the immunosuppressant drug rapamycin promotes rapid dephosphorylation of Npr1. As an alternative to peptide arrays, the substrate requirement of Npr1 was probed with a peptide library that was generated by cleaving yeast cell extracts with CNBr, and after reverse-phase chromatography, the individual fractions were phosphorylated in vitro with recombinant Npr1. In this way, the ribosomal protein Rpl24a was found to be an excellent in vitro substrate for Npr1. Synthetic peptides tailored around the phosphorylation site of Rpl24a show that Npr1 is a Ser/Thr protein kinase with an absolute requirement for a basic residue at the P-3 position and a strong preference for basic P + 1 residues, whereas proline at P + 1 is strongly disfavored. The results obtained with synthetic peptides suggest a (K/R)-X-X-S-(K/R) consensus sequence for Npr1. The availability of a consensus sequence allows a targeted search for physiologically relevant Npr1 substrates involved in the regulation of yeast amino acid permeases.

Combined Transcriptomic and Proteomic Analyses of Cerebral Frontal Lobe Tissue Identified RNA Metabolism Dysregulation as One Potential Pathogenic Mechanism in Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL).

BACKGROUND: Cerebral small vessel disease (SVD) is an important cause of stroke and vascular cognitive impairment (VCI), leading to subcortical ischemic vascular dementia. As a hereditary form of SVD with early onset, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) represents a pure form of SVD and may thus serve as a model disease for SVD. To date, underlying molecular mechanisms linking vascular pathology and subsequent neuronal damage in SVD are incompletely understood. OBJECTIVE: We performed comparative transcriptional profiling microarray and proteomic analyses on post-mortem frontal lobe specimen from 2 CADASIL patients and 5 non neurologically diseased controls in order to identify dysregulated pathways potentially involved in the development of tissue damage in CADASIL. METHODS: Transcriptional microarray analysis of material extracted from frontal grey and white matter (WM) identified subsets of up- or down-regulated genes enriched into biological pathways mostly in WM areas. Proteomic analysis of these regions also highlighted cellular processes identified by dysregulated proteins. RESULTS: Discrepancies between proteomic and transcriptomic data were observed, but a number of pathways were commonly associated with genes and corresponding proteins, such as: "ribosome" identified by upregulated genes and proteins in frontal cortex or "spliceosome" associated with down-regulated genes and proteins in frontal WM. CONCLUSION: This latter finding suggests that defective expression of spliceosomal components may alter widespread splicing profile, potentially inducing expression abnormalities that could contribute to cerebral WM damage in CADASIL.

Mapping and functional analysis of heterochromatin protein 1 phosphorylation in the malaria parasite Plasmodium falciparum.

Previous studies in model eukaryotes have demonstrated that phosphorylation of heterochromatin protein 1 (HP1) is important for dynamically regulating its various functions. However, in the malaria parasite Plasmodium falciparum both the function of HP1 phosphorylation and the identity of the protein kinases targeting HP1 are still elusive. In order to functionally analyze phosphorylation of P. falciparum HP1 (PfHP1), we first mapped PfHP1 phosphorylation sites by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of native PfHP1, which identified motifs from which potential kinases could be predicted; in particular, several phosphorylated residues were embedded in motifs rich in acidic residues, reminiscent of targets for P. falciparum casein kinase 2 (PfCK2). Secondly, we tested recombinant PfCK2 and a number of additional protein kinases for their ability to phosphorylate PfHP1 in in vitro kinase assays. These experiments validated our prediction that PfHP1 acts as a substrate for PfCK2. Furthermore, LC-MS/MS analysis showed that PfCK2 phosphorylates three clustered serine residues in an acidic motif within the central hinge region of PfHP1. To study the role of PfHP1 phosphorylation in live parasites we used CRISPR/Cas9-mediated genome editing to generate a number of conditional PfHP1 phosphomutants based on the DiCre/LoxP system. Our studies revealed that neither PfCK2-dependent phosphorylation of PfHP1, nor phosphorylation of the hinge domain in general, affect PfHP1's ability to localize to heterochromatin, and that PfHP1 phosphorylation in this region is dispensable for the proliferation of P. falciparum blood stage parasites.

Mitochondrial mediation of environmental osmolytes discrimination during osmoadaptation in the extremely halotolerant black yeast Hortaea werneckii.

We have investigated the mitochondrial responses to hyperosmotic environments of ionic (4.5 M NaCl) and non-ionic (3.0 M sorbitol) osmolytes in the most halo/osmo-tolerant black yeast, Hortaea werneckii. Adaptation to both types of osmolytes resulted in differential expression of mitochondria-related genes. Live-cell imaging has revealed a condensation of mitochondria in hyperosmotic media that depends on osmolyte type. In the hypersaline medium, this was accompanied by increased ATP synthesis and oxidative damage protection, whereas adaptation to the non-ionic osmolyte resulted in a decrease in ATP synthesis and lipid peroxidation level in mitochondria. A proteomic study of the mitochondria revealed preferential accumulation of energy metabolism enzymes in the hypersaline medium, and accumulation of protein chaperones in the non-ionic osmolyte. The HwBmh1/14-3-3 protein, localized to mitochondria in hypersaline conditions, and not at optimal salinity, suggesting its role in differential perception of ionic and non-ionic osmolytes in H. werneckii.

