The Alzhcimcr-like state of tau protein includes phosphorylation by a proline-directed Ser/Thr kinasc present in normal or pathological human brain. Extending earlier results on MAP kinase, WC show hcrc that the prolinc-dircctcd kinasc, GSK3, can induct an Alzhcimer-like immune response involving several distinct and phosborylalablc epitopcs at &-Pro motifs, as well as a gel mobility shift, similar 10 MAP kinasc. Both kinases bchavc like microtubule-nssociotcd proteins in that ~hcy co-purify through cycles of assembly and disassembly, and boih kinascs are directly associated with paired helical filaments.

Protein tau filaments in brain of patients suffering from Alzheimer's 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-3␤ (GSK-3␤) is an effective kinase for protein tau in cerebral neurons in vivo in adult GSK-3␤ and GSK-3␤ ؋ 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-3␤. Co-expression of GSK-3␤ 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.

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.

We have shown previously that the betagamma 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 Gbetagamma 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 Gbetagamma 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 Gbetagamma-mediated Golgi breakdown. Our findings suggest a possible mechanism by which the direct interaction of Gbetagamma with PKD regulates the dynamics of Golgi membranes and protein secretion.

Glycogen synthase kinase-3 (GSK3) is inactivated in vitro by p70 S6 kinase or MAP kinase-activated protein kinase-1 beta (MAPKAP kinase-1 beta; also known as Rsk-2). Here we show that GSK3 isoforms are inhibited by 40% within minutes after stimulation of the rat skeletal-muscle cell line L6 with insulin-like growth factor-1 (IGF-1) or insulin. GSK3 was similarly inhibited in rabbit skeletal muscle after an intravenous injection of insulin. Inhibition resulted from increased phosphorylation of GSK3, probably at a serine/threonine residue(s), because it was reversed by incubation with protein phosphatase-2A. Rapamycin blocked the activation of p70 S6 kinase by IGF-1 in L6 cells, but had no effect on the inhibition of GSK3 or the activation of MAPKAP kinase-1 beta. In contrast, wortmannin, a potent inhibitor of PtdIns 3-kinase, prevented the inactivation of GSK3 and the activation of MAPKAP kinase-1 beta and p70 S6 kinase by IGF-1 or insulin. Wortmannin also blocked the activation of p74raf-1. MAP kinase kinase and p42 MAP kinase, but not the formation of GTP-Ras by IGF-1. The results suggest that the stimulation of glycogen synthase by insulin/IGF-1 in skeletal muscle involves the MAP-KAP kinase-1-catalysed inhibition of GSK3, as well as the previously described activation of the glycogen-associated form of protein phosphatase-1.

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The protein kinase D family of enzymes consists of three isoforms: PKD1/PKCW W, PKD2 and PKD3/PKCX X. They all share a similar architecture with regulatory sub-domains that play speci¢c 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. ß

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 (