Phosphorylation Of Isoforms Research Articles

ArgBP2γ Interacts with Akt and p21-activated Kinase-1 and Promotes Cell Survival

Akt/protein kinase B is a major cell survival pathway through phosphorylation of proapoptotic proteins Bad and Bax and of additional apoptotic pathways linked to Forkhead proteins glycogen synthase kinase-3beta and ASK1. To further explore the mechanism by which Akt regulates cell survival, we identified an Akt interaction protein by yeast two-hybrid screening. It is highly homologous to ARG-binding protein 2 (ArgBP2) with splicing exon 8 of the coding region of the ArgBP2. As two splicing isoforms (ArgBP2alpha and -beta) of ArgBP2 have been identified (Wang, B., Golemis, E. A., and Kruh, G. D. (1997) J. Biol. Chem. 272, 17542-17550), it was named ArgBP2gamma. ArgBP2gamma contains four Akt phosphorylation consensus sites, a SoHo motif, and three Src homology (SH) 3 domains and binds to C-terminal proline-rich motifs of Akt through its first and second SH3 domains. It also interacts with p21-activated protein kinase (PAK1) via its first and third SH3 domains, indicating the SH3 domains of ArgBP2gamma as docking sites for Akt and PAK1. Akt phosphorylates ArgBP2gamma in vitro and in vivo. Expression of ArgBP2gamma induces PAK1 activity and overrides apoptosis induced by ectopic expression of Bad or DNA damage. Nonphosphorylatable ArgBP2gamma-4A and SH3 domain-truncated mutant ArgBP2gamma inhibit Akt-induced PAK1 activation and reduce Akt and PAK1 phosphorylation of Bad and antiapoptotic function. These data indicate that ArgBP2gamma is a physiological substrate of Akt, functions as an adaptor for Akt and PAK1, and plays a role in Akt/PAK1 cell survival pathway.

Valor pronóstico de la isoforma fosforilada de la proteína transportadora del factor de crecimiento similar a la insulina tipo 1, en el éxito de la inducción del parto

Objective To evaluate the relationship between successful induction of labor and the presence of the highly phosphorylated isoform of type-I insulinlike growth factor binding protein-1 (IGFBP-1) in the cervical fluid. Subjects and methods A total of 102 women with singleton vertex pregnancies undergoing induction of labor between 37 and 41 completed weeks of gestation were studied. Obstetric history, physical examination, transvaginal ultrasound and determination of the highly phosphorylated isoform of IGFBP-1 (Actim™TM PARTUS) were performed. Successful labor induction was defined as vaginal delivery within 24 hours of the procedure. Logistic regression modeling was performed to identify which variables were independently associated with successful induction. Results Labor induction was successful in 56 patients (65.1%). An Actim™ PARTUS positive result was observed in 67.4% of the patients, with a sensitivity of 76.8% and a specificity of 50%. On logistic regression, the only variable independently associated with the outcome of labor induction was parity. The other covariates, including the Actim™ PARTUS test, did not show a significant association. Conclusions The presence of the highly phosphorylated isoform of IGFBP-1 in the cervical fluid is not an independent factor associated with the success of labor induction.

Immunohistochemically determined total epidermal growth factor receptor levels not of prognostic value in newly diagnosed glioblastoma multiforme: Report from the Radiation Therapy Oncology Group

