Mie-Jae Im

Evidence That Phospholipase δ1 Is the Effector in the Gh (Transglutaminase II)-mediated Signaling

A new class of GTP-binding protein transglutaminase II (Gh) couples to a 69-kDa phospholipase C (PLC). An 8-amino acid region (Leu665-Lys672) of the α-subunit of Gh (Gαh) is involved in interaction and activation of PLC, an observation that has now been used to characterize the 69-kDa PLC further. A 20-amino acid peptide corresponding to Leu654-Leu673 of Gαh was used to prepare an affinity resin. On incubation with a partially purified PLC preparation from rat liver membranes, the affinity resin-bound ∼69- and 85-kDa proteins were recognized by an antibody to the 69-kDa PLC. Both purified 69-kDa PLC and PLC-δ1 bound to the affinity resin; moreover, antibodies to PLC-δ1 recognized the 69-kDa PLC, and antibodies to the 69-kDa PLC recognized PLC-δ1. A synthetic peptide corresponding to Leu661-Lys672 of Gαh inhibited the binding of PLC-δ1 to the affinity resin and also stimulated PLC-δ1. Reconstitution of PLC-δ1 with GTPγS (guanosine 5′-3-O-(thio)triphosphate)-activated Gh resulted in activation of PLC-δ1. Antibodies to Gαh also coimmunoprecipitated PLC-δ1 upon activation of Gh. These findings indicate that PLC-δ1 is the effector of Gh-mediated signaling.

Doxorubicin-induced reactive oxygen species generation and intracellular Ca2+ increase are reciprocally modulated in rat cardiomyocytes

Doxorubicin (DOX) is one of the most potent anticancer drugs and induces acute cardiac arrhythmias and chronic cumulative cardiomyopathy. Though DOX-induced cardiotoxicity is known to be caused mainly by ROS generation, a disturbance of Ca2+ homeostasis is also implicated one of the cardiotoxic mechanisms. In this study, a molecular basis of DOX-induced modulation of intracellular Ca2+ concentration ([Ca2+]i) was investigated. Treatment of adult rat cardiomyocytes with DOX increased [Ca2+]i irrespectively of extracellular Ca2+, indicating DOX-mediated Ca2+ release from intracellular Ca2+ stores. The DOX-induced Ca2+ increase was slowly processed and sustained. The Ca2+ increase was inhibited by pretreatment with a sarcoplasmic reticulum (SR) Ca2+ channel blocker, ryanodine or dantrolene, and an antioxidant, α-lipoic acid or α-tocopherol. DOX-induced ROS generation was observed immediately after DOX treatment and increased in a time-dependent manner. The ROS production was significantly reduced by the pretreatment of the SR Ca2+ channel blockers and the antioxidants. Moreover, DOX-mediated activation of caspase-3 was significantly inhibited by the Ca2+ channel blockers and a-lipoic acid but not a-tocopherol. In addition, cotreatment of ryanodine with α-lipoic acid resulted in further inhibition of the casapse-3 activity. These results demonstrate that DOX-mediated ROS opens ryanodine receptor, resulting in an increase in [Ca2+]i and that the increased [Ca2+]i induces ROS production. These observations also suggest that DOX/ROS-induced increase of [Ca2+]i plays a critical role in damage of cardiomyocytes.

Interaction Site of GTP Binding Gh (Transglutaminase II) with Phospholipase C

The GTP binding Gαh (transglutaminase II) mediates the α1B-adrenoreceptor signal to a 69-kDa phospholipase C (PLC). Thus, Gαh possesses both GTPase and transglutaminase activities with a signal transfer role. The recognition sites of this unique GTP binding protein for either the receptor or the effector are completely unknown. A site on human heart Gαh (hhGαh) has been identified that interacts with and stimulates PLC. Expressed mutants of hhGαh with deleted C-terminal regions lost the response to(-)-epinephrine and GTP and failed to coimmunoprecipitate PLC by the specific Gh7α antibody. The interaction regions were further defined by studies with synthetic peptides of hhGαh and a chimera in which residues Val665-Lys672 of hhGαh were substituted with Ile707-Ser714 residues of human coagulation factor XIIIa. Thus, eight amino acid residues near the C terminus of hhGαh are critical for recognition and stimulation of PLC.

