Gq protein-induced apoptosis is mediated by AKT kinase inhibition that leads to protein kinase C-induced c-Jun N-terminal kinase activation

G(q) protein-coupled receptors (G(q)PCRs) regulate various cellular processes, including mainly proliferation and differentiation. In a previous study we found that in prostate cancer cells, the G(q)PCR of gonadotropin-releasing hormone (GnRH) induces apoptosis by reducing the PKC-dependent AKT activity and elevating JNK phosphorylation. Because it was thought that G(q)PCRs mainly induce activation of AKT, we first undertook to examine how general this phenomenon is. In a screen of 21 cell lines we found that PKC activation results in the reduction of AKT activity, which correlates nicely with JNK activation and in some cases with apoptosis. To understand further the signaling pathways involved in this stimulation, we studied in detail SVOG-4O and αT3-1 cells. We found that prostaglandin F2α and GnRH agonist (GnRH-a) indeed induce significant Gα(q)- and PKC-dependent apoptosis in these cells. This is mediated by two signaling branches downstream of PKC, which converge at the level of MLK3 upstream of JNK. One branch consists of c-Src activation of the JNK cascade, and the second involves reduction of AKT activity that alleviates its inhibitory effect on MLK3 to allow the flow of the c-Src signal to JNK. At the MAPKK level, we found that the signal is transmitted by MKK7 and not MKK4. Our results present a general mechanism that mediates a G(q)PCR-induced, death receptor-independent, apoptosis in physiological, as well as cancer-related systems.     

Requirement for anti-dorsalizing morphogenetic protein in organizer patterning

The amphibian Spemann organizer is subdivided in trunk and head organizer and it is unclear how this division is regulated. The Xenopus trunk organizer expresses anti-dorsalizing morphogenetic protein (ADMP), a potent organizer antagonist. We show that ADMP represses head formation during gastrulation and that its expression is activated by BMP antagonists. A specifically acting dominant-negative ADMP anteriorizes embryos and its coexpression with BMP antagonists induces secondary embryonic axes with heads as well as expression of head inducers. Unlike other BMPs, ADMP is not inhibited by a dominant-negative BMP type I receptor, Noggin, Cerberus and Chordin but by Follistatin, suggesting that it utilizes a distinct TGF-β receptor pathway and displays differential sensitivity to BMP antagonists. The results indicate that ADMP functions in the trunk organizer to antagonize head formation, thereby regulating organizer patterning.     

Mechanism of calmodulin inhibition of cAMP-dependent protein kinase activation of phosphorylation kinase

The activation of phosphorylase kinase (EC 2.7.1.38; ATP:phosphorylase b phosphotransferase) by the catalytic subunit of cAMP-dependent protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) is inhibited by calmodulin. The mechanism of that inhibition has been studied by kinetic measurements of the interactions of the three proteins. The binding constant for calmodulin with phosphorylase kinase was found to be 90 nM when measured by fluorescence polarization spectroscopy. Glycerol gradient centrifugation studies indicated that 1 mol of calmodulin was bound to each phosphorylase kinase. Phosphorylation of the phosphorylase kinase did not reduce the amount of calmodulin bound. Kinetic studies of the activity of the catalytic subunit of cAMP-dependent protein kinase on phosphorylase kinase as a function of phosphorylase kinase and calmodulin concentrations were performed. The results of those studies were compared with mathematical models of four different modes of inhibition: competitive, noncompetitive, substrate depletion, and inhibition by a complex between phosphorylase kinase and calmodulin. The data conform best to the model in which the inhibitory species is a complex of phosphorylase kinase and calmodulin. The complex apparently competes with the substrate, phosphorylase kinase, which does not have exogenous calmodulin bound to it. In contrast, the phosphorylation of the synthetic phosphate acceptor peptide, Kemptide, is not inhibited by calmodulin.     

Requirement for RhoA kinase activation in leukocyte de-adhesion

Leukocyte migration from bloodstream to tissue requires rapid coordinated regulation of integrin-dependent adhesion and de-adhesion. Whether de-adhesion is an active process mediated by a distinct signaling pathway(s) or a passive decay of initial adhesion remains undetermined. We found that blockade of RhoA with C3 exoenzyme or inhibition of RhoA kinase by the specific inhibitor Y-27632 enhanced phorbol ester-stimulated alpha(4)beta(1)-dependent adhesion of Jurkat cells at 30 min. Similarly, Y-27632 treatment increased stimulated beta(2) integrin-dependent neutrophil adhesion at 30 min but not at 5 min. Because reduced de-adhesion could mimic augmentation of adhesion at later time points, we developed an assay to measure de-adhesion specifically. Treatment of phorbol ester-or bacterial chemoattractant peptide-but not Mn(2+)-stimulated neutrophils adherent to serum-coated plastic or endothelial cells with Y-27632 or C3 exoenzyme markedly reduced the rate of de-adhesion, while markedly increasing their spreading. RhoA kinase inhibitor effects on de-adhesion and spreading were reversed by treatment with the cytoskeletal-disrupting agent cytochalasin D. Treatment with Y-27632 influenced neither integrin activation epitope nor integrin clustering. We conclude that activation of RhoA kinase promotes leukocyte de-adhesion by inhibiting cytoskeletal-dependent spreading, and that these effects of RhoA kinase constitute a new mechanism for regulation of integrin receptor avidity.     

