Sun Sik Bae

Phospholipase C-γ Is Required for Agonist-Induced Ca2+ Entry

We report here that PLC-gamma isoforms are required for agonist-induced Ca2+ entry (ACE). Overexpressed wild-type PLC-gamma1 or a lipase-inactive mutant PLC-gamma1 each augmented ACE in PC12 cells, while a deletion mutant lacking the region containing the SH3 domain of PLC-gamma1 was ineffective. RNA interference to deplete either PLC-gamma1 or PLC-gamma2 in PC12 and A7r5 cells inhibited ACE. In DT40 B lymphocytes expressing only PLC-gamma2, overexpressed muscarinic M5 receptors (M5R) activated ACE. Using DT40 PLC-gamma2 knockout cells, M5R stimulation of ER Ca2+ store release was unaffected, but ACE was abolished. Normal ACE was restored by transient expression of PLC-gamma2 or a lipase-inactive PLC-gamma2 mutant. The results indicate a lipase-independent role of PLC-gamma in the physiological agonist-induced activation of Ca2+ entry.

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.

Src Homology Domains of Phospholipase C γ1 Inhibit Nerve Growth Factor‐Induced Differentiation of PC12 Cells

Abstract: Phospholipase C γ1 (PLC‐γ1) is phosphorylated on treatment of cells with nerve growth factor (NGF). To assess the role of PLC‐γ1 in mediating the neuronal differentiation induced by NGF treatment, we established PC12 cells that overexpress whole PLC‐γ1 (PLC‐γ1PC12), the SH2‐SH2‐SH3 domain (PLC‐γ1SH223PC12), SH2‐SH2‐deleted mutants (PLC‐γ1ΔSH22PC12), and SH3‐deleted mutants (PLC‐γ1ΔSH3PC12). Overexpressed whole PLC‐γ1 or the SH2‐SH2‐SH3 domain of PLC‐γ1 stimulated cell growth and inhibited NGF‐induced neurite outgrowth of PC12 cells. However, cells expressing PLC‐γ1 lacking the SH2‐SH2 domain or the SH3 domain had no effect on NGF‐induced neuronal differentiation. Overexpression of intact PLC‐γ1 resulted in a threefold increase in total inositol phosphate accumulation on treatment with NGF. However, overexpression of the SH2‐SH2‐SH3 domain of PLC‐γ1 did not alter total inositol phosphate accumulation. To investigate whether the SH2‐SH2‐SH3 domain of PLC‐γ1 can mediate the NGF‐induced signal, tyrosine phosphorylation of the SH2‐SH2‐SH3 domain of PLC‐γ1 on NGF treatment was examined. The SH2‐SH2‐SH3 domain of PLC‐γ1 as well as intact PLC‐γ1 could be tyrosine‐phosphorylated on NGF treatment. These results indicate that the overexpressed SH2‐SH2‐SH3 domain of PLC‐γ1 can block the differentiation of PC12 cells induced by NGF and that the inhibition appears not to be related to the lipase activity of PLC‐γ1 but to the SH2‐SH2‐SH3 domain of PLC‐γ1.

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.

Localization of phospholipase C-γ1 signaling in caveolae: importance in EGF-induced phosphoinositide hydrolysis but not in tyrosine phosphorylation

Upon epidermal growth factor treatment, phospholipase C‐γ1 (PLC‐γ1) translocates from cytosol to membrane where it is phosphorylated at tyrosine residues. Caveolae are small plasma membrane invaginations whose structural protein is caveolin. In this study, we show that the translocation of PLC‐γ1 and its tyrosine phosphorylation are localized in caveolae by caveolin‐enriched low‐density membrane (CM) preparation and immunostaining of cells. Pretreatment of cells with methyl‐β‐cyclodextrin (MβCD), a chemical disrupting caveolae structure, inhibits the translocation of PLC‐γ1 to CM as well as phosphatidylinositol (PtdIns) turnover. However, MβCD shows no effect on tyrosine phosphorylation level of PLC‐γ1. Our findings suggest that, for proper signaling, PLC‐γ1 phosphorylation has to occur at PtdInsP2‐enriched sites.

