Jang Hyun Choi

Proteolytic cleavage of phospholipase C-γ1 during apoptosis in Molt-4 cells

Apoptosis is a cell suicide mechanism that requires the activation of cellular death proteases for its induction. We examined whether the progress of apoptosis involves cleavage of phospho‐lipase C‐γΙ (PLC‐γΙ), which plays a pivotal role in mitogenic signaling pathway. Pretreatment of T leu‐kemic Molt‐4 cells with PLC inhibitors such as U‐73122 or ET‐18‐OCH3 potentiated etoposide‐in‐duced apoptosis in these cells. PLC‐γΙ was fragmented when Molt‐4 cells were treated with several apoptotic stimuli such as etoposide, ceramides, and tumor necrosis factor a. Cleavage of PLC‐γΙ was blocked by overexpression of Bcl‐2 and by specific inhibitors of caspases such as Z‐DEVD‐CH2F and YVAD‐cmk. Purified caspase‐3 and caspase‐7, group II caspases, cleaved PLC‐γΙ in vitro and generated a cleavage product of the same size as that observed in vivo, suggesting that PLC‐γΙ is cleaved by group II caspases in vivo. From point mutagenesis studies, Ala‐Glu‐Pro‐Asp 0 was identified to be a cleavage site within PLC‐γΙ. Epidermal growth factor receptor (EGFR)‐induced tyrosine phosphorylation of PLC‐γΙ resulted in resistance to cleavage by caspase‐3 in vitro. Furthermore, cleaved PLC‐γΙ could not be tyrosine‐phosphorylated by EGFR in vitro. In addition, tyrosine‐phosphorylated PLC‐γΙ was not significantly cleaved during etoposide‐in‐duced apoptosis in Molt‐4 cells. This suggests that the growth factor‐induced tyrosine phosphorylation may suppress apoptosis‐induced fragmentation of PLC‐γΙ. We provide evidence for the biochemical relationship between PLC‐γ1‐mediated signal pathway and apoptotic signal pathway, indicating that the defect of PLC‐γ1‐mediated signaling pathway can facilitate an apoptotic progression.—Bae, S. S., Perry, D. K., Oh, Y. S., Choi, J. H., Galadari, S. H., Ghayur, T., Ryu, S. H., Hannun, Y. A., Suh, P.‐G. Proteolytic cleavage of phospholipase C‐γΙ during apoptosis in Molt‐4 cells. FASEB J. 14, 1083–1092 (2000)

Phospholipase C-γ1 is a guanine nucleotide exchange factor for dynamin-1 and enhances dynamin-1-dependent epidermal growth factor receptor endocytosis

Phospholipase C-γ1 (PLC-γ1), which interacts with a variety of signaling molecules through its two Src homology (SH) 2 domains and a single SH3 domain has been implicated in the regulation of many cellular functions. We demonstrate that PLC-γ1 acts as a guanine nucleotide exchange factor (GEF) of dynamin-1, a 100 kDa GTPase protein, which is involved in clathrin-mediated endocytosis of epidermal growth factor (EGF) receptor. Overexpression of PLC-γ1 increases endocytosis of the EGF receptor by increasing guanine nucleotide exchange activity of dynamin-1. The GEF activity of PLC-γ1 is mediated by the direct interaction of its SH3 domain with dynamin-1. EGF-dependent activation of ERK and serum response element (SRE) are both up-regulated in PC12 cells stably overexpressing PLC-γ1, but knockdown of PLC-γ1 by siRNA significantly reduces ERK activation. These results establish a new role for PLC-γ1 in the regulation of endocytosis and suggest that endocytosis of activated EGF receptors may mediate PLC-γ1-dependent proliferation.

