The ß2-adrenergic receptor associates with CXCR4 multimers in human cancer cells.

While the existence and functional role of class C G-protein-coupled receptors (GPCR) dimers is well established, there is still a lack of consensus regarding class A and B GPCR multimerization. This lack of consensus is largely due to the inherent challenges of demonstrating the presence of multimeric receptor complexes in a physiologically relevant cellular context. The C-X-C motif chemokine receptor 4 (CXCR4) is a class A GPCR that is a promising target of anticancer therapy. Here, we investigated the potential of CXCR4 to form multimeric complexes with other GPCRs and characterized the relative size of the complexes in a live-cell environment. Using a bimolecular fluorescence complementation (BiFC) assay, we identified the ß2 adrenergic receptor (ß2AR) as an interaction partner. To investigate the molecular scale details of CXCR4-ß2AR interactions, we used a time-resolved fluorescence spectroscopy method called pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS can resolve membrane protein density, diffusion, and multimerization state in live cells at physiological expression levels. We probed CXCR4 and ß2AR homo- and heteromultimerization in model cell lines and found that CXCR4 assembles into multimeric complexes larger than dimers in MDA-MB-231 human breast cancer cells and in HCC4006 human lung cancer cells. We also found that ß2AR associates with CXCR4 multimers in MDA-MB-231 and HCC4006 cells to a higher degree than in COS-7 and CHO cells and in a ligand-dependent manner. These results suggest that CXCR4-ß2AR heteromers are present in human cancer cells and that GPCR multimerization is significantly affected by the plasma membrane environment.

ARV1 deficiency induces lipid bilayer stress and enhances rDNA stability by activating the unfolded protein response in Saccharomyces cerevisiae.

The stability of ribosomal DNA (rDNA) is maintained through transcriptional silencing by the NAD+-dependent histone deacetylase Sir2 in Saccharomyces cerevisiae. Alongside proteostasis, rDNA stability is a crucial factor regulating the replicative lifespan of S. cerevisiae. The unfolded protein response (UPR) is induced by misfolding of proteins or an imbalance of membrane lipid composition and is responsible for degrading misfolded proteins and restoring endoplasmic reticulum (ER) membrane homeostasis. Recent investigations have suggested that the UPR can extend the replicative lifespan of yeast by enhancing protein quality control mechanisms, but the relationship between the UPR and rDNA stability remains unknown. In this study, we found that the deletion of ARV1, which encodes an ER protein of unknown molecular function, activates the UPR by inducing lipid bilayer stress. In arv1Δ cells, the UPR and the cell wall integrity pathway are activated independently of each other, and the high osmolarity glycerol (HOG) pathway is activated in a manner dependent on Ire1, which mediates the UPR. Activated Hog1 translocates the stress response transcription factor Msn2 to the nucleus, where it promotes the expression of nicotinamidase Pnc1, a well-known Sir2 activator. Following Sir2 activation, rDNA silencing and rDNA stability are promoted. Furthermore, the loss of other ER proteins, such as Pmt1 or Bst1, and ER stress induced by tunicamycin or inositol depletion also enhance rDNA stability in a Hog1-dependent manner. Collectively, these findings suggest that the induction of the UPR enhances rDNA stability in S. cerevisiae by promoting the Msn2-Pnc1-Sir2 pathway in a Hog1-dependent manner.

The ubiquitin-proteasome system degrades fatty acid synthase under nitrogen starvation when autophagy is dysfunctional in Saccharomyces cerevisiae.

Autophagy and the ubiquitin-proteasome system (UPS) are two major protein quality control mechanisms maintaining cellular proteostasis. In Saccharomyces cerevisiae, the de novo synthesis of saturated fatty acids is performed by a multienzyme complex known as fatty acid synthase (FAS). A recent study reported that yeast FAS is preferentially degraded by autophagy under nitrogen starvation. In this study, we examined the fate of FAS during nitrogen starvation when autophagy is dysfunctional. We found that the UPS compensates for FAS degradation in the absence of autophagy. Additionally, we discovered that the UPS-dependent degradation of Fas2 requires the E3 ubiquitin ligase Ubr1. Our findings highlight the complementary relationship between autophagy and the UPS.

