Sukmook Lee

The Direct Interaction of Phospholipase C-γ1 with Phospholipase D2 Is Important for Epidermal Growth Factor Signaling

The epidermal growth factor (EGF) receptor has an important role in cellular proliferation, and the enzymatic activity of phospholipase C (PLC)-γ1 is regarded to be critical for EGF-induced mitogenesis. In this study, we report for the first time a phospholipase complex composed of PLC-γ1 and phospholipase D2 (PLD2). PLC-γ1 is co-immunoprecipitated with PLD2 in COS-7 cells. The results of in vitro binding analysis and co-immunoprecipitation analysis in COS-7 cells show that the Src homology (SH) 3 domain of PLC-γ1 binds to the proline-rich motif within the Phox homology (PX) domain of PLD2. The interaction between PLC-γ1 and PLD2 is EGF stimulation-dependent and potentiates EGF-induced inositol 1,4,5-trisphosphate (IP3) formation and Ca2+increase. Mutating Pro-145 and Pro-148 within the PX domain of PLD2 to leucines disrupts the interaction between PLC-γ1 and PLD2 and fails to potentiate EGF-induced IP3 formation and Ca2+ increase. However, neither PLD2 wild type nor PLD2 mutant affects the EGF-induced tyrosine phosphorylation of PLC-γ1. These findings suggest that, upon EGF stimulation, PLC-γ1 directly interacts with PLD2 and this interaction is important for PLC-γ1 activity.

Phospholipase D1 in caveolae: regulation by protein kinase Calpha and caveolin-1.

Caveolae are small plasma membrane invaginations that have been implicated in cell signaling, and caveolin is a principal structural component of the caveolar membrane. Previously we have demonstrated that protein kinase Calpha (PKCalpha) directly interacts with phospholipase D1 (PLD1), activating the enzymatic activity of PLD1 in the presence of phorbol 12-myristate 13-acetate (PMA) [Lee, T. G., et al. (1997) Biochim. Biophys. Acta 1347, 199-204]. In this study, using a detergent-free procedure for the purification of a caveolin-enriched membrane fraction (CEM) and immunoblot analysis, we show that PLD1 is enriched in the CEMs of 3Y1 rat fibroblasts. Purified PLD1 directly bound to a glutathione S-transferase-caveolin-1 fusion protein in in vitro binding assays. The association of PLD1 with caveolin-1 could be completely eliminated by preincubation of PLD1 with an oligopeptide corresponding to the scaffolding domain (amino acids 82-101) of caveolin-1, indicating that caveolin-1 interacts with PLD1 through the scaffolding domain. The peptide also inhibited PKCalpha-stimulated PLD1 activity and the interaction between PLD1 and PKCalpha with an IC50 of 0.5 microM. PMA elicits translocation of PKCalpha to the CEMs, inducing PLD activation through the interaction of PKCalpha with PLD1 in the CEMs. Caveolin-1 also coimmunoprecipitated with PLD1 in the absence of PMA, and the amounts of coimmunoprecipitated caveolin-1 decreased in response to treatment with PMA. Taken together, our results suggest a new mechanism for the regulation of the PKCalpha-dependent PLD activity through the molecular interaction between PLD1, PKCalpha, and caveolin-1 in caveolae.

Hydrogen peroxide induces association between glyceraldehyde 3-phosphate dehydrogenase and phospholipase D2 to facilitate phospholipase D2 activation in PC12 cells

Oxidative stress or signaling is widely implicated in apoptosis, ischemia and mitogenesis. Previously, our group reported that the hydrogen peroxide (H2O2)‐dependent activation of phospholipase D2 (PLD2) in PC12 cells is involved in anti‐apoptotic effect. However, the precise mechanism of PLD2 activation by H2O2 was not revealed. To find H2O2‐dependent PLD2‐regulating proteins, we immunoprecipitated PLD2 from PC12 cells and found that glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) coimmunoprecipitated with PLD2 upon H2O2 treatment. This interaction was found to be direct by in vitro reconstitution of purified GAPDH and PLD2. In vitro studies also indicated that PLD2‐associated GAPDH was modified on its reactive cysteine residues. Koningic acid, an alkylator of GAPDH on catalytic cysteine residue, also increased interaction between the two proteins in vitro and enhanced PLD2 activity in PC12 cells. Blocking H2O2‐dependent modification of GAPDH with 3‐aminobenzamide resulted in the inhibition of the GAPDH/PLD2 interaction and attenuated H2O2‐induced PLD2 activation in PC12 cells. From the results, we suggest that H2O2 modifies GAPDH on its catalytic cysteine residue not only to inactivate the dehydrogenase activity of GAPDH but also to endow GAPDH with the ability to bind PLD2 and the resulting association is involved in the regulation of PLD2 activity by H2O2.

Molecular shape effect in polyatomic ion–surface scattering. Surface-induced dissociation of Fe(C5H5)2+ and FeC5H5+ at Si(100)

Abstract Fe(C5H5)2+ and FeC5H5+ ions undergo ligand dissociation upon collision with an Si(100) surface at hyperthermal energy (5–120 eV). The dissociation starts at a collision energy of 5 eV for the both ions, in agreement with the reaction energetics. However, the dissociation yield for FeC5H5+ is much lower than for Fe(C5H5)2+ at the energies above the dissociation threshold. This result suggests that the molecular shape has a strong influence on the short-time dynamics of polyatomic ion dissociation at a surface.

GbetaL regulates TNFalpha-induced NF-kappaB signaling by directly inhibiting the activation of IkappaB kinase

The transcriptional activation of NF-kappaB, a critical player in both physiological and pathological cellular responses to diverse cytokines, is dependent on IKK activation. Although molecular mechanisms underlying IKK activation have been well elucidated, the processes that negatively regulate IKK activity are still largely unknown. Using yeast two-hybrid screening, we have identified GbetaL as an interacting partner of IKKbeta. In this study, we demonstrate that GbetaL interacts with IKKalpha and IKKbeta in vitro and in vivo. The C-terminal WD domains of GbetaL are required for the interaction with both the kinase domain and leucine zipper domain of IKKbeta. Overexpression of GbetaL inhibits TNFalpha-induced activation of NF-kappaB signaling, while down-regulation of GbetaL expression by small interfering RNA enhances NF-kappaB activity. GbetaL constitutively interacts with IKKbeta, and this interaction is enhanced by TNFalpha treatment. GbetaL also inhibits TNFalpha-induced phosphorylation of IKKs. Taken together, these data suggest that GbetaL is involved in the negative regulation of TNFalpha-stimulated NF-kappaB signaling through a direct interaction with IKK.