Jackie R. Vandenheede

Glycogen synthase kinase‐3 and the Alzheimer‐like state of microtubule‐associated protein tau

The Alzheimer‐like state of tau protein includes phosphorylation by a proline‐directed Ser/Thr kinase present in normal or pathological human brain. Extending earlier results on MAP kinase, we show here that the proline‐directed kinase, GSK3, can induce an Alzheimer‐like immune response involving several distinct and phoshorylatable epitopes at Ser—Pro motifs, as well as gel mobility shift, similar to MAP kinase. Both kinases behave like microtubule‐associated proteins in that they co‐purify through cycles of assembly and disassembly, and both kinases are directly associated with paired helical filaments.

Gβγ-Mediated Regulation of Golgi Organization Is through the Direct Activation of Protein Kinase D

Abstract We have shown previously that the βγ subunits of the heterotrimeric G proteins regulate the organization of the pericentriolarly localized Golgi stacks. In this report, evidence is presented that the downstream target of Gβγ is protein kinase D (PKD), an isoform of protein kinase C. PKD, unlike other members of this class of serine/threonine kinases, contains a pleckstrin homology (PH) domain. Our results demonstrate that Gβγ directly activates PKD by interacting with its PH domain. Inhibition of PKD activity through the use of pharmacological agents, synthetic peptide substrates, and, more specifically, the PH domain of PKD prevents Gβγ-mediated Golgi breakdown. Our findings suggest a possible mechanism by which the direct interaction of Gβγ with PKD regulates the dynamics of Golgi membranes and protein secretion.

Glycogen Synthase Kinase-3β Phosphorylates Protein Tau and Rescues the Axonopathy in the Central Nervous System of Human Four-repeat Tau Transgenic Mice

Protein tau filaments in brain of patients suffering from Alzheimers disease, frontotemporal dementia, and other tauopathies consist of protein tau that is hyperphosphorylated. The responsible kinases operating in vivo in neurons still need to be identified. Here we demonstrate that glycogen synthase kinase-3β (GSK-3β) is an effective kinase for protein tau in cerebral neurons in vivo in adult GSK-3β and GSK-3β × human tau40 transgenic mice. Phosphorylated protein tau migrates slower during electrophoretic separation and is revealed by phosphorylation-dependent anti-tau antibodies in Western blot analysis. In addition, its capacity to bind to re-assembled paclitaxel (Taxol®)-stabilized microtubules is reduced, compared with protein tau isolated from mice not overexpressing GSK-3β. Co-expression of GSK-3β reduces the number of axonal dilations and alleviates the motoric impairment that was typical for single htau40 transgenic animals (Spittaels, K., Van den Haute, C., Van Dorpe, J., Bruynseels, K., Vandezande, K., Laenen, I., Geerts, H., Mercken, M., Sciot, R., Van Lommel, A., Loos, R., and Van Leuven, F. (1999) Am. J. Pathol. 155, 2153–2165). Although more hyperphosphorylated protein tau is available, neither an increase in insoluble protein tau aggregates nor the presence of paired helical filaments or tangles was observed. These findings could have therapeutic implications in the field of neurodegeneration, as discussed.

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.

The activation of the c-Jun N-terminal kinase and p38 mitogen-activated protein kinase signaling pathways protects HeLa cells from apoptosis following photodynamic therapy with hypericin.

In this study, we elucidate signaling pathways induced by photodynamic therapy (PDT) with hypericin. We show that PDT rapidly activates JNK1 while irreversibly inhibiting ERK2 in several cancer cell lines. In HeLa cells, sustained PDT-induced JNK1 and p38 mitogen-activated protein kinase (MAPK) activations overlap the activation of a DEVD-directed caspase activity, poly(ADP-ribose) polymerase (PARP) cleavage, and the onset of apoptosis. The caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (zDEVD-fmk) protect cells against apoptosis and inhibit DEVD-specific caspase activity and PARP cleavage without affecting JNK1 and p38 MAPK activations. Conversely, stable overexpression of CrmA, the serpin-like inhibitor of caspase-1 and caspase-8, has no effect on PDT-induced PARP cleavage, apoptosis, or JNK1/p38 activations. Cell transfection with the dominant negative inhibitors of the c-Jun N-terminal kinase (JNK) pathway, SEK-AL and TAM-67, or pretreatment with the p38 MAPK inhibitor PD169316 enhances PDT-induced apoptosis. A similar increase in PDT-induced apoptosis was observed by expression of the dual specificity phosphatase MKP-1. The simultaneous inhibition of both stress kinases by pretreating cells with PD169316 after transfection with either TAM-67 or SEK-AL produces a more pronounced sensitizing effect. Cell pretreatment with the p38 inhibitor PD169316 causes faster kinetics of DEVD-caspase activation and PARP cleavage and strongly oversensitizes the cells to apoptosis following PDT. These observations indicate that the JNK1 and p38 MAPK pathways play an important role in cellular resistance against PDT-induced apoptosis with hypericin.

