Kyun Heo

NHERF2 Specifically Interacts with LPA2 Receptor and Defines the Specificity and Efficiency of Receptor-Mediated Phospholipase C-β3 Activation

ABSTRACT Lysophosphatidic acid (LPA) activates a family of cognate G protein-coupled receptors and is involved in various pathophysiological processes. However, it is not clearly understood how these LPA receptors are specifically coupled to their downstream signaling molecules. This study found that LPA2, but not the other LPA receptor isoforms, specifically interacts with Na+/H+ exchanger regulatory factor2 (NHERF2). In addition, the interaction between them requires the C-terminal PDZ domain-binding motif of LPA2 and the second PDZ domain of NHERF2. Moreover, the stable expression of NHERF2 potentiated LPA-induced phospholipase C-β (PLC-β) activation, which was markedly attenuated by either a mutation in the PDZ-binding motif of LPA2 or by the gene silencing of NHERF2. Using its second PDZ domain, NHERF2 was found to indirectly link LPA2 to PLC-β3 to form a complex, and the other PLC-β isozymes were not included in the protein complex. Consistently, LPA2-mediated PLC-β activation was specifically inhibited by the gene silencing of PLC-β3. In addition, NHERF2 increases LPA-induced ERK activation, which is followed by cyclooxygenase-2 induction via a PLC-dependent pathway. Overall, the results suggest that a ternary complex composed of LPA2, NHERF2, and PLC-β3 may play a key role in the LPA2-mediated PLC-β signaling pathway.

Hypoxia-induced up-regulation of apelin is associated with a poor prognosis in oral squamous cell carcinoma patients

Recently, apelin has been shown to be a novel angiogenic factor in various cancers including lung, breast and brain cancer. However, there is limited information regarding the expression and role of apelin in oral cavity cancer. In this study, we determined that apelin expression was localized in the cytoplasm of oral squamous cell carcinoma at various intensities. Strong apelin expression significantly correlated with tumor recurrence and disease-free survival. Using a multivariate analysis, we demonstrated that apelin was an independent prognostic factor for on disease-free survival, age, lymph node metastasis and CA9 expression. Moreover, apelin expression was up-regulated under hypoxic conditions, and exogenous apelin enhanced the proliferation and migration of oral cancer cells. Based on these results, we propose that the presence of hypoxia-induced apelin is a new prognostic factor and potential therapeutic target for oral squamous cell carcinoma.

Subtype-specific role of phospholipase C-β in bradykinin and LPA signaling through differential binding of different PDZ scaffold proteins

Among phospholipase C (PLC) isozymes (beta, gamma, delta, epsilon, zeta and eta), PLC-beta plays a key role in G-protein coupled receptor (GPCR)-mediated signaling. PLC-beta subtypes are often overlapped in their distribution, but have unique knock-out phenotypes in organism, suggesting that each subtype may have the different role even within the same type of cells. In this study, we examined the possibility of the differential coupling of each PLC-beta subtype to GPCRs, and explored the molecular mechanism underlying the specificity. Firstly, we found that PLC-beta1 and PLC-beta 3 are activated by bradykinin (BK) or lysophosphatidic acid (LPA), respectively. BK-triggered phosphoinositides hydrolysis and subsequent Ca(2+) mobilization were abolished specifically by PLC-beta1 silencing, whereas LPA-triggered events were by PLC-beta 3 silencing. Secondly, we showed the evidence that PDZ scaffold proteins is a key mediator for the selective coupling between PLC-beta subtype and GPCR. We found PAR-3 mediates physical interaction between PLC-beta1 and BK receptor, while NHERF2 does between PLC-beta 3 and LPA(2) receptor. Consistently, the silencing of PAR-3 or NHERF2 blunted PLC signaling induced by BK or LPA respectively. Taken together, these data suggest that each subtype of PLC-beta is selectively coupled to GPCR via PDZ scaffold proteins in given cell types and plays differential role in the signaling of various GPCRs.