Proteolytic cleavage of epidermal growth factor receptor by caspases

Apoptotic proteases cleave and inactivate survival signaling molecules such as Akt/PKB, phospholipase C (PLC)‐γ1, and Bcl‐2. We have found that treatment of A431 cells with tumor necrosis factor‐α in the presence of cycloheximide resulted in the cleavage of epidermal growth factor receptor (EGFR) as well as the activation of caspase‐3. Among various caspases, caspase‐1, caspase‐3 and caspase‐7 were most potent in the cleavage of EGFR in vitro. Proteolytic cleavage of EGFR was inhibited by both YVAD‐cmk and DEVD‐fmk in vitro. We also investigated the effect of caspase‐dependent cleavage of EGFR upon the mediation of signals to downstream signaling molecules such as PLC‐γ1. Cleavage of EGFR by caspase‐3 significantly impaired the tyrosine phosphorylation of PLC‐γ1 in vitro. Given these results, we suggest that apoptotic protease specifically cleaves and inactivates EGFR, which plays crucial roles in anti‐apoptotic signaling, to abrogate the activation of EGFR‐dependent downstream survival signaling molecules.

Pleckstrin Homology Domains of Phospholipase C-γ1 Directly Interact with β-Tubulin for Activation of Phospholipase C-γ1 and Reciprocal Modulation of β-Tubulin Function in Microtubule Assembly

Phosphoinositide-specific phospholipase C-γ1 (PLC-γ1) has two pleckstrin homology (PH) domains, an N-terminal domain and a split PH domain. Here we show that pull down of NIH3T3 cell extracts with PLC-γ1 PH domain-glutathione S-transferase fusion proteins, followed by matrix-assisted laser desorption ionization-time of flight-mass spectrometry, identified β-tubulin as a binding protein of both PLC-γ1 PH domains. Tubulin is a main component of microtubules and mitotic spindle fibers, which are composed of α- and β-tubulin heterodimers in all eukaryotic cells. PLC-γ1 and β-tubulin colocalized in the perinuclear region in COS-7 cells and cotranslocated to the plasma membrane upon agonist stimulation. Membrane-targeted translocation of depolymerized tubulin by agonist stimulation was also supported by immunoprecipitation analyses. The phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolyzing activity of PLC-γ1 was substantially increased in the presence of purified tubulin in vitro, whereas the activity was not promoted by bovine serum albumin, suggesting that β-tubulin activates PLC-γ1. Furthermore, indirect immunofluorescent microscopy showed that PLC-γ1 was highly concentrated in mitotic spindle fibers, suggesting that PLC-γ1 is involved in spindle fiber formation. The effect of PLC-γ1 in microtubule formation was assessed by overexpression and silencing PLC-γ1 in COS-7 cells, which resulted in altered microtubule dynamics in vivo. Cells overexpressing PLC-γ1 showed higher microtubule densities than controls, whereas PLC-γ1 silencing with small interfering RNAs led to decreased microtubule network densities as compared with control cells. Taken together, our results suggest that PLC-γ1 and β-tubulin transmodulate each other, i.e. that PLC-γ1 modulates microtubule assembly by β-tubulin, and β-tubulin promotes PLC-γ1 activity.

Sorting nexin 16 regulates EGF receptor trafficking by phosphatidylinositol-3-phosphate interaction with the Phox domain

Sorting nexins (SNXs) containing the Phox (PX) domain are implicated in the regulation of membrane trafficking and sorting processes of epithelial growth factor receptor (EGFR). In this study, we investigated whether SNX16 regulates EGF-induced cell signaling by regulating EGFR trafficking. SNX16 is localized in early and recycling endosomes via its PX domain. Mutation of the PX domain disrupted the association between SNX16 and phosphatidylinositol 3-phosphate [PtdIns(3)P]. Treatment with wortmannin, a PtdIns 3-kinase inhibitor, abolished the endosomal localization of SNX16, suggesting that the intracellular localization of SNX16 is regulated by PtdIns 3-kinase activity. SNX16 was found to associate with EGFR after stimulation with EGF in COS-7 cells. Moreover, overexpression of SNX16 increased the rate of EGF-induced EGFR degradation and inhibited the EGF-induced up-regulation of ERK and serum response element (SRE). In addition, mutation in the PX domain significantly blocked the inhibitory effect of SNX16 on EGF-induced activation of ERK and SRE. From these results, we suggest that SNX16 directs the sorting of EGFR to the endosomal compartment and thus regulates EGF-induced cell signaling.