Tear Film Proteomics Reveal Important Differences Between Patients With and Without Ocular GvHD After Allogeneic Hematopoietic Cell Transplantation.

Purpose: To date, no biomarkers for ocular graft versus host disease (GvHD), a frequent complication following allogeneic hematopoietic cell transplantation (HCT), exist. In this prospective study, we evaluated the potential of human tear proteins as biomarkers for ocular GvHD. Methods: Tears from 10 patients with moderate-to-severe ocular GvHD were compared to 10 patients without ocular GvHD. After a full ocular surface clinical examination, tears were collected onto Schirmer strips and protein composition was analyzed by liquid chromatography tandem mass spectrometry. Statistical evaluation was performed using the Mann-Whitney U test to compare means and the false discovery rate method to adjust for multiple comparisons. Functional annotation of differentially expressed proteins was done with the PANTHER classification system. Results: We identified 282 proteins in tryptic digests of Schirmer strips; 79 proteins were significantly differentially expressed between the two groups, from which 54 were up- and 25 downregulated. The most upregulated proteins were classified as nucleic acid binding and cytoskeletal proteins, while the most extensively downregulated proteins belong to an array of classes including transfer and receptor proteins, enzyme modulators, and hydrolases. In addition to proteins already confirmed as differentially expressed in dry eye disease, we report changes in 36 novel proteins. Conclusions: This study reports the proteomic profile of tears in ocular GvHD for the first time and identifies a number of unique differentially expressed proteins. Further studies with a higher number of participants are necessary to confirm these results and to evaluate the reliability of these expression patterns in longitudinal studies.

Correction: Diabetes-related changes in the protein composition and the biomechanical properties of human retinal vascular basement membranes.

[This corrects the article DOI: 10.1371/journal.pone.0189857.].

GDV1 induces sexual commitment of malaria parasites by antagonizing HP1-dependent gene silencing.

Malaria is caused by Plasmodium parasites that proliferate in the bloodstream. During each replication cycle, some parasites differentiate into gametocytes, the only forms able to infect the mosquito vector and transmit malaria. Sexual commitment is triggered by activation of AP2-G, the master transcriptional regulator of gametocytogenesis. Heterochromatin protein 1 (HP1)-dependent silencing of ap2-g prevents sexual conversion in proliferating parasites. In this study, we identified Plasmodium falciparum gametocyte development 1 (GDV1) as an upstream activator of sexual commitment. We found that GDV1 targeted heterochromatin and triggered HP1 eviction, thus derepressing ap2-g Expression of GDV1 was responsive to environmental triggers of sexual conversion and controlled via a gdv1 antisense RNA. Hence, GDV1 appears to act as an effector protein that induces sexual differentiation by antagonizing HP1-dependent gene silencing.

mTORC2 Promotes Tumorigenesis via Lipid Synthesis.

Dysregulated mammalian target of rapamycin (mTOR) promotes cancer, but underlying mechanisms are poorly understood. We describe an mTOR-driven mouse model that displays hepatosteatosis progressing to hepatocellular carcinoma (HCC). Longitudinal proteomic, lipidomics, and metabolomic analyses revealed that hepatic mTORC2 promotes de novo fatty acid and lipid synthesis, leading to steatosis and tumor development. In particular, mTORC2 stimulated sphingolipid (glucosylceramide) and glycerophospholipid (cardiolipin) synthesis. Inhibition of fatty acid or sphingolipid synthesis prevented tumor development, indicating a causal effect in tumorigenesis. Increased levels of cardiolipin were associated with tubular mitochondria and enhanced oxidative phosphorylation. Furthermore, increased lipogenesis correlated with elevated mTORC2 activity and HCC in human patients. Thus, mTORC2 promotes cancer via formation of lipids essential for growth and energy production.

Diabetes-related changes in the protein composition and the biomechanical properties of human retinal vascular basement membranes.

Basement membranes (BMs) are specialized sheets of extracellular matrix that outline epithelial cell layers, muscle fibers, blood vessels, and peripheral nerves. A well-documented histological hallmark of progressing diabetes is a major increase in vascular BM thickness. In order to investigate whether this structural change is accompanied by a change in the protein composition, we compared the proteomes of retinal vascular BMs from diabetic and non-diabetic donors by using LC-MS/MS. Data analysis showed that seventeen extracellular matrix (ECM)-associated proteins were more abundant in diabetic than non-diabetic vascular BMs. Four ECM proteins were more abundant in non-diabetic than in diabetic BMs. Most of the over-expressed proteins implicate a complement-mediated chronic inflammatory process in the diabetic retinal vasculature. We also found an up-regulation of norrin, a protein that is known to promote vascular proliferation, possibly contributing to the vascular remodeling during diabetes. Many of the over-expressed proteins were localized to microvascular aneurisms. Further, the overall stoichiometry of proteins was changed, such that the relative abundance of collagens in BMs from diabetic patients was higher than normal. Biomechanical measurements of vascular BM flat mounts using AFM showed that their outer surface was softer than normal.

