A rapid increase in the synthesis of lipid-derived second messengers is an important mechanism for transducing extracellular signals across the plasma membrane (1-3). The phospholipase C-mediated hydrolysis of inositol phospholipids is known to produce two second messengers: inositol 1,4,5-trisphosphate, which induces mobilization of calcium from intracellular stores (3), and diacylglycerol (DAG), which activates protein kinase C (PKC)-originally described as a Ca2+-activated, phospholipid-dependent protein kinase (4). The early findings that the potent tumor promoters of the phorbol ester family can substitute for DAG in PKC activation and that the phorbol ester receptor and PKC copurify supported the hypothesis that the cellular target of the phorbol esters is PKC (4). Subsequent studies revealed the diversity of the individual components of the DAG-PKC signal transduction pathway.

When a kinase inactive form of Protein Kinase D (PKD-K618N) was expressed in HeLa cells, it localized to the trans-Golgi network (TGN) and caused extensive tubulation. Cargo that was destined for the plasma membrane was found in PKD-K618N-containing tubes but the tubes did not detach from the TGN. As a result, the transfer of cargo from TGN to the plasma membrane was inhibited. We have also demonstrated the formation and subsequent detachment of cargo-containing tubes from the TGN in cells stably expressing low levels of PKD-K618N. Our results suggest that PKD regulates the fission from the TGN of transport carriers that are en route to the cell surface.

Protein kinase D (PKD) is a serine/threonine protein kinase that is directly stimulated in vitro by phorbol esters and diacylglycerol in the presence of phospholipids. Here, we examine the regulation of PKD in living cells. Our results demonstrate that tumour‐promoting phorbol esters, membrane‐permeant diacylglycerol and serum growth factors rapidly induced PKD activation in immortalized cell lines (e.g. Swiss 3T3 and Rat‐1 cells), in secondary cultures of mouse embryo fibroblasts and in COS‐7 cells transiently transfected with a PKD expression construct. PKD activation was maintained during cell disruption and immunopurification and was associated with an electrophoretic mobility shift and enhanced 32P incorporation into the enzyme, but was reversed by treatment with alkaline phosphatase. PKD was activated, deactivated and reactivated in response to consecutive cycles of addition and removal of PDB. PKD activation was completely abrogated by exposure of the cells to the protein kinase C inhibitors GF I and Ro 31–8220. In contrast, these compounds did not inhibit PKD activity when added directly in vitro. Co‐transfection of PKD with constitutively activated mutants of PKCs showed that PKCepsilon and eta but not PKCzeta strongly induced PKD activation in COS‐7 cells. Thus, our results indicate that PKD is activated in living cells through a PKC‐dependent signal transduction pathway.

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.

Activation of phosphatidylinositide 3-OH kinase (PI 3-kinase) is implicated in mediating a variety of growth factor-induced responses, among which are the inactivation of glycogen synthase kinase-3 (GSK-3) and the activation of the serine/threonine protein kinase B (PKB). GSK-3 inactivation occurs through phosphorylation of Ser-9, and several kinases, such as protein kinase C, mitogen-activated protein kinase-activated protein kinase-1 (p90 Rsk ), p70 S6kinase , and also PKB have been shown to phosphorylate this site in vitro. In the light of the many candidates to mediate insulin-induced GSK-3 inactivation we have investigated the role of PKB by constructing a PKB mutant that exhibits dominant-negative function (inhibition of growth factor-induced activation of PKB at expression levels similar to wild-type PKB), as currently no such mutant has been reported. We observed that the PKB mutant (PKB-CAAX) acts as an efficient inhibitor of PKB activation and also of insulin-induced GSK-3 regulation. Furthermore, it is shown that PKB and GSK-3 co-immunoprecipitate, indicating a direct interaction between GSK-3 and PKB. An additional functional consequence of this interaction is implicated by the observation that the oncogenic form of PKB, gagPKB induces a cellular relocalization of GSK-3 from the cytosolic to the membrane fraction. Our results demonstrate that PKB activation is both necessary and sufficient for insulin-induced GSK-3 inactivation and establish a linear pathway from insulin receptor to GSK-3. Regulation of GSK-3 by PKB is likely through direct interaction, as both proteins co-immunoprecipitate. This interaction also resulted in a translocation of GSK-3 to the membrane in cells expressing transforming gagPKB.Activation of membrane-bound receptors of both the serpentine and tyrosine kinase classes often results in the activation of the lipid kinase PI 1 3-kinase, of which several different isoforms have been described (1). Activation of PI 3-kinase results in the formation of 3Ј-phosphorylated phosphatidylinositol (PI-3P) lipids (i.e. PI 3,4-P 2 and PI 3,4,5-P 3 ). These lipids were long suspected to perform a second messenger function. However, until recently the nature of the PI-3P binding proteins and their role in cellular signaling remained unsolved.

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.

A novel protein kinase (named PKD) with an NH2-terminal region containing two cysteine-rich motifs has been expressed in COS-7 cells and identified as a receptor for phorbol esters. COS-7 cells transfected with a PKD cDNA construct (pcDNA3-PKD) exhibit a marked (4.8-fold) increase in [3H]phorbol 12,13-dibutyrate binding. An antiserum raised against the COOH-terminal 15 amino acids of PKD specifically recognized a single 110-kDa band in PKD-transfected cells. PKD prepared by elution from immunoprecipitates with the immunizing peptide efficiently phosphorylated the synthetic peptide syntide-2. The enzyme only poorly phosphorylated a variant syntide-2 where arginine 4 has been replaced by an alanine. The addition of [3H]phorbol 12,13-dibutyrate, 1-oleoyl-2-acetylglycerol, or 1,2-dioctanoyl-sn-glycerol in the presence of dioleoylphosphatidylserine stimulated the syntide-2 kinase activity of PKD in a synergistic fashion (4-6-fold). Furthermore, the autophosphorylation of PKD was strikingly stimulated by the same lipid activators (14-24-fold). Similar properties were found with PKD isolated from mouse lung. The substrate specificity of PKD is different from that of previously identified members of the protein kinase C family since it does not efficiently phosphorylate histone III-S, protamine sulfate, or a synthetic peptide based upon the conserved pseudosubstrate region of the protein kinase C family. Taken together, these data unambiguously establish PKD as a phorbol ester receptor and as a novel phospholipid/diacylglycerol-stimulated protein kinase.