Catherine J. Pears

Isoform specificity of activators and inhibitors of protein kinase C γ and δ

Expression of certain mammalian protein kinase C (PKC) isoforms inhibits the proliferation of Schizosaccharomyces pombe (Goode et al., Mol. Biol. Cell 5 (1994) 907–920). We have taken advantage of this fact to determine the in vivo isoform preference of a number of PKC inhibitors, using a microtitre plate assay which allows rapid screening. This in vivo model has revealed previously unreported preferences; calphostin C is a more efficient inhibitor of the novel PKCδ than chelerythrine chloride whereas the efficiencies are reversed for inhibition of the classical PKCγ. We have also shown that the anti‐leukaemic agent bryostatin 1 inhibits or activates in vivo in an isoform‐specific manner.

Identification of a DNA sequence element required for efficient expression of a developmentally regulated and cAMP-inducible gene of Dictyostelium discoideum.

The cysteine proteinase 2 gene of Dictyostelium encodes a developmentally regulated sulphydryl proteinase which is first expressed late during cellular aggregation. The mRNA is slightly enriched in pre‐stalk over pre‐spore cells but we show here that it is expressed at a somewhat higher level in mature spore cells than in stalk cells. The mRNA is induced to accumulate precociously in response to exogenous cAMP and we show that a fragment of DNA containing 921 nucleotides upstream of the major start site of transcription directs regulated expression of the gene. Approximately 200 nucleotides upstream of the cap site there are two, adjacent, homologous G‐rich regions of 9 nucleotides in length. We have constructed small internal deletions and point mutations which affect the distal element. We find a major reduction in regulated transcription indicating this element to be important in mediating cAMP induction of gene expression.

PARP regulates nonhomologous end joining through retention of Ku at double-strand breaks

Poly ADP-ribosylation polymerases are necessary for recruitment and/or retention of Ku at double-strand breaks during nonhomologous end-joining DNA repair.

DNA-PKcs-Dependent Signaling of DNA Damage in Dictyostelium discoideum

DNA double-strand breaks (DSBs) can be repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). In vertebrates, the first step in NHEJ is recruitment of the DNA-dependent protein kinase (DNA-PK) to DNA termini. DNA-PK consists of a catalytic subunit (DNA-PKcs) that is recruited to DNA ends by the Ku70/Ku80 heterodimer. Although Ku has been identified in a wide variety of organisms, to date DNA-PKcs has only been identified experimentally in vertebrates. Here, we report the identification of DNA-PK in the nonvertebrate Dictyostelium. Dictyostelium Ku80 contains a conserved domain previously implicated in recruiting DNA-PKcs to DNA and consistent with this observation, we have identified DNA-PKcs in the Dictyostelium genome. Disruption of the gene encoding Dictyostelium DNA-PKcs results in sensitivity to DNA DSBs and defective H2AX phosphorylation in response to this form of DNA damage. However, these phenotypes are only apparent when DNA damage is administered in G(1) phase of the cell cycle. These data illustrate a cell cycle-dependent requirement for Dictyostelium DNA-PK in signaling and combating DNA DSBs and represent the first experimental verification of DNA-PKcs in a nonvertebrate organism.

Differential Roles of the PKC Novel Isoforms, PKCδ and PKCε, in Mouse and Human Platelets

Background Increasing evidence suggests that individual isoforms of protein kinase C (PKC) play distinct roles in regulating platelet activation. Methodology/Principal Findings In this study, we focus on the role of two novel PKC isoforms, PKCδ and PKCε, in both mouse and human platelets. PKCδ is robustly expressed in human platelets and undergoes transient tyrosine phosphorylation upon stimulation by thrombin or the collagen receptor, GPVI, which becomes sustained in the presence of the pan-PKC inhibitor, Ro 31-8220. In mouse platelets, however, PKCδ undergoes sustained tyrosine phosphorylation upon activation. In contrast the related isoform, PKCε, is expressed at high levels in mouse but not human platelets. There is a marked inhibition in aggregation and dense granule secretion to low concentrations of GPVI agonists in mouse platelets lacking PKCε in contrast to a minor inhibition in response to G protein-coupled receptor agonists. This reduction is mediated by inhibition of tyrosine phosphorylation of the FcRγ-chain and downstream proteins, an effect also observed in wild-type mouse platelets in the presence of a PKC inhibitor. Conclusions These results demonstrate a reciprocal relationship in levels of the novel PKC isoforms δ and ε in human and mouse platelets and a selective role for PKCε in signalling through GPVI.

