PKC and the control of localized signal dynamics.

Networks of signal transducers determine the conversion of environmental cues into cellular actions. Among the main players in these networks are protein kinases, which can acutely and reversibly modify protein functions to influence cellular events. One group of kinases, the protein kinase C (PKC) family, have been increasingly implicated in the organization of signal propagation, particularly in the spatial distribution of signals. Examples of where and how various PKC isoforms direct this tier of signal organization are becoming more evident.

Binding of dynein intermediate chain 2 to paxillin controls focal adhesion dynamics and migration.

In migrating NRK cells, aPKCs control the dynamics of turnover of paxillin-containing focal adhesions (FA) determining migration rate. Using a proteomic approach (two-dimensional fluorescence difference gel electrophoresis), dynein intermediate chain 2 (dynein IC2) was identified as a protein that is phosphorylated inducibly during cell migration in a PKC-regulated manner. By gene silencing and co-immunoprecipitation studies, we show that dynein IC2 regulates the speed of cell migration through its interaction with paxillin. This interaction is controlled by serine 84 phosphorylation, which lies on the aPKC pathway. The evidence presented thus links aPKC control of migration to the dynein control of FA turnover through paxillin.

Molecular pharmacology in a simple model system: implicating MAP kinase and phosphoinositide signalling in bipolar disorder.

Understanding the mechanisms of drug action has been the primary focus for pharmacological researchers, traditionally using rodent models. However, non-sentient model systems are now increasingly being used as an alternative approach to better understand drug action or targets. One of these model systems, the social amoeba Dictyostelium, enables the rapid ablation or over-expression of genes, and the subsequent use of isogenic cell culture for the analysis of cell signalling pathways in pharmacological research. The model also supports an increasingly important ethical view of research, involving the reduction, replacement and refinement of animals in biomedical research. This review outlines the use of Dictyostelium in understanding the pharmacological action of two commonly used bipolar disorder treatments (valproic acid and lithium). Both of these compounds regulate mitogen activated protein (MAP) kinase and inositol phospholipid-based signalling by unknown means. Analysis of the molecular pathways targeted by these drugs in Dictyostelium and translation of discoveries to animal systems has helped to further understand the molecular mechanisms of these bipolar disorder treatments.

Manipulating signal delivery - plasma-membrane ERK activation in aPKC-dependent migration.

Members of the PKC superfamily have been implicated in various migratory models and in particular in spatially restricted processes. However, defining the precise local events that underlie the PKC-dependent processes is constrained by the unspecific nature of interventions. Here we address this problem in relation to atypical PKC (aPKC) action, which in conjunction with the exocyst complex controls the polarised delivery of promigratory signals. A drug-dependent recruitment approach was employed to manipulate the local recruitment of signals to the leading edge of migrating cells, under conditions where the aPKC-exocyst control is globally abrogated. We found that activation of ERK but not JNK at focal adhesions recovers the majority of the migratory loss attributed to ERK action, demonstrating a necessary role for active plasma membrane ERK in the downstream signalling of aPKC-dependent migration. The data further show that restored focal adhesion dynamics are a contributing mechanism through which localized ERK activity influences this aPKC-exocyst-dependent migration.

An aPKC-exocyst complex controls paxillin phosphorylation and migration through localised JNK1 activation.

Atypical protein kinase C (aPKC) isoforms have been implicated in cell polarisation and migration through association with Cdc42 and Par6. In distinct migratory models, the Exocyst complex has been shown to be involved in secretory events and migration. By RNA interference (RNAi) we show that the polarised delivery of the Exocyst to the leading edge of migrating NRK cells is dependent upon aPKCs. Reciprocally we demonstrate that aPKC localisation at the leading edge is dependent upon the Exocyst. The basis of this inter-dependence derives from two-hybrid, mass spectrometry, and co-immunoprecipitation studies, which demonstrate the existence of an aPKC-Exocyst interaction mediated by Kibra. Using RNAi and small molecule inhibitors, the aPKCs, Kibra, and the Exocyst are shown to be required for NRK cell migration and it is further demonstrated that they are necessary for the localized activation of JNK at the leading edge. The migration associated control of JNK by aPKCs determines JNK phosphorylation of the plasma membrane substrate Paxillin, but not the phosphorylation of the nuclear JNK substrate, c-jun. This plasma membrane localized JNK cascade serves to control the stability of focal adhesion complexes, regulating migration. The study integrates the polarising behaviour of aPKCs with the pro-migratory properties of the Exocyst complex, defining a higher order complex associated with the localised activation of JNK at the leading edge of migrating cells that determines migration rate.

