Biomedical subjects

Johan Van Lint

Publications and source records attributed to Johan Van Lint.

13 recordsLinked to original sources

PubMed2005

Role of the regulatory domain of protein kinase D2 in phorbol ester binding, catalytic activity, and nucleocytoplasmic shuttling.

Protein kinase D2 (PKD2) belongs to the PKD family of serine/threonine kinases that is activated by phorbol esters and G protein-coupled receptors (GPCRs). Its C-terminal regulatory domain comprises two cysteine-rich domains (C1a/C1b) followed by a pleckstrin homology (PH) domain. Here, we examined the role of the regulatory domain in PKD2 phorbol ester binding, catalytic activity, and subcellular localization: The PH domain is a negative regulator of kinase activity. C1a/C1b, in particular C1b, is required for phorbol ester binding and gastrin-stimulated PKD2 activation, but it has no inhibitory effect on the catalytic activity. Gastrin triggers nuclear accumulation of PKD2 in living AGS-B cancer cells. C1a/C1b, not the PH domain, plays a complex role in the regulation of nucleocytoplasmic shuttling: We identified a nuclear localization sequence in the linker region between C1a and C1b and a nuclear export signal in the C1a domain. In conclusion, our results define the critical components of the PKD2 regulatory domain controlling phorbol ester binding, catalytic activity, and nucleocytoplasmic shuttling and reveal marked differences to the regulatory properties of this domain in PKD1. These findings could explain functional differences between PKD isoforms and point to a functional role of PKD2 in the nucleus upon activation by GPCRs.

Catalysis↗

PubMed2005

Distinct transduction mechanisms of cyclooxygenase 2 gene activation in tumour cells after photodynamic therapy.

Photodynamic therapy (PDT) is a minimally invasive treatment for cancer and several noncancerous proliferating cell diseases. PDT relies on the uptake of a photosensitizing compound by the pathologic tissue followed by a selective irradiation with visible light, which leads to oxidative stress-mediated cell death. However, some studies showed that PDT induces the release of proangiogenic factors, such as vascular endothelial growth factor, and/or cyclooxygenase-2 (COX-2), thereby promoting cancer cell regrowth following PDT. In this work, we focused on the molecular mechanisms regulating COX-2 expression after low-dose PDT in two cancer cell lines, namely HeLa and T24. We report that PDT induces COX-2 expression in these cells and this expression is mainly due to nuclear factor kappa B (NF-kappaB)-dependent transcription of cox-2 gene without any post-transcriptional regulation. However, the transduction mechanism leading to NF-kappaB activation and subsequent cox-2 gene transcription differs in both cell types. In T24, NF-kappaB activation occurs through a protein kinase C (PKC)alpha- and phosphoinositide-3-kinase (PI3K)-dependent I kappa B kinase (IKK) complex activation, whereas in HeLa cells, NF-kappaB activation is mediated by PKC- and PI3K-independent IKK complex activation.

Cyclooxygenase 2↗

PubMed2005

Phosphorylation of histone deacetylase 7 by protein kinase D mediates T cell receptor-induced Nur77 expression and apoptosis.

The molecular basis of thymocyte negative selection, a crucial mechanism in establishing central tolerance, is not yet resolved. Histone deacetylases (HDACs) have emerged as key transcriptional regulators in several major developmental programs. Recently, we showed that the class IIa member, HDAC7, regulates negative selection by repressing expression of Nur77, an orphan nuclear receptor involved in antigen-induced apoptosis of thymocytes. Engagement of the T cell receptor (TCR) alleviates this repression through phosphorylation-dependent nuclear exclusion of HDAC7. However, the identity of the TCR-activated kinase that phosphorylates and inactivates HDAC7 was still unknown. Here, we demonstrate that TCR-induced nuclear export of HDAC7 and Nur77 expression is mediated by activation of protein kinase D (PKD). Indeed, active PKD stimulates HDAC7 nuclear export and Nur77 expression. In contrast, inhibition of PKD prevents TCR-mediated nuclear exclusion of HDAC7 and associated Nur77 activation. Furthermore, we show that HDAC7 is an interaction partner and a substrate for PKD. We identify four serine residues in the NH(2) terminus of HDAC7 as targets for PKD. More importantly, a mutant of HDAC7 specifically deficient in phosphorylation by PKD, inhibits TCR-mediated apoptosis of T cell hybridomas. These findings indicate that PKD is likely to play a key role in the signaling pathways controlling negative selection.