LPA1-mediated inhibition of CXCR4 attenuates CXCL12-induced signaling and cell migration.

BACKGROUND: G protein-coupled receptor heteromerization is believed to exert dynamic regulatory impact on signal transduction. CXC chemokine receptor 4 (CXCR4) and its ligand CXCL12, both of which are overexpressed in many cancers, play a pivotal role in metastasis. Likewise, lysophosphatidic acid receptor 1 (LPA1) is implicated in cancer cell proliferation and migration. In our preliminary study, we identified LPA1 as a prospective CXCR4 interactor. In the present study, we investigated in detail the formation of the CXCR4-LPA1 heteromer and characterized the unique molecular features and function of this heteromer. METHODS: We employed bimolecular fluorescence complementation, bioluminescence resonance energy transfer, and proximity ligation assays to demonstrate heteromerization between CXCR4 and LPA1. To elucidate the distinctive molecular characteristics and functional implications of the CXCR4-LPA1 heteromer, we performed various assays, including cAMP, BRET for G protein activation, ß-arrestin recruitment, ligand binding, and transwell migration assays. RESULTS: We observed that CXCR4 forms heteromers with LPA1 in recombinant HEK293A cells and the human breast cancer cell line MDA-MB-231. Coexpression of LPA1 with CXCR4 reduced CXCL12-mediated cAMP inhibition, ERK activation, Gαi/o activation, and ß-arrestin recruitment, while CXCL12 binding to CXCR4 remained unaffected. In contrast, CXCR4 had no impact on LPA1-mediated signaling. The addition of lysophosphatidic acid (LPA) further hindered CXCL12-induced Gαi/o recruitment to CXCR4. LPA or alkyl-OMPT inhibited CXCL12-induced migration in various cancer cells that endogenously express both CXCR4 and LPA1. Conversely, CXCL12-induced calcium signaling and migration were increased in LPAR1 knockout cells, and LPA1-selective antagonists enhanced CXCL12-induced Gαi/o signaling and cell migration in the parental MDA-MB-231 cells but not in LPA1-deficient cells. Ultimately, complete inhibition of cell migration toward CXCL12 and alkyl-OMPT was only achieved in the presence of both CXCR4 and LPA1 antagonists. CONCLUSIONS: The presence and impact of CXCR4-LPA1 heteromers on CXCL12-induced signaling and cell migration have been evidenced across various cell lines. This discovery provides crucial insights into a valuable regulatory mechanism of CXCR4 through heteromerization. Moreover, our findings propose a therapeutic potential in combined CXCR4 and LPA1 inhibitors for cancer and inflammatory diseases associated with these receptors, simultaneously raising concerns about the use of LPA1 antagonists alone for such conditions. Video Abstract.

Loss of Smi1, a protein involved in cell wall synthesis, extends replicative life span by enhancing rDNA stability in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, replicative life span (RLS) is primarily affected by the stability of ribosomal DNA (rDNA). The stability of the highly repetitive rDNA array is maintained through transcriptional silencing by the NAD+-dependent histone deacetylase Sir2. Recently, the loss of Smi1, a protein of unknown molecular function that has been proposed to be involved in cell wall synthesis, has been demonstrated to extend RLS in S. cerevisiae, but the mechanism by which Smi1 regulates RLS has not been elucidated. In this study, we determined that the loss of Smi1 extends RLS in a Sir2-dependent manner. We observed that the smi1Δ mutation enhances transcriptional silencing at the rDNA locus and promotes rDNA stability. In the absence of Smi1, the stress-responsive transcription factor Msn2 translocates from the cytoplasm to the nucleus, and nuclear-accumulated Msn2 stimulates the expression of nicotinamidase Pnc1, which serves as an activator of Sir2. In addition, we observed that the MAP kinase Hog1 is activated in smi1Δ cells and that the activation of Hog1 induces the translocation of Msn2 into the nucleus. Taken together, our findings suggest that the loss of Smi1 leads to the nuclear accumulation of Msn2 and stimulates the expression of Pnc1, thereby enhancing Sir2-mediated rDNA stability and extending RLS in S. cerevisiae.