Towards a molecular understanding of human diseases using Dictyostelium discoideum.

The social amoeba Dictyostelium discoideum is increasingly being used as a simple model for the investigation of problems that are relevant to human health. This article focuses on several recent examples of Dictyostelium-based biomedical research, including the analysis of immune-cell disease and chemotaxis, centrosomal abnormalities and lissencephaly, bacterial intracellular pathogenesis, and mechanisms of neuroprotective and anti-cancer drug action. The combination of cellular, genetic and molecular biology techniques that are available in Dictyostelium often makes the analysis of these problems more amenable to study in this system than in mammalian cell culture. Findings that have been made in these areas using Dictyostelium have driven research in mammalian systems and have established Dictyostelium as a powerful model for human-disease analysis.

The neuroprotective agent, valproic acid, regulates the mitogen-activated protein kinase pathway through modulation of protein kinase A signalling in Dictyostelium discoideum.

Activation of the mitogen-activated protein kinase (MAPK) cascade gives rise to a neuroprotective effect in a variety of cell types. The bipolar disorder treatment, valproic acid (VPA), increases the activity of this pathway by modulating extracellular signal-regulated kinase 2 (ERK2) phosphorylation through an unknown mechanism. To investigate the molecular basis of this effect, we have used the biomedical model system Dictyostelium discoideum to dissect this signalling pathway. We find that, similar to mammalian systems, VPA causes a transient increase in the activation of the MAPK signalling pathway, as shown by ERK2 phosphorylation. We show that the MAP kinase and phosphatase, protein kinase A (PKA) and glycogen synthase kinase signalling pathways all function in controlling the levels of phospho-ERK2 (pERK2). We find that VPA induces elevated pERK2 levels through attenuation of the PKA signalling pathway. Interestingly, pERK2 levels are also controlled by another bipolar disorder drug, lithium, providing a common effect of these two drugs. This work therefore suggests a conserved pathway in eukaryotes that is targeted by neuroprotective and bipolar disorder drugs and allows us to propose a model for this neuroprotective effect.

Aberrant stalk development and breakdown of tip dominance in Dictyostelium cell lines with RNAi-silenced expression of calcineurin B.

BACKGROUND: Calcineurin, the Ca2+/calmodulin-dependent protein phosphatase, plays important roles in various cellular processes in lower and higher eukaryotes. Here we analyze the role of calcineurin in the development of Dictyostelium discoideum by RNAi-mediated manipulation of its expression. RESULTS: The cnbA gene of Dictyostelium discoideum which encodes the regulatory B subunit (CNB) of calcineurin was silenced by RNAi. We found a variety of silencing levels of CNB in different recombinant cell lines. Reduction of CNB expression in a given cell line was correlated with developmental aberrations. Cell lines with strongly reduced protein levels developed slower than wild type cells and formed short stalks and spore heads with additional tips. Formation of short stalks results from incomplete vacuolization of prestalk cells during terminal differentiation. Expression of the stalk-specific gene ecmB was reduced in mutant cells. Aberrant stalk development is a cell autonomous defect, whereas the breakdown of tip dominance can be prevented by the presence of as low as 10% wild type cells in chimeras. CONCLUSION: Silencing of calcineurin B in Dictyostelium by expression of RNAi reveals an unexpected link between increased intracellular calcium levels, possibly triggered by the morphogen DIF, activation of calcineurin, and the terminal stage of morphogenesis.

The calcineurin inhibitor gossypol impairs growth, cell signalling and development in Dictyostelium discoideum.

The Dictyostelium genome harbors single copy genes for both the catalytic and regulatory subunits of the Ca2+/calmodulin-dependent protein phosphatase calcineurin. Since molecular genetic approaches to reduce the expression of these genes have failed so far, we attempted to pharmacologically target calcineurin activity in vivo by using the recently described calcineurin inhibitor, gossypol. Up-regulation of expression of the gene for the Ca2+-ATPase PAT1 in conditions of Ca2+ stress was reduced by gossypol. Dictyostelium wild-type cells treated with 12.5-100 microM gossypol showed reduced growth rates and impaired development. In addition, cell signalling was affected. A cell line that overproduces the catalytic subunit of calcineurin was more resistant to gossypol.