The Radiation Therapy Oncology Group (RTOG) performed an analysis of patterns of immunohistochemically detected total epidermal growth factor receptor (EGFR) protein expression levels and their prognostic significance on archival tissue in newly diagnosed glioblastoma multiforme (GBM) patients from prior prospective RTOG clinical trials. Patients in this study had been treated on previous RTOG GBM trials (RTOG 7401, 7918, 8302, 8409, 9006, 9305, 9602, and 9806). Tissue microarrays were prepared from 155 patients enrolled in these trials. These specimens were stained using a mouse monoclonal antibody specific for the extracellular binding domain of EGFR to detect total EGFR (including both wild-type phosphorylated and wild-type unphosphorylated isoforms with some cross-reactivity with EGFRvIII). The intensity of total EGFR protein expression was measured by computerized quantitative image analysis using the SAMBA 4000 Cell Image Analysis System. The parameters measured were the mean optical densities over the labeled areas and the staining index, which represents the proportion of stained area relative to the mean stain concentration. Both parameters were correlated with the clinical outcome. No differences in either overall or progression-free survival could be demonstrated by the mean optical density class or mean optical density quartile or the staining index of total EGFR immunostaining among the representative RTOG GBM cases. Total EGFR protein expression levels, as measured immunohistochemically, do not appear to be of prognostic value in newly diagnosed GBM patients. Given the accumulating clinical evidence of the activity of anti-EGFR agents in GBM and the preclinical data suggesting the important role of downstream mediators as effectors of EGFR signaling, the RTOG is conducting additional investigations into the prognostic value of activation patterns of EGFR signaling, both at the level of the receptor (e.g., EGFRvIII, phospho-EGFR) and at the level of downstream signal transduction pathways (e.g., PI3K, Ras/MAPK pathways).

Implication of STAT3 Signaling in Human Colonic Cancer Cells during Intestinal Trefoil Factor 3 (TFF3) – and Vascular Endothelial Growth Factor–Mediated Cellular Invasion and Tumor Growth

Signal transducer and activator of transcription (STAT) 3 is overexpressed or activated in most types of human tumors and has been classified as an oncogene. In the present study, we investigated the contribution of the STAT3s to the proinvasive activity of trefoil factors (TFF) and vascular endothelial growth factor (VEGF) in human colorectal cancer cells HCT8/S11 expressing VEGF receptors. Both intestinal trefoil peptide (TFF3) and VEGF, but not pS2 (TFF1), activate STAT3 signaling through Tyr(705) phosphorylation of both STAT3alpha and STAT3beta isoforms. Blockade of STAT3 signaling by STAT3beta, depletion of the STAT3alpha/beta isoforms by RNA interference, and pharmacologic inhibition of STAT3alpha/beta phosphorylation by cucurbitacin or STAT3 inhibitory peptide abrogates TFF- and VEGF-induced cellular invasion and reduces the growth of HCT8/S11 tumor xenografts in athymic mice. Differential gene expression analysis using DNA microarrays revealed that overexpression of STAT3beta down-regulates the VEGF receptors Flt-1, neuropilins 1 and 2, and the inhibitor of DNA binding/differentiation (Id-2) gene product involved in the neoplastic transformation. Taken together, our data suggest that TFF3 and the essential tumor angiogenesis regulator VEGF(165) exert potent proinvasive activity through STAT3 signaling in human colorectal cancer cells. We also validate new therapeutic strategies targeting STAT3 signaling by pharmacologic inhibitors and RNA interference for the treatment of colorectal cancer patients.

Interaction of TRAF6 with MAST205 Regulates NF-κB Activation and MAST205 Stability

The binding of immune complexes to macrophage Fcgamma receptor results in a subsequent inhibition of lipopolysaccharide-stimulated interleukin-12 synthesis without affecting the induction of tumor necrosis factor-alpha. RNA interference targeting MAST205, a 205-kDa serine/threonine kinase, and transfection of dominant negative MAST205 mutants also mimic this type II macrophage phenotype. Our previous epistasis experiments suggested that the position of MAST205 in the TLR4 signal pathway was proximal to the IkappaB kinase complex. We now report that MAST205 forms a complex with TRAF6, resulting in the inhibition of TRAF6 NF-kappaB activation. We have identified a peptide (residues 218-233) from the N terminus of MAST205 that, when coupled to a protein transduction domain, inhibits the lipopolysaccharide-stimulated activation of NF-kappaB, modulates the size of the MAST205.TRAF6 complex, and inhibits ubiquitination of TRAF6. A dominant negative N-terminal MAST205 deletion mutant also inhibits TRAF6 ubiquitination. The domain required for degradation of MAST205 after Fcgamma receptor activation resides within the N-terminal 261 residues, and degradation is triggered by protein kinase C isoform phosphorylation of Ser/Thr residues. These results suggest that MAST205 functions as a scaffolding protein controlling TRAF6 activity and, therefore, plays an important role in regulating inflammatory responses.