Loss of Smi1, a protein involved in cell wall synthesis, extends replicative lifespan by enhancing rDNA stability in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, replicative lifespan (RLS) is primarily affected by the stability of ribosomal DNA (rDNA). The stability of the highly repetitive rDNA array is maintained through transcriptional silencing by the NAD+-dependent histone deacetylase Sir2. Recently, the loss of Smi1, a protein of unknown molecular function that has been proposed to be involved in cell wall synthesis, has been demonstrated to extend RLS in S. cerevisiae, but the mechanism by which Smi1 regulates RLS has not been elucidated. In this study, we determined that the loss of Smi1 extends RLS in a Sir2-dependent manner. We observed that the smi1D mutation enhances transcriptional silencing at the rDNA locus and promotes rDNA stability. In the absence of Smi1, the stress-responsive transcription factor Msn2 translocates from the cytoplasm to the nucleus, and nuclear-accumulated Msn2 stimulates the expression of nicotinamidase Pnc1, which serves as an activator of Sir2. In addition, we observed that the MAP kinase Hog1 is activated in smi1D cells and that the activation of Hog1 induces the translocation of Msn2 into the nucleus. Taken together, our findings suggest that the loss of Smi1 leads to the nuclear accumulation of Msn2 and stimulates the expression of Pnc1, thereby enhancing Sir2-mediated rDNA stability and extending RLS in S. cerevisiae.

Global analysis of protein homomerization in Saccharomyces cerevisiae.

In vivo analyses of the occurrence, subcellular localization, and dynamics of protein-protein interactions (PPIs) are important issues in functional proteomic studies. The bimolecular fluorescence complementation (BiFC) assay has many advantages in that it provides a reliable way to detect PPIs in living cells with minimal perturbation of the structure and function of the target proteins. Previously, to facilitate the application of the BiFC assay to genome-wide analysis of PPIs, we generated a collection of yeast strains expressing full-length proteins tagged with the N-terminal fragment of Venus (VN), a yellow fluorescent protein variant, from their own native promoters. In the present study, we constructed a VC (the C-terminal fragment of Venus) fusion library consisting of 5671 MATα strains expressing C-terminally VC-tagged proteins (representing ∼91% of the yeast proteome). For genome-wide analysis of protein homomer formation, we mated each strain in the VC fusion library with its cognate strain in the VN fusion library and performed the BiFC assay. From this analysis, we identified 186 homomer candidates. We further investigated the functional relevance of the homomerization of Pln1, a yeast perilipin. Our data set provides a useful resource for understanding the physiological roles of protein homomerization. Furthermore, the VC fusion library together with the VN fusion library will provide a valuable platform to systematically analyze PPIs in the natural cellular context.

G2A Protects Mice against Sepsis by Modulating Kupffer Cell Activation: Cooperativity with Adenosine Receptor 2b.

G2A is a GPCR abundantly expressed in immune cells. G2A-/- mice showed higher lethality, higher plasma cytokines, and an impaired bacterial clearance in response to a murine model of sepsis (cecal ligation and puncture), which were blocked by GdCl3, an inhibitor of Kupffer cells. Anti-IL-10 Ab reversed the impaired bacterial clearance in G2A-/- mice. Indomethacin effectively blocked both the increased i.p. IL-10 levels and the impaired bacterial clearance, indicating that disturbed PG system is the proximal cause of these phenomena. Stimulation with LPS/C5a induced an increase in Escherichia coli phagocytosis and intracellular cAMP levels in G2A+/+ peritoneal macrophages but not G2A-/- cells, which showed more PGE2/nitrite release and intracellular reactive oxygen species levels. Heterologous coexpression of G2A and adenosine receptor type 2b (A2bAR) induced a synergistic increase in cAMP signaling in a ligand-independent manner, with the evidence of physical interaction of G2A with A2bAR. BAY 60-6583, a specific agonist for A2bAR, increased intracellular cAMP levels in Kupffer cells from G2A+/+ but not from G2A-/- mice. Both G2A and A2bAR were required for antiseptic action of lysophosphatidylcholine. These results show inappropriate activation of G2A-/- Kupffer cells to septic insults due to an impaired cAMP signaling possibly by lack of interaction with A2bAR.