Active Transport, Cell Nucleus↗

PubMed2005

PKCepsilon-PKD1 signaling complex at Z-discs plays a pivotal role in the cardiac hypertrophy induced by G-protein coupling receptor agonists.

Cardiac hypertrophy is triggered in response to mechanical stress and various neurohumoral factors, such as G-protein coupling receptor (GPCR) and gp130 cytokine receptor agonists. Recent studies have suggested cardiac Z-disc plays a pivotal role to regulate these cellular responses. Here, we demonstrate stimulations with GPCR agonists (norepinephrine, angiotensin II, and endothelin 1) and phorbol ester activated and translocated protein kinase D1 (PKD1) to the Z-discs in neonatal rat cardiomyocytes in a protein kinase C (PKC)-dependent manner, whereas gp130 agonist did not. Especially, upon the alpha-adrenergic receptor agonist stimulations, following the PKCepsilon-PKD1 complex formation, PKCepsilon-dependent activation of PKD1 was essential to induce hypertrophic responses. Constitutively active mutant of either PKD1 or PKCepsilon also induced cardiac hypertrophy ex vivo. Taken together, the PKCepsilon-PKD1 complex at Z-discs could play a pivotal role in the cardiac hypertrophy induced by GPCR agonists, at least alpha-adrenergic receptor agonist.

Adrenergic alpha-Agonists↗

PubMed2005

Activation of hematopoietic progenitor kinase 1 involves relocation, autophosphorylation, and transphosphorylation by protein kinase D1.

Adaptive immune signaling can be coupled to stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and NF-kappaB activation by the hematopoietic progenitor kinase 1 (HPK1), a mammalian hematopoiesis-specific Ste20 kinase. To gain insight into the regulation of leukocyte signal transduction, we investigated the molecular details of HPK1 activation. Here we demonstrate the capacity of the Src family kinase Lck and the SLP-76 family adaptor protein Clnk (cytokine-dependent hematopoietic cell linker) to induce HPK1 tyrosine phosphorylation and relocation to the plasma membrane, which in lymphocytes results in recruitment of HPK1 to the contact site of antigen-presenting cell (APC)-T-cell conjugates. Relocation and clustering of HPK1 cause its enzymatic activation, which is accompanied by phosphorylation of regulatory sites in the HPK1 kinase activation loop. We show that full activation of HPK1 is dependent on autophosphorylation of threonine 165 and phosphorylation of serine 171, which is a target site for protein kinase D (PKD) in vitro. Upon T-cell receptor stimulation, PKD robustly augments HPK1 kinase activity in Jurkat T cells and enhances HPK1-driven SAPK/JNK and NF-kappaB activation; conversely, antisense down-regulation of PKD results in reduced HPK1 activity. Thus, activation of major lymphocyte signaling pathways via HPK1 involves (i) relocation, (ii) autophosphorylation, and (iii) transphosphorylation of HPK1 by PKD.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Protein kinase D2 mediates activation of nuclear factor kappaB by Bcr-Abl in Bcr-Abl+ human myeloid leukemia cells.