PKC-epsilon is upstream and PKC-alpha is downstream of mitoKATP channels in the signal transduction pathway of ischemic preconditioning of human myocardium.

Protein kinase C (PKC) is involved in the process of ischemic preconditioning (IPC), although the precise mechanism is still a subject of debate. Using specific PKC inhibitors, we investigated which PKC isoforms were involved in IPC of the human atrial myocardium sections and to determine their temporal relationship to the opening of mitochondrial potassium-sensitive ATP (mitoKATP) channels. Right atrial muscles obtained from patients undergoing elective cardiac surgery were equilibrated and then randomized to receive any of the following protocols: aerobic control, 90-min simulated ischemia/120-min reoxygenation, IPC using 5-min simulated ischemia/5-min reoxygenation followed by 90-min simulated ischemia/120-min reoxygenation and finally, PKC inhibitors were added 10 min before and 10 min during IPC followed by 90-min simulated ischemia/120-min reoxygenation. The PKC isoforms inhibitors investigated were V1-2 peptide, GO-6976, rottlerin, and LY-333531 for PKC-epsilon, -alpha, -delta and -beta, respectively. To investigate the relation of PKC isoforms to mitoKATP channels, PKC inhibitors found to be involved in IPC were added 10 min before and 10 min during preconditioning by diazoxide followed by 90-min simulated ischemia/120-min reoxygenation in a second experiment. Creatine kinase leakage and methylthiazoletetrazolium cell viability were measured. Phosphorylation of PKC isoforms after activation of the sample by either diazoxide or IPC was detected by using Western blot analysis and then analyzed by using Scion image software. PKC-alpha and -epsilon inhibitors blocked IPC, whereas PKC-delta and -beta inhibitors did not. The protection elicited by diazoxide, believed to be via mitoKATP channels opening, was blocked by the inhibition of PKC-alpha but not -epsilon isoforms. In addition, diazoxide caused increased phosphorylation of PKC-alpha to the same extent as IPC but did not affect the phosphorylation of PKC-epsilon, a process believed to be critical in PKC activation. The results demonstrate that PKC-alpha and -epsilon are involved in IPC of the human myocardium with PKC-epsilon being upstream and PKC-alpha being downstream of mitoKATP channels.

Activation of Vascular Endothelial Growth Factor Receptor-3 and Its Downstream Signaling Promote Cell Survival under Oxidative Stress

Reactive oxygen species (ROS) mediate cell damage and have been implicated in the pathogenesis of diseases that involve endothelial injury. Cells possess antioxidant systems, including intracellular antioxidants and ROS scavenging enzymes, that control the redox state and prevent cell damage. In addition to intracellular antioxidants, certain growth factor receptors can be activated under oxidative stress and trigger downstream cell survival signaling cascades. Vascular endothelial growth factor receptor-3 (VEGFR-3) is a primary modulator of lymphatic endothelial proliferation and survival. Here, we provide evidence that activation of VEGFR-3 signaling in response to hydrogen peroxide (H(2)O(2)) promotes endothelial cell survival. Treatment with H(2)O(2) induced the tyrosine phosphorylation of VEGFR-3 and its association with the signaling adaptor proteins Shc, growth factor receptor binding protein 2, Sos, p85, SHP-2, and phospholipase C-gamma. Of note, a hereditary lymphoedema-linked mutant of VEGFR-3 was not phosphorylated by H(2)O(2) treatment. Isoforms of protein kinase C (PKC), alpha and delta, were also tyrosine-phosphorylated after H(2)O(2) stimulation. However, only the delta isoform of PKC was required for H(2)O(2)-induced phosphorylation of VEGFR-3. The tyrosine phosphorylation of VEGFR-3 or isoforms of PKC was completely inhibited by treatment with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, a specific inhibitor for Src family kinases, indicating that Src family kinases are upstream of PKC and VEGFR-3. Furthermore, expression of the wild-type but not the lymphoedema-linked mutant form of VEGFR-3 in porcine artery endothelial cells significantly enhanced the activation of Akt after H(2)O(2) stimulation. Consistent with these biochemical changes, we observed that expression and activation of the wild-type but not the mutant form of VEGFR-3 inhibited H(2)O(2)-induced apoptosis. These studies suggest that VEGFR-3 protects against oxidative damage in endothelial cells, and that patients with hereditary lymphoedema may be susceptible to ROS-induced cell damage.