Targeted knockout of a chemokine-like gene increases anxiety and fear responses.

Emotional responses, such as fear and anxiety, are fundamentally important behavioral phenomena with strong fitness components in most animal species. Anxiety-related disorders continue to represent a major unmet medical need in our society, mostly because we still do not fully understand the mechanisms of these diseases. Animal models may speed up discovery of these mechanisms. The zebrafish is a highly promising model organism in this field. Here, we report the identification of a chemokine-like gene family, samdori (sam), and present functional characterization of one of its members, sam2 We show exclusive mRNA expression of sam2 in the CNS, predominantly in the dorsal habenula, telencephalon, and hypothalamus. We found knockout (KO) zebrafish to exhibit altered anxiety-related responses in the tank, scototaxis and shoaling assays, and increased crh mRNA expression in their hypothalamus compared with wild-type fish. To investigate generalizability of our findings to mammals, we developed a Sam2 KO mouse and compared it to wild-type littermates. Consistent with zebrafish findings, homozygous KO mice exhibited signs of elevated anxiety. We also found bath application of purified SAM2 protein to increase inhibitory postsynaptic transmission onto CRH neurons of the paraventricular nucleus. Finally, we identified a human homolog of SAM2, and were able to refine a candidate gene region encompassing SAM2, among 21 annotated genes, which is associated with intellectual disability and autism spectrum disorder in the 12q14.1 deletion syndrome. Taken together, these results suggest a crucial and evolutionarily conserved role of sam2 in regulating mechanisms associated with anxiety.

Ksp1-dependent phosphorylation of eIF4G modulates post-transcriptional regulation of specific mRNAs under glucose deprivation conditions.

Post-transcriptional regulation is an important mechanism for modulating gene expression and is performed by numerous mRNA-binding proteins. To understand the mechanisms underlying post-transcriptional regulation, we investigated the phosphorylation status of 32 mRNA-binding proteins under glucose deprivation conditions in Saccharomyces cerevisiae. We identified 17 glucose-sensitive phosphoproteins and signal pathways implicated in their phosphorylation. Notably, phosphorylation of the eukaryotic translation initiation factor 4G (eIF4G) was regulated by both the Snf1/AMPK pathway and the target of rapamycin complex 1 (TORC1) pathway. The serine/threonine protein kinase Ksp1 has previously been suggested to be a downstream effector of TORC1, but its detailed function has rarely been discussed. We identified that Snf1/AMPK and TORC1 signalings converge on Ksp1, which phosphorylates eIF4G under glucose deprivation conditions. Ksp1-dependent phosphorylation of eIF4G regulates the degradation of specific mRNAs (e.g. glycolytic mRNAs and ribosomal protein mRNAs) under glucose deprivation conditions likely through the recruitment of Dhh1. Taken together, our results suggest that Ksp1 functions as a novel modulator of post-transcriptional regulation in yeast.

Rad52 phosphorylation by Ipl1 and Mps1 contributes to Mps1 kinetochore localization and spindle assembly checkpoint regulation.

Rad52 is well known as a key factor in homologous recombination. Here, we report that Rad52 has functions unrelated to homologous recombination in Saccharomyces cerevisiae; it plays a role in the recruitment of Mps1 to the kinetochores and the maintenance of spindle assembly checkpoint (SAC) activity. Deletion of RAD52 causes various phenotypes related to the dysregulation of chromosome biorientation. Rad52 directly affects efficient operation of the SAC and accurate chromosome segregation. Remarkably, by using an in vitro kinase assay, we found that Rad52 is a substrate of Ipl1/Aurora and Mps1 in yeast and humans. Ipl1-dependent phosphorylation of Rad52 facilitates the kinetochore accumulation of Mps1, and Mps1-dependent phosphorylation of Rad52 is important for the accurate regulation of the SAC under spindle damage conditions. Taken together, our data provide detailed insights into the regulatory mechanism of chromosome biorientation by mitotic kinases.