Potential Inhibition of PDK1/Akt Signaling by Phenothiazines Suppresses Cancer Cell Proliferation and Survival

3′‐Phosphoinositide‐dependent kinase‐1 (PDK1) has been identified for its ability to phosphorylate and activate Akt. Accumulated studies have shown that the activation of the PDK1/Akt pathway plays a pivotal role in cell survival, proliferation, and tumorigenesis. Therefore, the PDK1/Akt pathway is believed to be a critical target for cancer intervention. In this paper, we report the discovery of a new function of phenothiazines, widely known as antipsychotics, inhibiting PDK1/Akt pathway. Upon epidermal growth factor (EGF) stimulation, phenothiazines specifically suppressed the kinase activity of PDK1 and the phosphorylation level of Akt. The inhibition of PDK1/Akt kinase resulted in suppression of EGF‐induced cell growth and induction of apoptosis in human ovary cancer cells. In particular, phenothiazines were highly selective for downstream targets of PDK1/Akt and did not inhibit the activation of phosphatidylinositol 3‐kinase (PI3K), EGFR, or extracellular signal‐regulated kinase 1/2 (ERK1/2). In particular, phenothiazines effectively suppressed tumor growth in nude mice of human cancer cells. Taken together, these findings provide strong evidence for novel function of phenothiazines, pharmacologically targeting PDK1/Akt for anticancer drug discovery.

Phospholipase Cγ1 negatively regulates growth hormone signalling by forming a ternary complex with Jak2 and protein tyrosine phosphatase-1B

Growth hormone binds to its membrane receptor (GHR), whereby it regulates many cellular functions, including proliferation, differentiation and chemotaxis. However, although the activation of growth hormone-mediated signalling is well understood, the precise mechanism responsible for its regulation has not been elucidated. Here, we demonstrate that phospholipase Cγ1 (PLCγ1) modulates the action of growth hormone-mediated signalling by interacting with tyrosine kinase Jak2 (janus kinase 2) in a growth hormone-dependent manner. In the absence of PLCγ1 (PLCγ1−/−), growth hormone-induced JAK2 and STAT5 phosphorylation significantly increased in mouse embryonic fibroblasts (MEFs). Furthermore, the re-expression of PLCγ1 reduced growth hormone-induced Jak2 activation. Growth hormone-induced Jak2 phosphorylation was enhanced by siRNA-specific knockdown of PLCγ1. Interestingly, PLCγ1 physically linked Jak2 and protein tyrosine phosphatase-1B (PTP-1B) by binding to both using different domains, and this process was implicated in the modulation of cytokine signalling through Jak2. In addition, in PLCγ1−/− MEFs, growth hormone-dependent c-Fos activation was upregulated and growth hormone-induced proliferation was potentiated. These results suggest that PLCγ1 has a key function in the regulation of growth hormone-mediated signalling by negatively regulating Jak2 activation.

Pleckstrin homology domain of phospholipase C-γ1 directly binds to 68-kDa neurofilament light chain