P38 mitogen-activated protein kinase contributes to the diminished aortic contraction by endothelin-1 in DOCA-salt hypertensive rats.

We investigated whether the diminished contractile responsiveness to endothelin-1 (ET-1) is associated with the altered activation of mitogen-activated protein kinase (MAPK) in aortic smooth muscles from deoxycorticosterone acetate (DOCA)-salt hypertensive rats. ET-1 dose-dependently increased contractions in aortic smooth muscle strips, and the contractions were significantly attenuated in tissues from DOCA-salt hypertensive rats compared with those from sham-operated rats. The phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was elevated by ET-1, with the magnitude and time-course being similar between strips. Although ET-1 also increased the phosphorylation of p38 MAPK in both strips, the increment was markedly lower in the strips from DOCA-salt hypertensive rats compared with sham-operated controls. 5-hydroxytryptamine (5-HT) increased vascular contraction and phosphorylation of both MAPK isoforms; these were greater in DOCA-salt hypertensive rats than in sham-operated rats. ET-1 also increased the phosphorylation of caldesmon, an actin-binding protein, in sham-operated and DOCA-salt hypertensive rats. However, the increment was markedly lower in the strips from DOCA-salt hypertensive rats compared with sham-operated controls. The phosphorylation of MAPK isoforms and caldesmon elevated by ET-1 was inhibited by PD098059, an inhibitor of ERK1/2 kinase, and SB203580, an inhibitor of p38 MAPK, respectively. These results suggest that ET-1 and 5-HT induce contraction by activating the MAPK pathway in rat aortic smooth muscle and that the diminished responsiveness to ET-1 in the DOCA-salt hypertensive rat may be, in part, mediated by the decrease of caldesmon phosphorylation after the decreased activation of p38 MAPK.

Neuroprotective effects of resveratrol against beta-amyloid-induced neurotoxicity in rat hippocampal neurons: involvement of protein kinase C.

1. Resveratrol, an active ingredient of red wine extracts, has been shown to exhibit neuroprotective effects in several experimental models. 2. The present study evaluated the neuroprotective effects of resveratrol against amyloid beta(Abeta)-induced toxicity in cultured rat hippocampal cells and examined the role of the protein kinase C (PKC) pathway in this effect. 3. Pre-, co- and post-treatment with resveratrol significantly attenuated Abeta-induced cell death in a concentration-dependent manner, with a concentration of 25 microm being maximally effective. 4. Pretreatment (1 h) of hippocampal cells with phorbol-12-myristate-13-acetate, a PKC activator, at increasing concentrations (1-100 ng x ml(-1)), resulted in a dose-dependent reduction in Abeta-induced toxicity, whereas the inactive 4alpha-phorbol had no effect. 5. Pretreatment (30 min) of hippocampal cells with GF 109203X (1 microm), a general PKC inhibitor, significantly attenuated the neuroprotective effect of resveratrol against Abeta-induced cell death. 6. Treatment of hippocampal cells with resveratrol (20 microm) also induced the phosphorylation of various isoforms of PKC leading to activation. 7. Taken together, the present results indicate that PKC is involved in the neuroprotective action of resveratrol against Abeta-induced toxicity.

Creatine supplementation increases glucose oxidation and AMPK phosphorylation and reduces lactate production in L6 rat skeletal muscle cells.