On the role of calcium as second messenger in liver for the hormonally induced activation of glycogen phosphorylase

We have studied the mode of action of three hormones (angiotensin, vasopressin and phenylephrine, an alpha-adrenergic agent) which promote liver glycogenolysis in a cyclic AMP-independent way, in comparison with that of glucagon, which is known to act essentially via cyclic AMP. The following observations were made using isolated rat hepatocytes: (a) In the normal Krebs-Henseleit bicarbonate medium, the hormones activated glycogen phosphorylase (EC 2.4.1.1) to about the same degree. In contrast to glucagon, the cyclic AMP-independent hormones did not activate either protein kinase (EC 2.7.1.37) or phosphorylase b kinase (EC 2.7.1.38). (b) The absence of Ca2+ from the incubation medium prevented the activation of glycogen phosphorylase by the cyclic AMP-independent agents and slowed down that induced by glucagon. (c) The ionophore A 23187 produced the same degree of activation of glycogen phosphorylase, provided that Ca2+ was present in the incubation medium. (d) Glucagon, cyclic AMP and three cyclic AMP-dependent hormones caused an enhanced uptake of 45Ca; it was verified that concentrations of angiotensin and of vasopressin known to occur in haemorrhagic conditions were able to produce phosphorylase activation and stimulate 45Ca uptake. (e) Appropriate antagonists (i.e. phentolamine against phenylephrine and an angiotensin analogue against angiotensin) prevented both the enhanced 45Ca uptake and the phosphorylase activation. We interpret our data in favour of a role of calcium (1) as the second messenger in liver for the three cyclic AMP-independent glycogenolytic hormones and (2) as an additional messenger for glucagon which, via cyclic AMP, will make calcium available to the cytoplasm either from extracellular or from intracellular pools. The target enzyme for Ca2+ is most probably phosphorylase b kinase.

Protein kinase D: a family affair.

The protein kinase D family of enzymes consists of three isoforms: PKD1/PKCμ, PKD2 and PKD3/PKCν. 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.

Multiple and cooperative phosphorylation events regulate the CREM activator function.

Phosphorylation is one of the major mechanisms by which the activity of transcription factors can be regulated. We have investigated the role of phosphorylation in the regulation of the transcription factor CREM. We show that the CREM tau activator is phosphorylated on multiple serine and threonine residues in vivo. Stimulation of various signal transduction pathways by forskolin, TPA or Ca2+ ionophore leads to enhanced phosphorylation of serine 117, concomitant with an increase in the transactivation potential of CREM tau. We have identified multiple kinases that can also phosphorylate S117 in vitro. Moreover, we show that casein kinase I and II cooperatively phosphorylate CREM tau on multiple residues, eliciting enhanced DNA binding. Cooperative phosphorylation is also observed with other kinases. These results show that the activity of CREM tau is regulated by multiple phosphorylation events, suggesting that CREM could be considered as a nuclear effector where signalling pathways may converge and/or cross‐talk.

Role of endoplasmic reticulum depletion and multidomain proapoptotic BAX and BAK proteins in shaping cell death after hypericin-mediated photodynamic therapy

Both the commitment event and the modality of cell death in photodynamic therapy (PDT) remain poorly defined. We report that PDT with endoplasmic reticulum (ER)‐associating hypericin leads to an immediate loss of SERCA2 protein levels, causing disruption of Ca2+ homeostasis and cell death. Protection of SERCA2 protein rescues ER‐Ca2+ levels and prevents cell death, suggesting that SERCA2 photodestruction with consequent incapability of the ER to maintain intracellular Ca2+ homeostasis is causal to cell killing. Apoptosis is rapidly initiated after ER‐Ca2+ depletion and strictly requires the BAX/BAK gateway at the mitochondria. Bax−/− Bak−/− double‐knockout (DKO) cells are protected from apoptosis but undergo autophagy‐associated cell death as revealed by electron microscopy and biochemical analysis. Autophagy inhibitors, but not caspase antagonists, significantly reduce death of DKO cells, suggesting that sustained autophagy is lethal. Thus, following ER photodamage and consequent disruption of Ca2+ homeostasis, BAX and BAK proteins model PDT‐mediated cell killing, which is executed through apoptosis in their presence or via an autophagic pathway in their absence.

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 NH2 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.