Proteome-wide remodeling of protein location and function by stress.

Protein location and function can change dynamically depending on many factors, including environmental stress, disease state, age, developmental stage, and cell type. Here, we describe an integrative computational framework, called the conditional function predictor (CoFP; http://nbm.ajou.ac.kr/cofp/), for predicting changes in subcellular location and function on a proteome-wide scale. The essence of the CoFP approach is to cross-reference general knowledge about a protein and its known network of physical interactions, which typically pool measurements from diverse environments, against gene expression profiles that have been measured under specific conditions of interest. Using CoFP, we predict condition-specific subcellular locations, biological processes, and molecular functions of the yeast proteome under 18 specified conditions. In addition to highly accurate retrieval of previously known gold standard protein locations and functions, CoFP predicts previously unidentified condition-dependent locations and functions for nearly all yeast proteins. Many of these predictions can be confirmed using high-resolution cellular imaging. We show that, under DNA-damaging conditions, Tsr1, Caf120, Dip5, Skg6, Lte1, and Nnf2 change subcellular location and RNA polymerase I subunit A43, Ino2, and Ids2 show changes in DNA binding. Beyond specific predictions, this work reveals a global landscape of changing protein location and function, highlighting a surprising number of proteins that translocate from the mitochondria to the nucleus or from endoplasmic reticulum to Golgi apparatus under stress.

Genome-wide bimolecular fluorescence complementation analysis of SUMO interactome in yeast.

The definition of protein-protein interactions (PPIs) in the natural cellular context is essential for properly understanding various biological processes. So far, however, most large-scale PPI analyses have not been performed in the natural cellular context. Here, we describe the construction of a Saccharomyces cerevisiae fusion library in which each endogenous gene is C-terminally tagged with the N-terminal fragment of Venus (VN) for a genome-wide bimolecular fluorescence complementation assay, a powerful technique for identifying PPIs in living cells. We illustrate the utility of the VN fusion library by systematically analyzing the interactome of the small ubiquitin-related modifier (SUMO) and provide previously unavailable information on the subcellular localization, types, and protease dependence of SUMO interactions. Our data set is highly complementary to the existing data sets and represents a useful resource for expanding the understanding of the physiological roles of SUMO. In addition, the VN fusion library provides a useful research tool that makes it feasible to systematically analyze PPIs in the natural cellular context.

The ß-1,3-glucanosyltransferase Gas1 regulates Sir2-mediated rDNA stability in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the stability of highly repetitive rDNA array is maintained through transcriptional silencing. Recently, a ß-1,3-glucanosyltransferase Gas1 has been shown to play a significant role in the regulation of transcriptional silencing in S. cerevisiae. Here, we show that the gas1Δ mutation increases rDNA silencing in a Sir2-dependent manner. Remarkably, the gas1Δ mutation induces nuclear localization of Msn2/4 and stimulates the expression of PNC1, a gene encoding a nicotinamidase that functions as a Sir2 activator. The lack of enzymatic activity of Gas1 or treatment with a cell wall-damaging agent, Congo red, exhibits effects similar to those of the gas1Δ mutation. Furthermore, the loss of Gas1 or Congo red treatment lowers the cAMP-dependent protein kinase (PKA) activity in a cell wall integrity MAP kinase Slt2-dependent manner. Collectively, our results suggest that the dysfunction of Gas1 plays a positive role in the maintenance of rDNA integrity by decreasing PKA activity and inducing the accumulation of Msn2/4 in the nucleus. It seems that nuclear-localized Msn2/4 stimulate the expression of Pnc1, thereby enhancing the association of Sir2 with rDNA and promoting rDNA stability.

UDP-glucose pyrophosphorylase Ugp1 is involved in oxidative stress response and long-term survival during stationary phase in Saccharomyces cerevisiae.