Malaria parasites possess a telomere repeat-binding protein that shares ancestry with transcription factor IIIA.

Telomere repeat-binding factors (TRFs) are essential components of the molecular machinery that regulates telomere function. TRFs are widely conserved across eukaryotes and bind duplex telomere repeats via a characteristic MYB-type domain. Here, we identified the telomere repeat-binding protein PfTRZ in the malaria parasite Plasmodium falciparum, a member of the Alveolate phylum for which TRFs have not been described so far. PfTRZ lacks an MYB domain and binds telomere repeats via a C2H2-type zinc finger domain instead. In vivo, PfTRZ binds with high specificity to the telomeric tract and to interstitial telomere repeats upstream of subtelomeric virulence genes. Conditional depletion experiments revealed that PfTRZ regulates telomere length homeostasis and is required for efficient cell cycle progression. Intriguingly, we found that PfTRZ also binds to and regulates the expression of 5S rDNA genes. Combined with detailed phylogenetic analyses, our findings identified PfTRZ as a remote functional homologue of the basic transcription factor TFIIIA, which acquired a new function in telomere maintenance early in the apicomplexan lineage. Our work sheds unexpected new light on the evolution of telomere repeat-binding proteins and paves the way for dissecting the presumably divergent mechanisms regulating telomere functionality in one of the most deadly human pathogens.

Neospora caninum protein disulfide isomerase is involved in tachyzoite-host cell interaction.

We have previously shown that treatment of Neospora caninum tachyzoites with the aspartyl protease inhibitor pepstatin A reduces host cell invasion [Naguleswaran, A., Muller, N., Hemphill, A., 2003. Neospora caninum and Toxoplasma gondii: a novel adhesion/invasion assay reveals distinct differences in tachyzoite-host cell interactions. Exp. Parasitol. 104, 149-158]. Pepstatin A-affinity-chromatography led to the isolation of a major band of approximately 52 kDa which was identified as a homologue of a previously described Toxoplasma gondii putative protein disulfide isomerase (TgPDI) through tandem mass spectrometry. A BLAST search against N. caninum expressed sequence tags (ESTs) on the ApiDots server using TgPDI cDNA as query sequence revealed a 2251 bp PDI-like consensus (NcPDI), which shows 94% identity to the T. gondii homologue. In N. caninum tachyzoites, NcPDI was found mainly in the soluble hydrophilic fraction. Immunofluorescence showed that expression of NcPDI was dramatically down-regulated in the bradyzoite stage, and immunogold-EM on tachyzoites localised the protein to the cytoplasm, mostly in close vicinity to the nuclear membrane, to the micronemes, and to the parasite cell surface. However, NcPDI was absent in rhoptries and dense granules. Preincubation of tachyzoites with the sulfhydryl blocker 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), p-chloromercuribenzoic acid (pCMBA), and with the PDI inhibitor bacitracin reduced adhesion of parasites to host cells. In addition, incubation of N. caninum tachyzoites with affinity-purified anti-NcPDI antibodies reduced host cell adhesion. PDIs catalyse the formation, reduction or isomerisation of disulfide bonds. Many major components of the adhesion and invasion machinery of apicomplexan parasites are cysteine-rich and dependent on correct folding via disulfide bond formation. Thus, our data points towards an important role for surface-associated NcPDI in Neospora-host cell interaction.

Cytokine-induced macropinocytosis in macrophages is regulated by 14-3-3ζ through its interaction with serine-phosphorylated coronin 1.

The induction of macropinocytosis in macrophages during an inflammatory response is important for clearance of pathogenic microbes as well as the generation of appropriate immune responses. Recent data suggest that cytokine stimulation of macrophages induces macropinocytosis through phosphorylation of the protein coronin 1, thereby redistributing coronin 1 from the cell cortex to the cytoplasm followed by the activation of phosphoinositol-3 (PI-3) kinase. However, how coronin 1 phosphorylation regulates these processes remains unclear. We here define an essential role for 14-3-3ζ in cytokine-induced and coronin-1-dependent macropinocytosis in macrophages. We found that, upon stimulation, phosphorylated coronin 1 transiently associated with 14-3-3ζ and receptor of activated C kinase 1 (RACK1). Importantly, downregulation of 14-3-3ζ, but not RACK1, prevented relocation of coronin 1, as well as the induction of PI-3 kinase activity and thereby macropinocytosis upon cytokine stimulation. Together these data define an essential role for 14-3-3ζ in the regulation of macropinocytosis in macrophages upon cytokine stimulation through modulation of the localization of coronin 1.