Therapeutic aptamers: developmental potential as anticancer drugs.

Aptamers, composed of single-stranded DNA or RNA oligonucleotides that interact with target molecules through a specific three-dimensional structure, are selected from pools of combinatorial oligonucleotide libraries. With their high specificity and affinity for target proteins, ease of synthesis and modification, and low immunogenicity and toxicity, aptamers are considered to be attractive molecules for development as anticancer therapeutics. Two aptamers - one targeting nucleolin and a second targeting CXCL12 - are currently undergoing clinical trials for treating cancer patients, and many more are under study. In this mini-review, we present the current clinical status of aptamers and aptamer-based cancer therapeutics. We also discuss advantages, limitations, and prospects for aptamers as cancer therapeutics. [BMB Reports 2015; 48(4): 234-237]

A myristoylated pseudosubstrate peptide of PKC-ζ induces degranulation in HMC-1 cells independently of PKC-ζ activity

Mast cells play a central role in allergic disease and host defense against several pathogens through the release of various bioactive compounds via degranulation. In this study, we found that a myristoylated pseudosubstrate of PKC-zeta (zeta-PS; myristoyl-SIYRRGARRWRKL, a PKC-zeta inhibitor) regulates mast cell degranulation. zeta-PS increased [Ca+2]i level at nanomolar concentrations in a PKC-zeta activity-independent manner in HMC-1 cells. Moreover, zeta-PS-induced [Ca+2]i generation was completely abrogated by phospholipase C (PLC), IP3 receptor or Galpha i/o inhibitor and zeta-PS potently induced degranulation in HMC-1 cells which was significantly inhibited by pretreating PLC inhibitors or a calcium chelator. Therefore, our results suggest that zeta-PS can induce degranulation in HMC-1 cells by triggering the calcium signal via a PKC-zeta-independent but Galpha i/o, PLC and IP3-dependent pathways.

Inhibition of phospholipase C-β1-mediated signaling by O-GlcNAc modification

Here we report inhibition of phospholipase C‐β1 (PLC‐β1)‐mediated signaling by post‐translational glycosylation with β‐N‐acetylglucosamine (O‐GlcNAc modification). In C2C12 myoblasts, isoform‐specific knock‐down experiments using siRNA showed that activation of bradykinin (BK) receptor led to stimulation of PLC‐β1 and subsequent intracellular Ca2+ mobilization. In C2C12 myotubes, O‐GlcNAc modification of PLC‐β1 was markedly enhanced in response to treatment with glucosamine (GlcNH2), an inhibitor of O‐GlcNAase (PUGNAc) and hyperglycemia. This was associated with more than 50% inhibition of intracellular production of IP3 and Ca2+ mobilization in response to BK. Since the abundance of PLC‐β1 remained unchanged, these data suggest that O‐GlcNAc modification of PLC‐β1 led to inhibition of its activity. Moreover, glucose uptake stimulated by BK was significantly blunted by treatment with PUGNAc. These data support the notion that O‐GlcNAc modification negatively modulates the activity of PLC‐β1. J. Cell. Physiol.

G-protein-coupled receptor 81 promotes a malignant phenotype in breast cancer through angiogenic factor secretion

G-protein-coupled receptor 81 (GPR81) functions as a receptor for lactate and plays an important role in the regulation of anti-lipolytic effects in adipocytes. However, to data, a role for GPR81 in the tumor microenvironment has not been clearly defined. Here, GPR81 expression in breast cancer patients and several breast cancer cell lines was significantly increased compared with normal mammary tissues and cells. GPR81 knockdown resulted in impaired breast cancer growth and led to apoptosis both in vitro and in vivo. Furthermore, the inhibition of GPR81 signaling suppressed angiogenesis through a phosphoinositide 3-OH kinase (PI3K)/Akt-cAMP response element binding protein (CREB) pathway, which led to decreased production of the pro-angiogenic mediator amphiregulin (AREG). Overall, these findings identify GPR81 as a tumor-promoting receptor in breast cancer progression and suggest a novel mechanism that regulates GPR81-dependent activation of the PI3K/Akt signaling axis in tumor microenvironment.

