Protein kinase C molecules regulate both positive and negative signal transduction pathways essential for the initiation and homeostasis of immune responses. There are multiple isoforms of protein kinase C that are activated differently by calcium and diacylglycerol, and these are activated mainly by antigen receptors in T cells, B cells and mast cells. Additionally, mammals express several other diacylglycerol binding proteins that are linked to a network of key signal transduction pathways that control lymphocyte biology. Diacylglycerol and protein kinase C regulate a broad range of gene transcription programs but also modulate integrins, chemokine responses and antigen receptors, thereby regulating lymphocyte adhesion, migration, differentiation and proliferation.

We have taken a knockout approach to interrogate the function of protein kinase D (PKD) serine/threonine kinases in lymphocytes. DT40 B cells express two PKD family members, PKD1 and PKD3, which are both rapidly activated by the B-cell antigen receptor (BCR). DT40 cells with single or dual deletions of PKD1 and/or PKD3 were viable, allowing the role of individual PKD isoforms in BCR signal transduction to be assessed. One proposed downstream target for PKD1 in lymphocytes is the class II histone deacetylases (HDACs). Regulation of chromatin accessibility via class II histone deacetylases is an important mechanism controlling gene expression patterns, but the molecules that control this key process in B cells are not known. Herein, we show that phosphorylation and nuclear export of the class II histone deacetylases HDAC5 and HDAC7 are rapidly induced following ligation of the BCR or after treatment with phorbol esters (a diacylglycerol mimetic). Loss of either PKD1 or PKD3 had no impact on HDAC phosphorylation, but loss of both PKD1 and PKD3 abrogated antigen receptor-induced class II HDAC5/7 phosphorylation and nuclear export. These studies reveal an essential and redundant role for PKD enzymes in controlling class II HDACs in B lymphocytes and suggest that PKD serine kinases are a critical link between the BCR and epigenetic control of chromatin.The protein kinase D (PKD) family comprises three different but closely related serine kinases, PKD1, PKD2, and PKD3, all of which have a highly conserved N-terminal regulatory domain containing two cysteine-rich diacylglycerol (DAG) binding domains and an autoinhibitory pleckstrin homology (PH) domain. PKD enzymes are highly expressed in hematopoietic cells, and they are selectively activated by the engagement of antigen receptors, including the B-cell antigen receptor (BCR), the T-cell receptor, and the FcεR1 in B cells, T cells, and mast cells, respectively (22,24). PKD family members are activated by a signaling pathway involving gamma phospholipase C activation, production of diacylglycerol, and activation of classical/novel PKCs. PKC-mediated phosphorylation of two conserved serine residues in the catalytic domains of PKDs is essential for their activation (13,22,45,48,49). In addition, binding of DAG to the regulatory domain of PKD contributes both to PKD1 activation (50) and to PKD subcellular localization (21,23,(38)(39)(40).PKD enzymes are predicted to play important functions in controlling lymphocyte biology, but most of the evidence that supports this hypothesis is indirect. For example, studies in transgenic mice have shown that constitutively active PKD1 can substitute for the pre-T-cell antigen receptor complex to regulate early thymocyte differentiation and proliferation (20). In other cell types, PKD enzymes have also been implicated in the regulation of Golgi organization and protein trafficking to the cell surface (1,8,12,14,18,53), cell survival (44), 43,44), glucose transport (5), and integrin activation/recycling (29, 51).In addition, it has been ...

SummarySignalling molecules integrate, codify and transport information in cells. Organisation of these molecules in complexes and clusters improves the efficiency, fidelity and robustness of cellular signalling. Here, we summarise current views on how signalling molecules assemble into macromolecular complexes and clusters and how they use their physical properties to transduce environmental information into a variety of cellular processes. In addition, we discuss recent innovations in live-cell imaging at the sub-micrometer scale and the challenges of object (particle) tracking, both of which help us to observe signalling complexes and clusters and to examine their dynamic character.

The serine kinase protein kinase D (PKD) has a cysteine-rich domain (CRD) that binds diacylglycerol (DAG) with high affinity. PKD is cytosolic in unstimulated T cells, but it rapidly polarizes to the immunological synapse in response to antigen/antigen presenting cells (APCs). PKD repositioning is determined by the accumulation of DAG at the immunological synapse and changes in DAG accessibility of the PKD-CRD. Unstimulated T cells are shown to have a uniform distribution of DAG at the plasma membrane, whereas after T cell activation, a gradient of DAG is created with a persistent focus of DAG at the center of the synapse. PKD is only transiently associated with the immune synapse, indicating a fine tuning of PKD responsiveness to DAG by additional regulatory mechanisms. These results reveal the immune synapse as a focal point for DAG and PKD as an immediate and dynamic DAG effector during T cell activation.

Expression of transforming Ha-Ras L61 in NIH3T3 cells causes profound morphological alterations which include a disassembly of actin stress fibers. The Ras-induced dissolution of actin stress fibers is blocked by the specific PKC inhibitor GF109203X at concentrations which inhibit the activity of the atypical aPKC isotypes λ and ζ, whereas lower concentrations of the inhibitor which block conventional and novel PKC isotypes are ineffective. Coexpression of transforming Ha-Ras L61 with kinase-defective, dominant-negative (DN) mutants of aPKC-λ and aPKC-ζ, as well as antisense constructs encoding RNA-directed against isotype-specific 5′ sequences of the corresponding mRNA, abrogates the Ha-Ras–induced reorganization of the actin cytoskeleton. Expression of a kinase-defective, DN mutant of cPKC-α was unable to counteract Ras with regard to the dissolution of actin stress fibers. Transfection of cells with constructs encoding constitutively active (CA) mutants of atypical aPKC-λ and aPKC-ζ lead to a disassembly of stress fibers independent of oncogenic Ha-Ras. Coexpression of (DN) Rac-1 N17 and addition of the phosphatidylinositol 3′-kinase (PI3K) inhibitors wortmannin and LY294002 are in agreement with a tentative model suggesting that, in the signaling pathway from Ha-Ras to the cytoskeleton aPKC-λ acts upstream of PI3K and Rac-1, whereas aPKC-ζ functions downstream of PI3K and Rac-1.This model is supported by studies demonstrating that cotransfection with plasmids encoding L61Ras and either aPKC-λ or aPKC-ζ results in a stimulation of the kinase activity of both enzymes. Furthermore, the Ras-mediated activation of PKC-ζ was abrogated by coexpression of DN Rac-1 N17.