The Bcr-Abl tyrosine kinase activates various signaling pathways including nuclear factor kappaB that mediate proliferation, transformation, and apoptosis resistance in Bcr-Abl(+) myeloid leukemia cells. Here we report that protein kinase (PK) D2, a serine threonine kinase of the PKD family, is a novel substrate of Bcr-Abl. PKD2 was found to be the major isoform of the PKD family expressed in chronic myeloid leukemia cells and is tyrosine phosphorylated by Bcr-Abl in its pleckstrin homology domain. A mutant that mimicks tyrosine phosphorylation of PKD2 in the pleckstrin homology domain activates nuclear factor kappaB independently of its catalytic activity. Furthermore, our data show that Bcr-Abl-induced activation of the nuclear factor kappaB cascade in LAMA84 cells is largely mediated by tyrosine-phosphorylated PKD2. These data present a novel mechanism of Bcr-Abl-induced nuclear factor kappaB activation in myeloid leukemia. Targeting PKD2 tyrosine phosphorylation, not its kinase activity, could be a novel therapeutic approach for the treatment of Bcr-Abl(+) myeloid leukemia.

Cell Line, Tumor↗

PubMed2004

Doxorubicin-induced activation of protein kinase D1 through caspase-mediated proteolytic cleavage: identification of two cleavage sites by microsequencing.

Recent studies have demonstrated the importance of protein kinase D (PKD) in cell proliferation and apoptosis. Here, we report that in vitro cleavage of recombinant PKD1 by caspase-3 generates two alternative active PKD fragments. N-terminal sequencing of these fragments revealed two distinct caspase-3 cleavage sites located between the acidic and pleckstrin homology (PH) domains of PKD1. Moreover, we present experimental evidence that PKD1 is an in vitro substrate for both initiator and effector caspases. During doxorubicin-induced apoptosis, a zVAD-sensitive caspase induces cleavage of PKD1 at two sites, generating fragments with the same molecular masses as those determined in vitro. The in vivo caspase-dependent generation of the PKD1 fragments correlates with PKD1 kinase activation. Our results indicate that doxorubicin-mediated apoptosis induces activation of PKD1 through a novel mechanism involving the caspase-mediated proteolysis.

Amino Acid Sequence↗

PubMed2003

Protein kinase D: a family affair.

The protein kinase D family of enzymes consists of three isoforms: PKD1/PKCmu PKD2 and PKD3/PKCnu. They all share a similar architecture with regulatory sub-domains that play specific roles in the activation, translocation and function of the enzymes. The PKD enzymes have recently been implicated in very diverse cellular functions, including Golgi organization and plasma membrane directed transport, metastasis, immune responses, apoptosis and cell proliferation.

Animals↗

PubMed2003

In vitro polarized transport of L-phenylalanine in human nasal epithelium and partial characterization of the amino acid transporters involved.

PURPOSE: The purpose of this study was to provide functional and molecular evidence to support the existence of large neutral amino acid transporters in human nasal epithelium using nasal primary cell culture model. METHODS: L-Phenylalanine was used as a model substrate to characterize carrier-mediated permeation of amino acids across human nasal epithelium. The influence of temperature, concentration, other amino acids, metabolic/transport inhibitors, and polarity/stereo-selectivity on transport of the model compound was investigated. Reverse transcriptase polymerase chain reaction was used for molecular characterization of the existence of the transporters. RESULTS: The transport of L-phenylalanine across the human nasal epithelium was polarized (apical --> basolateral >> basolateral --> apical), saturable (Km = 1.23 mM; Vmax = 805.1 nmol/mg protein/min) and stereo-selective (permeation of L-phenylalanine >> D-Phenylalanine). Its permeation was significantly (< 0.05) reduced by cationic, small and large neutral amino acids, oubain, amiloride, sodium-free medium, and temperature lowering. Reverse transcriptase polymerase chain reaction revealed the presence of the broad-scope cationic-dependent amino acid transporter gene (y+LAT-2) in the human nasal epithelium. CONCLUSIONS: Based on the results of this study, one may postulate that the human nasal epithelium expresses L-amino acid transporters. More studies are necessary for detailed characterization of the transporters.

Amino Acid Transport Systems↗

PubMed2003

Ligand structure-activity requirements and phospholipid dependence for the binding of phorbol esters to protein kinase D.

