Carolyn Keenan

Protein Kinase C and the Cytoskeleton

The protein kinase C family of serine-threonine kinases are important signal transducers participating in many different agonist-induced signalling cascades. PKC is activated by increases in diacylglycerol produced in response to agonist-induced hydrolysis of inositol phospholipids. PKC is thought to reside in the cytosol in an inactive conformation and translocate to the plasma membrane upon cell activation where it modifies various cellular functions through phosphorylation of target substrates. Increasing evidence has illustrated that this family of enzymes is capable of translocating to other subcellular sites than the plasma membrane. A key to understanding the functions of the members of this family is identifying their physiological substrates and their relationship with those target substrates. The idea that PKC may be an important regulator of cytoskeletal function has been suggested by numerous studies. Activation of PKC in a variety of different cell types leads to changes in the cell cytoskeleton including lymphocyte surface receptor capping, smooth muscle contraction and actin rearrangement in T cells and neutrophils. Given the ubiquitous expression of PKC and the diversity of cytoskeletons in different cell types it is not surprising that PKC has been shown to be associated with and/or phosphorylate a wide range of cytoskeletal components. This review examines the interaction of PKC with the cytoskeleton and discusses some of the cytoskeletal functions ascribed to PKC to date.

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.

Protein kinase C isotypes theta, delta and eta in human lymphocytes: differential responses to signalling through the T-cell receptor and phorbol esters

The repertoire of novel and atypical protein kinase C (PKC) isotypes present in human T cells and their subcellular localization have not been fully characterized. We detected calcium‐independent PKC activity in whole cell fractions from unstimulated peripheral blood lymphocytes (PBL). Towards an understanding of the role of PKC isoforms in lymphocyte activation we have studied the expression of calcium‐independent PKC isoforms θ, δ and η in PBL. With isoform‐specific antibodies we detected the presence of PKC θ and δ in whole cell fractions from unstimulated human PBL by Western blot analysis. In addition, immunocytochemical analysis confirmed the presence of the novel PKC isoform PKC η in PBL. Using immunocytochemistry, PKC θ, δ and η had distinct patterns of redistribution following activation by phorbol myristate acetate (PMA). However, signalling through the T‐cell receptor (TCR) did not appear to induce such changes in isoenzyme redistribution. These findings indicate that activation of lymphocytes either through the TCR–CD3 complex or with PMA induces different signalling pathways with respect to calcium‐independent isoenzymes. Signalling through receptors other than the CD3 complex may be involved in activation of these isotypes.