Phosphoinositide-specific phospholipase C-γ1 (PLC-γ1) has two pleckstrin homology (PH) domains: an amino-terminal domain (PH1) and a split PH domain (PH2). Here, we show that overlay assay of bovine brain tubulin pool with glutathione-S-transferase (GST)-PLC-γ1 PH domain fusion proteins, followed by matrix-assisted laser-desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), identified 68-kDa neurofilament light chain (NF-L) as a binding protein of amino-terminal PH domain of PLC-γ1. NF-L is known as a component of neuronal intermediate filaments, which are responsible for supporting the structure of myelinated axons in neuron. PLC-γ1 and NF-L colocalized in the neurite in PC12 cells upon nerve growth factor stimulation. In vitro binding assay and immunoprecipitation analysis also showed a specific interaction of both proteins in differentiated PC12 cells. The phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P2] hydrolyzing activity of PLC-γ1 was slightly decreased in the presence of purified NF-L in vitro, suggesting that NF-L inhibits PLC-γ1. Our results suggest that PLC-γ1-associated NF-L sequesters the phospholipid from the PH domain of PLC-γ1.

PDZ Domain-containing 1 (PDZK1) Protein Regulates Phospholipase C-β3 (PLC-β3)-specific Activation of Somatostatin by Forming a Ternary Complex with PLC-β3 and Somatostatin Receptors

Background: The four PLC-β subtypes (β1–β4) have different roles in GPCR-mediated signaling despite having similar structures and regulatory modes. Results: PDZK1 mediates the physical coupling of PLC-β3 to SSTRs using different PDZ domains. Conclusion: PLC-β3 is specifically involved in SSTR-mediated signaling via its interaction with PDZK1. Significance: The subtype-specific role of PLC-β is mediated by differential interactions with PDZ proteins and GPCRs. Phospholipase C-β (PLC-β) is a key molecule in G protein-coupled receptor (GPCR)-mediated signaling. Many studies have shown that the four PLC-β subtypes have different physiological functions despite their similar structures. Because the PLC-β subtypes possess different PDZ-binding motifs, they have the potential to interact with different PDZ proteins. In this study, we identified PDZ domain-containing 1 (PDZK1) as a PDZ protein that specifically interacts with PLC-β3. To elucidate the functional roles of PDZK1, we next screened for potential interacting proteins of PDZK1 and identified the somatostatin receptors (SSTRs) as another protein that interacts with PDZK1. Through these interactions, PDZK1 assembles as a ternary complex with PLC-β3 and SSTRs. Interestingly, the expression of PDZK1 and PLC-β3, but not PLC-β1, markedly potentiated SST-induced PLC activation. However, disruption of the ternary complex inhibited SST-induced PLC activation, which suggests that PDZK1-mediated complex formation is required for the specific activation of PLC-β3 by SST. Consistent with this observation, the knockdown of PDZK1 or PLC-β3, but not that of PLC-β1, significantly inhibited SST-induced intracellular Ca2+ mobilization, which further attenuated subsequent ERK1/2 phosphorylation. Taken together, our results strongly suggest that the formation of a complex between SSTRs, PDZK1, and PLC-β3 is essential for the specific activation of PLC-β3 and the subsequent physiologic responses by SST.

Phospholipase C-epsilon augments epidermal growth factor-dependent cell growth by inhibiting epidermal growth factor receptor down-regulation

The down-regulation of the epidermal growth factor (EGF) receptor is critical for the termination of EGF-dependent signaling, and the dysregulation of this process can lead to oncogenesis. In the present study, we suggest a novel mechanism for the regulation of EGF receptor down-regulation by phospholipase C-ϵ. The overexpression of PLC-ϵ led to an increase in receptor recycling and decreased the down-regulation of the EGF receptor in COS-7 cells. Adaptor protein complex 2 (AP2) was identified as a novel binding protein that associates with the PLC-ϵ RA2 domain independently of Ras. The interaction of PLC-ϵ with AP2 was responsible for the suppression of EGF receptor down-regulation, since a perturbation in this interaction abolished this effect. Enhanced EGF receptor stability by PLC-ϵ led to the potentiation of EGF-dependent growth in COS-7 cells. Finally, the knockdown of PLC-ϵ in mouse embryo fibroblast cells elicited a severe defect in EGF-dependent growth. Our results indicated that PLC-ϵ could promote EGF-dependent cell growth by suppressing receptor down-regulation.