Ugp1, UDP-glucose pyrophosphorylase, plays an important role in carbohydrate metabolism because it provides UDP-glucose that is a pivotal metabolite in several metabolic pathways in Saccharomyces cerevisiae. In this study, we show that a considerable reduction of glycogen and trehalose content in ugp1 knockdown cells is rescued by complementing the expression of Ugp1, indicating that Ugp1 is required for the production of storage carbohydrates. Because of the specific function of trehalose as a stress protectant, Ugp1 expression contributed to oxidative stress response and long-term cell survival during stationary phase. Furthermore, the modulation of Ugp1 level readjusted glycogen and trehalose accumulation in the protein kinase A (PKA)-related gene mutants. The PKA-dependent phenotypes of oxidative stress resistance and long-term cell survival were also alleviated via adjustment of Ugp1 level. Collectively, our data suggest that the regulation of UPG1 influences several PKA-dependent processes by adjusting the levels of various carbohydrates.

Monitoring G protein-coupled receptor activation using an adenovirus-based ß-arrestin bimolecular fluorescence complementation assay.

G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors and are involved in a variety of pathological conditions including cancer and cardiovascular, metabolic, neurological, and autoimmune diseases. GPCRs are being intensively investigated as targets for therapeutic intervention, and the ß-arrestin recruitment assay has become a popular tool for analyzing GPCR activation. Here, we report a high-throughput method for cloning GPCR cDNAs into adenoviral bimolecular fluorescence complementation (BiFC) vectors and performing the ß-arrestin BiFC assay in cells transduced with recombinant adenoviruses. An analysis of the activation of somatostatin receptor 2 (SSTR2) with the adenovirus-based ß-arrestin BiFC assay showed that the assay is suitable for quantifying SSTR2 activation in response to specific agonists or antagonists. Furthermore, the adenovirus-based ß-arrestin BiFC assay was able to detect the activation of a broad range of GPCRs. Collectively, our data indicate that the adenovirus-based ß-arrestin BiFC assay can serve as a simple and universal platform for studying GPCR activation and thus will be useful for high-throughput screening of drugs that target GPCRs.

Nsi1 plays a significant role in the silencing of ribosomal DNA in Saccharomyces cerevisiae.

In eukaryotic cells, ribosomal DNA (rDNA) forms the basis of the nucleolus. In Saccharomyces cerevisiae, 100-200 copies of a 9.1-kb rDNA repeat exist as a tandem array on chromosome XII. The stability of this highly repetitive array is maintained through silencing. However, the precise mechanisms that regulate rDNA silencing are poorly understood. Here, we report that S. cerevisiae Ydr026c, which we name NTS1 silencing protein 1 (Nsi1), plays a significant role in rDNA silencing. By studying the subcellular localization of 159 nucleolar proteins, we identified 11 proteins whose localization pattern is similar to that of Net1, a well-established rDNA silencing factor. Among these proteins is Nsi1, which is associated with the NTS1 region of rDNA and is required for rDNA silencing at NTS1. In addition, Nsi1 physically interacts with the known rDNA silencing factors Net1, Sir2 and Fob1. The loss of Nsi1 decreases the association of Sir2 with NTS1 and increases histone acetylation at NTS1. Furthermore, Nsi1 contributes to the longevity of yeast cells. Taken together, our findings suggest that Nsi1 is a new rDNA silencing factor that contributes to rDNA stability and lifespan extension in S. cerevisiae.

Rapamycin increases rDNA stability by enhancing association of Sir2 with rDNA in Saccharomyces cerevisiae.

The target of rapamycin (TOR) kinase is an evolutionarily conserved key regulator of eukaryotic cell growth and proliferation. Recently, it has been reported that inhibition of TOR signaling pathway can delay aging and extend lifespan in several eukaryotic organisms, but how lifespan extension is mediated by inhibition of TOR signaling is poorly understood. Here we report that rapamycin treatment and nitrogen starvation, both of which cause inactivation of TOR complex 1 (TORC1), lead to enhanced association of Sir2 with ribosomal DNA (rDNA) in Saccharomyces cerevisiae. TORC1 inhibition increases transcriptional silencing of RNA polymerase II-transcribed gene integrated at the rDNA locus and reduces homologous recombination between rDNA repeats that causes formation of toxic extrachromosomal rDNA circles. In addition, TORC1 inhibition induces deacetylation of histones at rDNA. We also found that Pnc1 and Net1 are required for enhancement of association of Sir2 with rDNA under TORC1 inhibition. Taken together, our findings suggest that inhibition of TORC1 signaling stabilizes the rDNA locus by enhancing association of Sir2 with rDNA, thereby leading to extension of replicative lifespan in S. cerevisiae.