Recent observations have suggested that creatine supplementation might have a beneficial effect on glucoregulation in skeletal muscle. However, conclusive studies on the direct effects of creatine on glucose uptake and metabolism are lacking. The objective of this study was to investigate the effects of creatine supplementation on basal and insulin-stimulated glucose transporter (GLUT4) translocation, glucose uptake, glycogen content, glycogen synthesis, lactate production, glucose oxidation and AMP-activated protein kinase (AMPK) phosphorylation in L6 rat skeletal muscle cells. Four treatment groups were studied: control, insulin (100 nM), creatine (0.5 mM) and creatine + insulin. After 48 h of creatine supplementation the creatine and phosphocreatine contents of L6 myoblasts increased by approximately 9.3- and approximately 5.1-fold, respectively, but the ATP content of the cells was not affected. Insulin significantly increased 2-deoxyglucose uptake ( approximately 1.9-fold), GLUT4 translocation ( approximately 1.8-fold), the incorporation of D-[U-(14)C]glucose into glycogen ( approximately 2.3-fold), lactate production ( approximately 1.5-fold) and (14)CO(2) production ( approximately 1.5-fold). Creatine neither altered the glycogen and GLUT4 contents of the cells nor the insulin-stimulated rates of 2-DG uptake, GLUT4 translocation, glycogen synthesis and glucose oxidation. However, creatine significantly reduced by approximately 42% the basal rate of lactate production and increased by approximately 40% the basal rate of (14)CO(2) production. This is in agreement with the approximately 35% increase in citrate synthase activity and also with the approximately 2-fold increase in the phosphorylation of both alpha-1 and alpha-2 isoforms of AMPK after creatine supplementation. We conclude that 48 h of creatine supplementation does not alter insulin-stimulated glucose uptake and glucose metabolism; however, it activates AMPK, shifts basal glucose metabolism towards oxidation and reduces lactate production in L6 rat skeletal muscle cells.

PKC-epsilon regulation of extracellular signal-regulated kinase: a potential role in phenylephrine-induced cardiocyte growth.

Hypertrophic growth of cardiac muscle is dependent on activation of the PKC-epsilon isoform. To define the effectors of PKC-epsilon involved in growth regulation, recombinant adenoviruses were used to overexpress either wild-type PKC-epsilon (PKC-epsilon/WT) or dominant negative PKC-epsilon (PKC-epsilon/DN) in neonatal rat cardiocytes. PKC-epsilon/DN inhibited acute activation of PKC-epsilon produced in response to phorbol ester and reduced ERK1/2 activity as measured by the phosphorylation of p42 and p44 isoforms. The inhibitory effects were specific to PKC-epsilon because PKC-epsilon/DN did not prevent translocation of either PKC-alpha or PKC-delta. Overexpression of PKC-epsilon/DN blunted the acute increase in ERK1/2 phorphorylation induced by the alpha(1)-adrenergic agonist phenylephrine (PE ). Inhibition of PKC-delta with rottlerin potentiated the effects of PE on ERK1/2 phosphorylation. PKC-epsilon/DN adenovirus also blocked cardiocyte growth as measured after 48 h of PE treatment, although the multiplicity of infection was lower than that required to block acute ERK1/2 activation. PE activated p38 mitogen-activated protein kinase as measured by its phosphorylation, but the response was not blocked by PKC inhibitors or by overexpression of PKC-epsilon/DN. Taken together, these studies show that the hypertrophic agonist PE regulates ERK1/2 activity in cardiocytes by a pathway dependent on PKC-epsilon and that PE-induced growth is mediated by PKC-epsilon.

The mitochondrial K(ATP) channel opener BMS-191095 induces neuronal preconditioning.

BMS-191095, reportedly a selective mitoK(ATP) channel opener which is free from the known side effects of the prototype mitoK(ATP) channel opener diazoxide, induced acute and delayed preconditioning against glutamate excitotoxicity and delayed preconditioning against oxygen-glucose deprivation in primary cultures of rat cortical neurons. BMS-191095 dose dependently depolarized the mitochondria, increased the phosphorylation of PKC isoforms, but had no detectable effects on the activation of MAP kinases and did not influence the expressions of HSP70 and Mn-SOD. In BMS-191095-preconditioned neurons the glutamate-induced free-radical production was abolished. Our data give the first evidence that selective opening of mitoK(ATP) channels with BMS-191095 leads to remarkable neuroprotection via mechanisms that involve mitochondrial depolarization, PKC activation and attenuated free radical production during neuronal stress.