Inhibition of Phospholipase D by a Protein Factor from Bovine Brain Cytosol PARTIAL PURIFICATION AND CHARACTERIZATION OF THE INHIBITION MECHANISM

A specific protein inhibitor of partially purified bovine brain phospholipase D (PLD) was identified from bovine brain cytosol. The PLD inhibitor has been enriched through several chromatographic steps and characterized with respect to size and mechanism of inhibition. The inhibitor showed an apparent molecular mass of 30 kDa by Superose 12 gel exclusion chromatography and inhibited PLD activity with an IC50 of 7 nM. The inhibitor had neither proteolytic activity nor phospholipid-hydrolyzing activity. Because phosphatidylinositol 4,5-bisphosphate (PIP2), which is included in substrate vesicles, is an essential cofactor for PLD, we examined whether the inhibition might be mediated by sequestration of PIP2. PIP2 hydrolysis by phospholipase C (PLC)-β1 was not affected by the inhibitor and the inhibitor did not bind to substrate vesicles containing PIP2. In contrast, a PH domain derived from PLC-δ1, which could bind to PIP2, showed a nearly identical inhibition of both PLC-β1 and PLD activities. Thus, the PLD inhibition by the inhibitor is due to the specific interaction with not PIP2 but PLD. The suppression of PLD activity by the inhibitor was largely eliminated by the addition of ADP-ribosylation factor (ARF) and GTPγS. We propose that the inhibitor plays a negative role in regulation of PLD activity by PIP2 and ARF.

Molecular Events of Insulin Action Occur at Lipid Raft/Caveolae in Adipocytes

Insulin stimulates the fusion of intracellular vesicles containing glucose transporter 4 (GLUT4) with plasma membrane in adipocytes and muscle cells. Here we show that adipocyte differentiation results in enhanced insulin sensitivity of glucose uptake. On the other hand, glucose uptake in response to platelet-derived growth factor (PDGF) stimulation was markedly reduced by adipocyte differentiation. Expression level of insulin receptor and caveolin-1 was dramatically increased during adipocyte differentiation. Adipocyte differentiation caused slightly enhanced activation of acutely transforming retrovirus AKT8 in rodent T cell lymphoma (Akt) by insulin stimulation. However, activation of Akt by PDGF stimulation was largely reduced. Activation of ERK was not detected in both fibroblasts and adipocytes after stimulation with insulin. PDGF-dependent activation of ERK was reduced by adipocyte differentiation. Insulin-dependent glucose uptake was abrogated by LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, in both fibroblasts and adipocytes. Also disassembly of caveolae structure by methyl-β-cyclodextrin caused impairment of Akt activation and glucose uptake. Finally, insulin receptor, Akt, SH2-domain-containing inositol 5-phosphatase 2 (SHIP2), and regulatory subunit of PI3K are localized at lipid raft domain and the translocation was facilitated upon insulin stimulation. Given these results, we suggest that lipid raft provide proper site for insulin action for glucose uptake.

Negative regulatory role of overexpression of PLCγ1 in the expression of early growth response 1 gene in rat 3Y1 fibroblasts

The early growth response 1 (Egr‐1) gene product is a transcription factor that functions as an oikis factor. Loss of Egr‐1 expression is closely associated with tumor formation. Phospholipase Cγ1 (PLCγ1) is overexpressed in some tumors, and its overexpression causes anchorage‐independent growth. Here we report that overexpression of PLCγ1 and SH2‐SH3 domain of PLCγ1 decreased induction of Egr‐1 and the Egr‐1‐regulated genes TSP‐1 and PAI‐1. Results from the nuclear run‐on assay and transfection experiment with the proximal 455 base pair region of the Egr‐1 promoter (‐454 to +1) showed that Egr‐1 transcriptional activity was suppressed in PLCγ1–3Y1 cells whereas decay of Egr‐1 mRNA was similar in both cell lines. Serum response element‐and ternary complex factor Elk‐1‐mediated transcriptional activation of the reporter gene in response to EGF were also inhibited in PLCγ1–3Y1 cells. Pretreatment with the protein synthesis inhibitor cycloheximide (CHX) partially abrogated the serum‐induced suppression of Egr‐1 transcription in PLCγ1–3Y1 cells, suggesting that a CHX‐sensitive factor(s) is involved in the suppression of Egr‐1 transcription in PLCγ1–3Y1 cells. Our results demonstrated that overexpression of PLCγ1 functions as a negative modulator of the tumor suppressor Egr‐1 gene expression, possibly through inhibition of Elk‐1‐dependent transcriptional activity.—Shin, S. Y., Ko, J., Chang, J.‐S., Min, D. S., Choi, C., Bae, S. S., Kim, M. J., Hyun, D. S., Kim, J.‐H., Han, M. Y., Kim, Y.‐H., Kim, Y. S., Na, D. S., Suh, P.‐G., Lee, Y. H. Negative regulatory role of overexpression of PLCγ1 in the expression of early growth response 1 gene in rat 3Y1 fibroblasts. FASEB J. 16, 1504–1514 (2002)