Atg1-dependent phosphorylation of Vps34 is required for dynamic regulation of the phagophore assembly site and autophagy in Saccharomyces cerevisiae.

Macroautophagy/autophagy is a key catabolic pathway in which double-membrane autophagosomes sequester various substrates destined for degradation, enabling cells to maintain homeostasis and survive under stressful conditions. Several autophagy-related (Atg) proteins are recruited to the phagophore assembly site (PAS) and cooperatively function to generate autophagosomes. Vps34 is a class III phosphatidylinositol 3-kinase, and Atg14-containing Vps34 complex I plays essential roles in autophagosome formation. However, the regulatory mechanisms of yeast Vps34 complex I are still poorly understood. Here, we demonstrate that Atg1-dependent phosphorylation of Vps34 is required for robust autophagy activity in Saccharomyces cerevisiae. Following nitrogen starvation, Vps34 in complex I is selectively phosphorylated on multiple serine/threonine residues in its helical domain. This phosphorylation is important for full autophagy activation and cell survival. The absence of Atg1 or its kinase activity leads to complete loss of Vps34 phosphorylation in vivo, and Atg1 directly phosphorylates Vps34 in vitro, regardless of its complex association type. We also demonstrate that the localization of Vps34 complex I to the PAS provides a molecular basis for the complex I-specific phosphorylation of Vps34. This phosphorylation is required for the normal dynamics of Atg18 and Atg8 at the PAS. Together, our results reveal a novel regulatory mechanism of yeast Vps34 complex I and provide new insights into the Atg1-dependent dynamic regulation of the PAS.Abbreviations: ATG: autophagy-related; BARA: the repeated, autophagy-specific Co-IP: co-immunoprecipitation; GFP: green fluorescent protein; IP-MS: immunoprecipitation followed by tandem mass spectrometry; NTD: the N-terminal domain; PAS: phagophore assembly site; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns3K: phosphatidylinositol 3-kinase; SUR: structurally uncharacterized region; Vps34[KD]: Vps34D731N.

Simultaneous activation of CXC chemokine receptor 4 and histamine receptor H1 enhances calcium signaling and cancer cell migration.

C-X-C chemokine receptor 4 (CXCR4) is widely overexpressed in various types of cancer and is involved in several cancer phenotypes including tumor growth, survival, and metastasis. The roles of histamine and histamine receptor H1 (HRH1) in cancer pathogenesis remain controversial. Here, we show that HRH1 is widely expressed in various cancer cell lines and cancer tissues and that coexpression of CXCR4 and HRH1 is associated with poor prognosis in breast cancer. Using bimolecular fluorescence complementation and bioluminescence resonance energy transfer donor saturation assays, we demonstrate that CXCR4 and HRH1 can assemble into a heteromeric complex. Simultaneous activation of CXCR4 and HRH1 synergistically increases calcium flux in MDA-MB-231 cells that endogenously express CXCR4 and HRH1 but not in cells deficient in CXCR4 or HRH1. Costimulation of CXCR4 and HRH1 also significantly enhances CXCL12-induced MDA-MB-231 cell migration, while histamine alone does not induce cell migration. Synergistic effects on calcium flux and cell migration are inhibited by the Gαi inhibitor pertussis toxin and the Gαq inhibitor YM254890, suggesting that the Gαi and Gαq pathways are involved in the synergy. Enhanced calcium signaling and cell migration are also observed in NCI-H23 and HeLa cells, which coexpress CXCR4 and HRH1. Taken together, our findings demonstrate an interplay between CXCR4 and HRH1, and suggest the possibility of the CXCR4-HRH1 heteromer as a potential therapeutic target for anticancer therapy.