Differential involvement and heterogeneous phosphorylation of tau isoforms in progressive supranuclear palsy

We found previously that aggregated insoluble tau protein in progressive supranuclear palsy (PSP) brains exhibits a heterogeneous pattern that is not segregated by the type of clinical presentation. Here we have investigated tau isoform composition from 20 PSP cases and found marked variation between different brains. Cases were classified into three groups, each comprising essentially of (1) 1N4R; (2) 1N4R and 1N3R; or (3) 1N4R, 1N3R and 0N4R tau isoforms. There was also an absence of a simple relationship between isoform composition and the pattern of insoluble tau before dephosphorylation. We conclude that there is distinct molecular heterogeneity in the involvement of tau isoforms in the tau pathology in PSP.

Tyrosine phosphorylation of QKI mediates developmental signals to regulate mRNA metabolism.

The selective RNA-binding protein QKI is essential for myelination in the central nervous system (CNS). QKI belongs to the family of signal transduction activators of RNA (STARs), characteristic of binding RNA and signaling molecules, therefore is postulated to regulate RNA homeostasis in response to developmental signals. Here we report that QKI acts downstream of the Src family protein tyrosine kinases (Src-PTKs) during CNS myelination. QKI selectively interacted with the mRNA encoding the myelin basic protein (MBP). Such interaction stabilized MBP mRNA and was required for the rapid accumulation of MBP mRNA during active myelinogenesis. We found that the interaction between QKI and MBP mRNA was negatively regulated by Src-PTK-dependent phosphorylation of QKI. During early myelin development, tyrosine phosphorylation of QKI in the developing myelin drastically declined, presumably leading to enhanced interactions between QKI and MBP mRNA, which was associated with the rapid accumulation of MBP mRNA and accelerated myelin production. Therefore, developmental regulation of Src-PTK-dependent tyrosine phosphorylation of QKI suggests a novel mechanism for accelerating CNS myelinogenesis via regulating mRNA metabolism.

Bile Acids Stimulate cFLIP Phosphorylation Enhancing TRAIL-mediated Apoptosis

Bile acids induce hepatocyte injury by enhancing death receptor-mediated apoptosis. In this study, bile acid effects on TRAIL-mediated apoptosis were examined to gain insight into bile acid potentiation of death receptor signaling. TRAIL-induced apoptosis of HuH-7 cells, stably transfected with a bile acid transporter, was enhanced by bile acids. Caspase 8 and 10 activation, bid cleavage, cytosolic cytochrome c, and caspase 3 activation by TRAIL were all increased by the bile acid glycochenodeoxycholate (GCDCA). GCDCA (100 microm) did not alter expression of TRAIL-R1/DR4, TRAIL-R2/DR5, procaspase 8, cFLIP-L, cFLIP-s, Bax, Bcl-xL, or Bax. However, both caspase 8 and caspase 10 recruitment and processing within the TRAIL death-inducing signaling complex (DISC) were greater in GCDCA-treated cells whereas recruitment of cFLIP long and short was reduced. GCDCA stimulated phosphorylation of both cFLIP isoforms, which was associated with decreased binding to GST-FADD. The protein kinase C antagonist chelerythrine prevented bile acid-stimulated cFLIP-L and -s phosphorylation, restored cFLIP binding to GST-FADD, and attenuated bile acid potentiation of TRAIL-induced apoptosis. These results provide new insights into the mechanisms of bile acid cytotoxicity and the proapoptotic effects of cFLIP phosphorylation in TRAIL signaling.

Tau protein expression in adult bovine oligodendrocytes: functional and pathological significance.