Although protein kinase D (PKD), like protein kinase C (PKC), possesses a C1 domain that binds phorbol esters and diacylglycerol, the structural differences from PKC within this and other domains of PKD imply differential regulation by lipids and ligands. We characterized the phorbol ester and phospholipid binding properties of a glutathione S-transferase-tagged full-length PKD and compared them with those of PKC-alpha and -delta. We found that PKD is a high-affinity phorbol ester receptor for a range of structurally and functionally divergent phorbol esters and analogs and showed both similarities and differences in structure-activity relations compared with the PKCs examined. In particular, PKD had lower affinity than PKC for certain diacylglycerol analogs, which might be caused by a lysine residue at the 22 position of the PKD-C1b domain in place of the tryptophan residue at this position conserved in the PKCs. The membrane-targeting domains in PKD are largely different from those in PKC; among these differences, PKD contains a pleckstrin homology (PH) domain that is absent in PKC. However, phosphatidylinositol-4,5-bisphosphate PIP2, a lipid ligand for some PH domains, reconstitutes phorbol 12,13-dibutyrate (PDBu) binding to PKD similarly as it does to PKC-alpha and -delta, implying that the PH domain in PKD may not preferentially interact with PIP2. Overall, the requirement of anionic phospholipids for the reconstitution of [3H]PDBu binding to PKD was intermediate between those of PKC-alpha and -delta. We conclude that PKD is a high-affinity phorbol ester receptor; its lipid requirements for ligand binding are approximately comparable with those of PKC but may be differentially regulated in cells through the binding of diacylglycerol to the C1 domain.

Animals↗

PubMed2002

Mechanism of activation of protein kinase D2(PKD2) by the CCK(B)/gastrin receptor.

Recently, we cloned a novel serine/threonine kinase termed protein kinase D2 (PKD2). PKD2 can be activated by phorbol esters both in vivo and in vitro but also by gastrin via the cholecystokinin/CCK(B) receptor in human gastric cancer cells stably transfected with the CCK(B)/gastrin receptor (AGS-B cells). Here we identify the mechanisms of gastrin-induced PKD2 activation in AGS-B cells. PKD2 phosphorylation in response to gastrin was rapid, reaching a maximum after 10 min of incubation. Our data demonstrate that gastrin-stimulated PKD2 activation involves a heterotrimeric G alpha(q) protein as well as the activation of phospholipase C. Furthermore, we show that PKD2 can be activated by classical and novel members of the protein kinase C (PKC) family such as PKC alpha, PKC epsilon, and PKC eta. These PKCs are activated by gastrin in AGS-B cells. Thus, PKD2 is likely to be a novel downstream target of specific PKCs upon the stimulation of AGS-B cells with gastrin. Our data suggest a two-step mechanism of activation of PKD2 via endogenously produced diacylglycerol and the activation of PKCs.

Cell Line↗

PubMed2002

Protein kinase D: an intracellular traffic regulator on the move.

Recent research has identified protein kinase D (PKD, also called PKCmu) as a serine/threonine kinase with potentially important roles in growth factor signaling as well as in stress-induced signaling. Moreover, PKD has emerged as an important regulator of plasma membrane enzymes and receptors, in some cases mediating cross-talk between different signaling systems. The recent discovery of two additional kinases belonging to the PKD family and the plethora of proteins that interact with PKD point to a multifaceted regulation and a multifunctional role for these enzymes, with functions in processes as diverse as cell proliferation, apoptosis, immune cell regulation, tumor cell invasion and regulation of Golgi vesicle fission.

Animals↗

PubMed2002

Getting to know protein kinase D.

The protein kinase D (PKD) enzymes represent a new family of second messenger stimulated kinases, with diacylglycerol as a prime, but not the sole, mediator of activation. Their molecular architecture features a catalytic domain, unrelated to that of all PKC family members, and a large inhibitory, regulatory domain, comprised of two Zinc fingers, and a pleckstrin homology domain. These different sub-domains play distinctive roles in the activation, translocation and biological functions of the kinase. The enzymes have been implicated in signalling mechanisms controlling cell proliferation and programmed cell death and in metastasis, immune responses, and Golgi restructuring and function. A variety of proteins specifically interact with the different sub-domains of the enzymes and direct their wide range of cellular functions.

Amino Acid Sequence↗