Modulation of intracellular Ca2+ via α1B-adrenoreceptor signaling molecules, Gαh (transglutaminase II) and phospholipase C-δ1

Abstract We characterized the α1B-adrenoreceptor (α1B-AR)-mediated intracellular Ca2+ signaling involving Gαh (transglutaminase II, TGII) and phospholipase C (PLC)-δ1 using DDT1-MF2 cell. Expression of wild-type TGII and a TGII mutant lacking transglutaminase activity resulted in significant increases in a rapid peak and a sustained level of intracellular Ca2+ concentration ([Ca2+]i) in response to activation of the α1B-AR. Expression of a TGII mutant lacking the interaction with the receptor or PLC-δ1 substantially reduced both the peak and sustained levels of [Ca2+]i. Expression of TGII mutants lacking the interaction with PLC-δ1 resulted in a reduced capacitative Ca2+ entry. Reduced expression of PLC-δ1 displayed a transient elevation of [Ca2+]i and a reduction in capacitative Ca2+ entry. Expression of the C2-domain of PLC-δ1, which contains the TGII interaction site, resulted in reduction of the α1B-AR-evoked peak increase in [Ca2+]i, while the sustained elevation in [Ca2+]i and capacitative Ca2+ entry remained unchanged. These findings demonstrate that stimulation of PLC-δ1 via coupling of the α1B-AR with TGII evokes both Ca2+ release and capacitative Ca2+ entry and that capacitative Ca2+ entry is mediated by the interaction of TGII with PLC-δ1.

A novel signaling pathway of ADP-ribosyl cyclase activation by angiotensin II in adult rat cardiomyocytes

ADP-ribosyl cyclase (ADPR-cyclase) produces a Ca(2+)-mobilizing second messenger, cADP-ribose (cADPR), from NAD(+). In this study, we investigated the molecular basis of ADPR-cyclase activation in the ANG II signaling pathway and cellular responses in adult rat cardiomyocytes. The results showed that ANG II generated biphasic intracellular Ca(2+) concentration increases that include a rapid transient Ca(2+) elevation via inositol trisphosphate (IP(3)) receptor and sustained Ca(2+) rise via the activation of L-type Ca(2+) channel and opening of ryanodine receptor. ANG II-induced sustained Ca(2+) rise was blocked by a cADPR antagonistic analog, 8-bromo-cADPR, indicating that sustained Ca(2+) rise is mediated by cADPR. Supporting the notion, ADPR-cyclase activity and cADPR production by ANG II were increased in a time-dependent manner. Application of pharmacological inhibitors and immunological analyses revealed that cADPR formation was activated by sequential activation of Src, phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (Akt), phospholipase C (PLC)-gamma1, and IP(3)-mediated Ca(2+) signal. Inhibitors of these signaling molecules not only completely abolished the ANG II-induced Ca(2+) signals but also inhibited cADPR formation. Application of the cADPR antagonist and inhibitors of upstream signaling molecules of ADPR-cyclase inhibited ANG II-stimulated hypertrophic responses, which include nuclear translocation of Ca(2+)/calcineurin-dependent nuclear factor of activated T cells 3, protein expression of transforming growth factor-beta1, and incorporation of [(3)H]leucine in cardiomyocytes. Taken together, these findings suggest that activation of ADPR-cyclase by ANG II entails a novel signaling pathway involving sequential activation of Src, PI 3-kinase/Akt, and PLC-gamma1/IP(3) and that the activation of ADPR-cyclase can lead to cardiac hypertrophy.

Association of CD38 with Nonmuscle Myosin Heavy Chain IIA and Lck Is Essential for the Internalization and Activation of CD38

Activation of CD38 in lymphokine-activated killer (LAK) cells involves interleukin-8 (IL8)-mediated protein kinase G (PKG) activation and results in an increase in the sustained intracellular Ca2+ concentration ([Ca2+]i), cADP-ribose, and LAK cell migration. However, direct phosphorylation or activation of CD38 by PKG has not been observed in vitro. In this study, we examined the molecular mechanism of PKG-mediated activation of CD38. Nonmuscle myosin heavy chain IIA (MHCIIA) was identified as a CD38-associated protein upon IL8 stimulation. The IL8-induced association of MHCIIA with CD38 was dependent on PKG-mediated phosphorylation of MHCIIA. Supporting these observations, IL8- or cell-permeable cGMP analog-induced formation of cADP-ribose, increase in [Ca2+]i, and migration of LAK cells were inhibited by treatment with the MHCIIA inhibitor blebbistatin. Binding studies using purified proteins revealed that the association of MHCIIA with CD38 occurred through Lck, a tyrosine kinase. Moreover, these three molecules co-immunoprecipitated upon IL8 stimulation of LAK cells. IL8 treatment of LAK cells resulted in internalization of CD38, which co-localized with MHCIIA and Lck, and blebbistatin blocked internalization of CD38. These findings demonstrate that the association of phospho-MHCIIA with Lck and CD38 is a critical step in the internalization and activation of CD38.