C-terminally mutated tubby protein accumulates in aggresomes

The tubby protein (Tub), a putative transcription factor, plays important roles in the maintenance and function of neuronal cells. A splicing defect-causing mutation in the 3′-end of the tubby gene, which is predicted to disrupt the carboxy-terminal region of the Tub protein, causes maturity-onset obesity, blindness, and deafness in mice. Although this pathological Tub mutation leads to a loss of function, the precise mechanism has not yet been investigated. Here, we found that the mutant Tub proteins were mostly localized to puncta found in the perinuclear region and that the C-terminus was important for its solubility. Immunocytochemical analysis revealed that puncta of mutant Tub co-localized with the aggresome. Moreover, whereas wild-type Tub was translocated to the nucleus by extracellular signaling, the mutant forms failed to undergo such translocation. Taken together, our results suggest that the malfunctions of the Tub mutant are caused by its misfolding and subsequent localization to aggresomes.

Dynamic relocalization of NHERF1 mediates chemotactic migration of ovarian cancer cells toward lysophosphatidic acid stimulation

NHERF1/EBP50 (Na+/H+ exchanger regulating factor 1; Ezrin-binding phosphoprotein of 50 kDa) organizes stable protein complexes beneath the apical membrane of polar epithelial cells. By contrast, in cancer cells without any fixed polarity, NHERF1 often localizes in the cytoplasm. The regulation of cytoplasmic NHERF1 and its role in cancer progression remain unclear. In this study, we found that, upon lysophosphatidic acid (LPA) stimulation, cytoplasmic NHERF1 rapidly translocated to the plasma membrane, and subsequently to cortical protrusion structures, of ovarian cancer cells. This movement depended on direct binding of NHERF1 to C-terminally phosphorylated ERM proteins (cpERMs). Moreover, NHERF1 depletion downregulated cpERMs and further impaired cpERM-dependent remodeling of the cell cortex, suggesting reciprocal regulation between these proteins. The LPA-induced protein complex was highly enriched in migratory pseudopodia, whose formation was impaired by overexpression of NHERF1 truncation mutants. Consistent with this, NHERF1 depletion in various types of cancer cells abolished chemotactic cell migration toward a LPA gradient. Taken together, our findings suggest that the high dynamics of cytosolic NHERF1 provide cancer cells with a means of controlling chemotactic migration. This capacity is likely to be essential for ovarian cancer progression in tumor microenvironments containing LPA.

CLEC14a-HSP70-1A interaction regulates HSP70-1A-induced angiogenesis

CLEC14a (C-type lectin domain family 14 member) is a tumor endothelial cell marker protein that is known to play an important role in tumor angiogenesis, but the basic molecular mechanisms underlying this function have not yet been clearly elucidated. In this study, using various proteomic tools, we isolated a 70-kDa protein that interacts with the C-type lectin-like domain of CLEC14a (CLEC14a-CTLD) and identified it as heat shock protein 70-1A (HSP70-1A). Co-immunoprecipitation showed that HSP70-1A and CLEC14a interact on endothelial cells. In vitro binding analyses identified that HSP70-1A specifically associates with the region between amino acids 43 and 69 of CLEC14a-CTLD. Competitive blocking experiments indicated that this interacting region of CLEC14a-CTLD significantly inhibits HSP70-1A-induced extracellular signal-regulated kinase (ERK) phosphorylation and endothelial tube formation by directly inhibiting CLEC14a-CTLD-mediated endothelial cell-cell contacts. Our data suggest that the specific interaction of HSP70-1A with CLEC14a may play a critical role in HSP70-1A-induced angiogenesis and that the HSP70-1A-interacting region of CLEC14a-CTLD may be a useful tool for inhibiting HSP70-1A-induced angiogenesis.