Tumor necrosis factor alpha-induced desumoylation and cytoplasmic translocation of homeodomain-interacting protein kinase 1 are critical for apoptosis signal-regulating kinase 1-JNK/p38 activation

The apoptosis signal-regulating kinase 1 (ASK1)-JNK/p38 signaling pathway is pivotal component in cell apoptosis and can be activated by a variety of death stimuli including tumor necrosis factor (TNF) alpha and oxidative stress (reactive oxygen species). However, the mechanism for ASK1 activation is not fully understood. We have recently identified ASK1-interacting protein (AIP1) as novel signal transducer in TNFalpha-induced ASK1 activation by facilitating dissociation of ASK1 from its inhibitor 14-3-3. In the present study, we employed yeast two-hybrid system using the N-terminal domain of AIP1 as bait and identified homeodomain-interacting protein kinase 1 (HIPK1) as an AIP1-associated protein. Interestingly, we showed that TNFalpha induced HIPK1 desumoylation concomitant with a translocation from nucleus to cytoplasm at 15 min followed by a return to nucleus by 60 min. The kinetics of HIPK1 translocation correlates with those of stress-induced ASK1-JNK/P38 activation. A specific JNK inhibitor blocked the reverse but not the initial translocation of HIPK1, suggesting that the initial translocation is an upstream event of ASK1-JNK/p38 signaling and JNK activation regulates the reverse translocation as a feedback mechanism. Consistently, expression of HIPK1 increased, whereas expression of a kinase-inactive form (HIPK1-D315N) or small interference RNA of HIPK1 decreased stress-induced ASK1-JNK/P38 activation without effects on IKK-NF-kappaB signaling. Moreover, a sumoylation-defective mutant of HIPK1 (KR5) localizes to the cytoplasm and is constitutively active in ASK1-JNK/P38 activation. Furthermore, HIPK1-KR5 induces dissociation of ASK1 from its inhibitors 14-3-3 and thioredoxin and synergizes with AIP1 to induce ASK1 activation. Our study suggests that TNFalpha-induced desumoylation and cytoplasmic translocation of HIPK1 are critical in TNFalpha-induced ASK1-JNK/p38 activation.     

Ubiquitination and degradation of homeodomain-interacting protein kinase 2 by WD40 repeat/SOCS box protein WSB-1

Homeodomain-interacting protein kinase 2 (HIPK2) is a member of the nuclear protein kinase family, which induces both p53- and CtBP-mediated apoptosis. Levels of HIPK2 were increased by UV irradiation and cisplatin treatment, thereby implying the degradation of HIPK2 in cells under normal conditions. Here, we indicate that HIPK2 is ubiquitinated and degraded by the WD40-repeat/SOCS box protein WSB-1, a process that is blocked under DNA damage conditions. Yeast two-hybrid screening was conducted to identify the proteins that interact with HIPK2. WSB-1, an E3 ubiquitin ligase, was characterized as an HIPK2-interacting protein. The coexpression of WSB-1 resulted in the degradation of HIPK2 via its C-terminal region. Domain analysis of WSB-1 showed that WD40-repeats and the SOCS box were required for its interaction with and degradation of HIPK2, respectively. In support of the degradation of HIPK2 by WSB-1, HIPK2 was polyubiquitinated by WSB-1 in vitro and in vivo. The knockdown of endogenous WSB-1 with the expression of short hairpin RNA against WSB-1 increases the stability of endogenous HIPK2 and resulted in the accumulation of HIPK2. The ubiquitination and degradation of HIPK2 by WSB-1 was inhibited completely via the administration of DNA damage reagents, including Adriamycin and cisplatin. These findings effectively illustrate the regulatory mechanisms by which HIPK2 is maintained at a low level, by WSB-1 in cells under normal conditions, and stabilized by genotoxic stresses.     

Matrix GLA protein, a regulatory protein for bone morphogenetic protein-2.

Matrix GLA protein (MGP) has been identified as a calcification inhibitor in cartilage and vasculature. Part of this effect may be attributed to its influence on osteoinductive activity of bone morphogenetic protein-2 (BMP-2). To detect binding between MGP and BMP-2, we performed immunoprecipitation using MGP and BMP-2 tagged with FLAG and c-Myc. The results showed co-precipitation of BMP-2 with MGP. To quantify the effect of MGP on BMP-2 activity, we assayed for alkaline phosphatase activity and showed a dose-dependent effect. Low levels of MGP relative to BMP-2 (<1-fold excess) resulted in mild enhancement of osteoinduction, whereas intermediate levels (1-15-fold excess) resulted in strong inhibition. High levels of MGP (>15-fold excess), however, resulted in pronounced enhancement of the osteoinductive effect of BMP-2. Cross-linking studies showed that inhibitory levels of MGP abolished BMP-2 receptor binding. Immunoblotting showed a corresponding decrease in activation of Smad1, part of the BMP signaling system. Enhancing levels of MGP resulted in increased Smad1 activation. To determine the cellular localization of BMP-2 in the presence of MGP, binding assays were performed on whole cells and cell-synthesized matrix. Inhibitory levels of MGP yielded increased matrix binding of BMP-2, suggesting that MGP inhibits BMP-2 in part via matrix association. These results suggest that MGP is a BMP-2 regulatory protein.