In tauopathies, overexpression of tau exon 10 is linked to degeneration and abnormal tau deposition in neurons and oligodendroglia (OLGs). To compare exon 10 expression in normal neurons and OLGs, adult bovine brain was examined for the expression of tau in gray matter and cultured OLGs isolated from white matter. Using exon-specific antibodies, we found that both types of tissues abundantly expressed exon 2 but isolated OLGs had a lower expression of exons 3 and 10 when compared to gray matter. Relative expression of exons 3 and 10 did not change significantly during the in vitro maturation of OLGs for 39 days. Using a panel of well-characterized antibodies against tau, we determined that isolated OLGs contained tau phosphorylated at the Tau-1, 12E8, and PHF-1 but not the AT8, AT100, AT180, and AT270 epitopes. Tau phosphorylation status diminished during in vitro maturation, suggesting that healthy OLG processes require regulated phosphorylation of tau at specific sites. We propose that the tau isoform profile and phosphorylation status contribute to the vulnerability of OLGs in degenerative diseases linked to overexpression of exon 10.

Polar Expression and Phosphorylation of Human Leptin Receptor Isoforms in Paired, Syncytial, Microvillous and Basal Membranes from Human Term Placenta

The hormone leptin (OB) and its receptor (OB-R) are key homeostatic regulators of mammalian body weight. Two predominant isoforms of OB-R are expressed by alternative splicing: the long form, OB-RL, with full signalling capacity is highly expressed in the hypothalamus and the short, signalling-defective form, OB-Rs, is ubiquitously expressed.In a previous study we detected expression of OB-RL and OB-Rs in human syncytiotrophoblast cells using in situ hybridization and immunohistochemistry (Bodner et al., 1999).The aim of this study was to investigate leptin receptor isoform expression and phosphorylation in paired, syncytial, microvillous and basal membranes from human term placenta by Western blot analysis. Both the OB-RL and the OB-Rs isoforms were detected in the syncytial membrane preparations. The OB-RL isoform was observed exclusively in microvillous membranes, whereas the OB-Rs isoform was found in both microvillous and basal membrane preparations. No significant differences were observed between syncytial membranes from normal and type 1 diabetic pregnancies. To test the phosphorylation capacity of the OB-R isoforms, microvillous and basal membrane vesicles loaded with ATP were stimulated with leptin and the phosphorylation status of the OB-R at the tyrosine 985 (Y985) was determined. A single band at the molecular weight corresponding to the molecular weight of the OB-RL isoform was detected exclusively in the ATP-loaded microvillous vesicles.We conclude that the long form OB-RL is expressed exclusively in the microvillous membrane of the syncytiotrophoblast and is capable of being phosphorylated, suggesting that it has signal transduction capacity.

Long PDE4 cAMP specific phosphodiesterases are activated by protein kinase A-mediated phosphorylation of a single serine residue in Upstream Conserved Region 1 (UCR1).

1. Challenge of COS1 cells with the adenylyl cyclase activator forskolin led to the activation of recombinant PDE4A8, PDE4B1, PDE4C2 and PDE4D5 cAMP-specific phosphodiesterase long isoforms. 2. Forskolin challenge did not activate mutant long PDE4 isoforms where the serine target residue (STR) within the protein kinase A (PKA) consensus phosphorylation site in Upstream Conserved Region 1 (UCR1) was mutated to alanine. 3. The PKA inhibitor, H89, ablated forskolin activation of wild-type long PDE4 isoforms. 4. Activated PKA caused the in vitro phosphorylation of recombinant wild-type long PDE4 isoforms, but not those where the STR was mutated to alanine. 5. An antiserum specific for the phosphorylated form of the STR detected a single immunoreactive band for recombinant long PDE4 isoforms expressed in COS1 cells challenged with forskolin. This was not evident in forskolin-challenged cells treated with H89. Neither was it evident in forskolin-challenged cells expressing long isoforms where the STR had been mutated to alanine. 6. In transfected COS cells challenged with forskolin, only the phosphorylated PDE4D3 long form showed a decrease in mobility in Western blotting analysis. This decreased mobility of PDE4D3 was ablated upon mutation of either of the two serine targets for PKA phosphorylation in this isoform, namely Ser54 in UCR1 and Ser13 in the isoform-specific N-terminal region. 7. Activation by forskolin challenge did not markedly alter the sensitivity of PDE4A8, PDE4B1, PDE4C2 and PDE4D5 to inhibition by rolipram. 8. Long PDE4 isoforms from all four sub-families can be phosphorylated by protein kinase A (PKA). This leads to an increase in their activity and may thus contribute to cellular desensitization processes in cells where these isoforms are selectively expressed.