The role of ADP-ribosylation in regulating DNA double-strand break repair

ADP-ribosylation is the post translational modification of proteins catalysed by ADP-ribosyltransferases (ARTs). ADP-ribosylation has been implicated in a wide variety of cellular processes including cell growth and differentiation, apoptosis and transcriptional regulation. Perhaps the best characterised role, however, is in DNA repair and genome stability where ADP-ribosylation promotes resolution of DNA single strand breaks. Although ADP-ribosylation also occurs at DNA double strand breaks (DSBs), which ARTs catalyse this reaction and the molecular basis of how this modification regulates their repair remains a matter of debate. Here we review recent advances in our understanding of how ADP-ribosylation regulates DSB repair. Specifically, we highlight studies using the genetic model organism Dictyostelium, in addition to vertebrate cells that identify a third ART that accelerates DSB repair by non-homologous end-joining through promoting the interaction of repair factors with DNA lesions. The implications of these data with regards to how ADP-ribosylation regulates DNA repair and genome stability are discussed.

Down‐regulation of a kinase defective PKC‐α

A kinase defective mutant of PKC‐α down‐regulates in response to phorboI esters as effectively as the wild‐type protein when introduced into COS‐1 cells. This demonstrates that intramolecular autophosphorylation is not a prerequisite for down‐regulation.

Distinct but critical roles for integrin αIIbβ3 in platelet lamellipodia formation on fibrinogen, collagen‐related peptide and thrombin

Integrins are the major receptor type known to facilitate cell adhesion and lamellipodia formation on extracellular matrix proteins. However, collagen‐related peptide and thrombin have recently been shown to mediate platelet lamellipodia formation when presented as immobilized surfaces. The aims of this study were to establish if there exists a role for the platelet integrin αIIbβ3 in this response; and if so, whether signalling from the integrin is required for lamellipodia formation on these surfaces. Real‐time analysis was used to compare platelet morphological changes on surfaces of fibrinogen, collagen‐related peptide or thrombin in the presence of various pharmacological inhibitors and platelets from ‘knockout’ mice. We demonstrate that collagen‐related peptide and thrombin stimulate distinct patterns of platelet lamellipodia formation and elevation of intracellular Ca2+ to that induced by the integrin αIIbβ3 ligand, fibrinogen. Nevertheless, lamellipodia formation on collagen‐related peptide and thrombin is dependent upon engagement of αIIbβ3, consistent with release of αIIbβ3 ligand(s) from platelet granules. However, the requirement for signalling by the integrin on fibrinogen can be bypassed by the addition of thrombin to the solution. These observations reveal a critical role for αIIbβ3 in forming lamellipodia on collagen‐related peptide and thrombin which is dependent on its ability to function as an adhesive receptor but not necessarily on its ability to signal. These results suggest that integrins may play an important role in lamellipodia formation triggered by nonintegrin ligands in platelets and possibly in other cell types.

Proteomic and Microarray Analyses of the Dictyostelium Zak1-GSK-3 Signaling Pathway Reveal a Role in Early Development

ABSTRACT GskA, the Dictyostelium GSK-3 orthologue, is modified and activated by the dual-specificity tyrosine kinase Zak1, and the two kinases form part of a signaling pathway that responds to extracellular cyclic AMP. We identify potential cellular effectors for the two kinases by analyzing the corresponding null mutants. There are proteins and mRNAs that are altered in abundance in only one or the other of the two mutants, indicating that each kinase has some unique functions. However, proteomic and microarray analyses identified a number of proteins and genes, respectively, that are similarly misregulated in both mutant strains. The positive correlation between the array data and the proteomic data is consistent with the Zak1-GskA signaling pathways functioning by directly or indirectly regulating gene expression. The discoidin 1 genes are positively regulated by the pathway, while the abundance of the H5 protein is negatively regulated. Two of the targets, H5 and discoidin 1, are well-characterized markers for early development, indicating that the Zak1-GskA pathway plays a role in development earlier than previously observed.

Submaximal inhibition of protein kinase C restores ADP-induced dense granule secretion in platelets in the presence of Ca2+

Protein kinase C (PKC) is a family of serine/threonine kinases that play isoform-specific inhibitory and stimulatory roles in platelet activation. We show here that the pan-PKC inhibitor Ro31-8220 can be used to dissect these events following platelet activation by ADP. Submaximal concentrations of Ro31-8220 potentiated aggregation and dense granule secretion to ADP in plasma anticoagulated with citrate, in d-Phe-Pro-Arg-chloromethyl ketone-anticoagulated plasma, which has physiological levels of Ca2+, and in washed platelets. Potentiation was retained on inhibition of cyclooxygenase and was associated with an increase in intracellular Ca2+. Potentiation of aggregation and secretion was abolished by a maximally effective concentration of Ro31-8220, consistent with a critical role of PKC in secretion. ADP-induced secretion was potentiated in the presence of an inhibitor of PKCβ but not in the presence of available inhibitors of other PKC isoforms in human and mouse platelets. ADP-induced secretion was also potentiated in mouse platelets deficient in PKCϵ but not PKCθ. These results demonstrate that partial blockade of PKC potentiates aggregation and dense granule secretion by ADP in association with increased Ca2+. This provides a molecular explanation for the inability of ADP to induce secretion in plasma in the presence of physiological Ca2+ concentrations, and it reveals a novel role for PKC in inhibiting platelet activation by ADP in vivo. These results also demonstrate isoform-specific inhibitory effects of PKC in platelets.