Diacylglycerol-dependent Binding Recruits PKCθ and RasGRP1 C1 Domains to Specific Subcellular Localizations in Living T Lymphocytes

Section: Resultssupporting

confidence: 88%

Section: Discussionmentioning

confidence: 99%

TCR-Induced Activation of Ras Proceeds at the Plasma Membrane and Requires Palmitoylation of N-Ras

Rubio¹,

Grund²,

Song³

et al. 2010

“…Hypotonicity induced a small, but inconsistent change in DAG levels, and following BCR engagement little, if any, measurable steady state increase (data not shown). These results suggested that either the rate of DAG metabolism is increased by the BCR and mechanical stimuli in proportion to any increase in production rate and/or that cellular localization of DAG, or enzymes that metabolize DAG, is more critical to its action than changes in total cellular steady state concentration, as demonstrated previously in T cells (10). Because we were unable to detect consistent changes in DAG concentration, we used an alternative strategy to assess its production.…”

Section: Mechanism Of Hypotonicity-induced Ca 2ϩ Mobilization In B Cellsmentioning

confidence: 73%

Mechanisms of Hypotonicity-Induced Calcium Signaling and Integrin Activation by Arachidonic Acid-Derived Inflammatory Mediators in B Cells

Zhu

Liu

LaBelle

et al. 2005

We previously characterized the initial steps in the activation of novel (calcium-permeant) nonselective cation channels (NSCCs) and calcium release-activated calcium channels in primary murine B lymphocytes. Phospholipase C products, namely diacylglycerol and d-myo-inositol 1,4,5-trisphosphate, were identified as proximal intracellular agonists of these respective channels following mechanical stimulation of B cells. However, neither the distal steps in NSCC activation nor the contribution of these channels to sustained mechanical signaling were defined in these previous studies. In this study, single cell measurements of intracellular Ca2+ were used to define the mechanisms of NSCC activation and demonstrate a requirement for arachidonic acid liberated from diacylglycerol. Several arachidonic acid-derived derivatives were identified that trigger Ca2+ entry into B cells, including the lipoxygenase product 5-hydroperoxyeicosatetranenoic acid and the cytochrome P450 hydroxylase product 20-hydroxyeicosatetraenoic; however, the cytochrome P450 epoxygenase product 5,6-epoxyeicosatrienoic acid is primarily responsible for hypotonicity-induced responses. In addition to regulating calcium entry, our data suggest that eicosanoid-activated NSCCs have a separate and direct role in regulating the avidity of integrins on B cells for extracellular matrix proteins, including ICAM-1 and VCAM-1. Thus, in addition to defining a novel osmotically activated signal transduction pathway in B cells, our results have broad implications for understanding how inflammatory mediators dynamically and rapidly regulate B cell adhesion and trafficking.

“…IP 3 mediates an increase in intracellular Ca 2ϩ ([Ca 2ϩ ] i ) levels and ensures accurate activation of NF-AT-modulated genes (11,12), whereas DAG generation at the membrane regulates localization and activation of several signaling molecules, including protein kinase C (PKC), Ras guanyl nucleotide-releasing protein 1 (Ras-GRP1), and protein kinase D (3,13). DAG-and [Ca 2ϩ ] i -regulated signals are both necessary for an appropriate immune response; in addition, an adequate balance between these two messengers guarantees correct initiation and termination of the T cell activation program.…”

Section: The Src Kinase Family Members P56mentioning

confidence: 99%

Lck-Dependent Tyrosine Phosphorylation of Diacylglycerol Kinase α Regulates Its Membrane Association in T Cells

Merino

Ávila-Flores

Shirai³

et al. 2008

TCR engagement triggers phospholipase Cγ1 activation through the Lck-ZAP70-linker of activated T cell adaptor protein pathway. This leads to generation of diacylglycerol (DAG) and mobilization of intracellular Ca2+, both essential for TCR-dependent transcriptional responses. TCR ligation also elicits transient recruitment of DAG kinase α (DGKα) to the lymphocyte plasma membrane to phosphorylate DAG, facilitating termination of DAG-regulated signals. The precise mechanisms governing dynamic recruitment of DGKα to the membrane have not been fully elucidated, although Ca2+ influx and tyrosine kinase activation were proposed to be required. We show that DGKα is tyrosine phosphorylated, and identify tyrosine 335 (Y335), at the hinge between the atypical C1 domains and the catalytic region, as essential for membrane localization. Generation of an Ab that recognizes phosphorylated Y335 demonstrates Lck-dependent phosphorylation of endogenous DGKα during TCR activation and shows that pY335DGKα is a minor pool located exclusively at the plasma membrane. Our results identify Y335 as a residue critical for DGKα function and suggest a mechanism by which Lck-dependent phosphorylation and Ca2+ elevation regulate DGKα membrane localization. The concerted action of these two signals results in transient, receptor-regulated DGKα relocalization to the site at which it exerts its function as a negative modulator of DAG-dependent signals.