Phospholipase D1 is threonine-phosphorylated in human-airway epithelial cells stimulated by sphingosine-1-phosphate by a mechanism involving Src tyrosine kinase and protein kinase Cdelta.

The regulatory role of protein kinase C (PKC) delta isoform in the stimulation of phospholipase D (PLD) by sphingosine-1-phosphate (SPP) in a human-airway epithelial cell line (CFNPE9o(-)) was revealed by using antisense oligodeoxynucleotide to PKCdelta, in combination with the specific inhibitor rottlerin. Cell treatment with antisense oligodeoxynucleotide, but not with sense oligodeoxynucleotide, completely eliminated PKCdelta expression and resulted in the strong inhibition of SPP-stimulated phosphatidic acid formation. Indeed, among the PKCalpha, beta, delta, epsilon and zeta isoforms expressed in these cells, only PKCdelta was activated on cell stimulation with SPP, as indicated by translocation into the membrane fraction. Furthermore, pertussis toxin and genistein eliminated both PKCdelta translocation and PLD activation. In particular, a significant reduction in phosphatidylbutanol formation by SPP was observed in the presence of 4-amino-5-(4-methylphenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine (PP1), an inhibitor of Src tyrosine kinase. Furthermore, the activity of Src kinase was slightly increased by SPP and inhibited by PP1. However, the level of PKCdelta tyrosine phosphorylation was not increased in SPP-stimulated cells, suggesting that Src did not directly phosphorylate PKCdelta. Finally, the level of serine phosphorylation of PLD1 and PLD2 isoforms was not changed, whereas the PLD1 isoform alone was threonine-phosphorylated in SPP-treated cells. PLD1 threonine phosphorylation was strongly inhibited by rottlerin, by anti-PKCdelta oligodeoxynucleotide and by PP1. In conclusion, in CFNPE9o(-) cells, SPP interacts with a membrane receptor linked to a G(i) type of G-protein, leading to activation of PLD, probably the PLD1 isoform, by a signalling pathway involving Src and PKCdelta.

Calcium sensitivity of force in human ventricular cardiomyocytes from donor and failing hearts.

In failing human myocardium changes occur, in particular, in isoform composition and phosphorylation level of the troponin T (TnT) and troponin I (TnI) subunits of the actin filament and the myosin light chains (MLC-1 and -2), but it is unclear to what extent they influence cardiac performance. This overview concentrates on the relation between contractile function, contractile protein composition and phosphorylation levels in small biopsies from control (donor) hearts, from biopsies obtained during open heart surgery (NYHA Class I-IV) and from end-stage failing (explanted, NYHA class IV) hearts. Furthermore, attention is paid to the effect of the catalytic subunit of protein kinase A on isometric force development in single Triton-skinned human cardiomyocytes isolated from donor and end-stage failing left ventricular myocardium at different resting sarcomere lengths. A reduction in sarcomere length from 2.2 to 1.8 microm caused reductions in maximum isometric force by approximately 35% both in donor and in failing cardiomyocytes. The midpoints of the calcium sensitivity curves (pCa50) of donor and end-stage failing hearts differed markedly at all sarcomere lengths (mean delta pCa50 = 0.22). Our findings indicate that 1) TnI phosphorylation contributes to the differences in calcium sensitivity between donor and end-stage failing hearts, 2) human ventricular myocardium is heterogeneous with respect of the phosphorylation of TnT, MLC-2 and the isoform distribution of MLC-1 and MLC-2, and 3) the Frank-Starling mechanism is preserved in end-stage failing myocardium.