Diacylglycerol kinase ζ controls diacylglycerol metabolism at the immunological synapse

Diacylglycerol (DAG) generation at the T cell immunological synapse (IS) determines the correct activation of antigen-specific immune responses. DAG kinases (DGKs) α and ζ act as negative regulators of DAG-mediated signals by catalyzing DAG conversion to phosphatidic acid (PA). Nonetheless, the specific input of each enzyme and their spatial regulation during IS formation remain uncharacterized. Here we report recruitment of endogenous DGKα and DGKζ to the T cell receptor (TCR) complex following TCR/CD28 engagement. Specific DGK gene silencing shows that PA production at the activated complex depends mainly on DGKζ, indicating functional differences between these proteins. DGKζ kinase activity at the TCR is enhanced by phorbol-12-myristate-13-acetate cotreatment, suggesting DAG-mediated regulation of DGKζ responsiveness. We used GFP-DGKζ and -DGKα chimeras to assess translocation dynamics during IS formation. Only GFP-DGKζ translocated rapidly to the plasma membrane at early stages of IS formation, independent of enzyme activity. Finally, use of a fluorescent DAG sensor confirmed rapid, sustained DAG accumulation at the IS and allowed us to directly correlate membrane translocation of active DGKζ with DAG consumption at the IS. This study highlights a DGKζ-specific function for local DAG metabolism at the IS and offers new clues to its mode of regulation.     

Diacylglycerol kinase ζ generates dipalmitoyl-phosphatidic acid species during neuroblastoma cell differentiation

Phosphatidic acid (PA) is one of the phospholipids composing the plasma membrane and acts as a second messenger to regulate a wide variety of important cellular events, including mitogenesis, migration and differentiation. PA consists of various molecular species with different acyl chains at the sn-1 and sn-2 positions. However, it has been poorly understood what PA molecular species are produced during such cellular events. Here we identified the PA molecular species generated during retinoic acid (RA)-induced neuroblastoma cell differentiation using a newly established liquid chromatography/mass spectrometry (LC/MS) method. Intriguingly, the amount of 32:0-PA species was dramatically and transiently increased in Neuro-2a neuroblastoma cells 24–48 h after RA-treatment. In addition, 30:0- and 34:0-PA species were also moderately increased. Moreover, similar results were obtained when Neuro-2a cells were differentiated for 24 h by serum starvation. MS/MS analysis revealed that 32:0-PA species contains two palmitic acids (16:0 s). RT-PCR analysis showed that diacylglycerol kinase (DGK) δ and DGKζ were highly expressed in Neuro-2a cells. The silencing of DGKζ expression significantly decreased the production of 32:0-PA species, whereas DGKδ-siRNA did not. Moreover, neurite outgrowth was also markedly attenuated by the deficiency of DGKζ. Taken together, these results indicate that DGKζ exclusively generates very restricted PA species, 16:0/16:0-PA, and up-regulates neurite outgrowth during the initial/early stage of neuroblastoma cell differentiation.     

Diacylglycerol activation of protein kinase Cε and hepatic insulin resistance

Nonalcoholic fatty liver disease (NAFLD) is now the most frequent chronic liver disease in Western societies, affecting one in four adults in the USA, and is strongly associated with hepatic insulin resistance, a major risk factor in the pathogenesis of type 2 diabetes. Although the cellular mechanisms underlying this relationship are unknown, hepatic accumulation of diacylglycerol (DAG) in both animals and humans has been linked to hepatic insulin resistance. In this Perspective, we discuss the role of DAG activation of protein kinase Cε as the mechanism responsible for NAFLD-associated hepatic insulin resistance seen in obesity, type 2 diabetes, and lipodystrophy.     

Diacylglycerol kinase: a key modulator of signal transduction?

Diacylglycerol kinase (DGK) plays a central role in the metabolism of diacylglycerol released as a second messenger in agonist-stimulated cells. The major purified form of the enzyme (80 kDa DGK) is highly abundant in lymphocyte cytosol and may become membrane-associated via phosphorylation by protein kinase C. In addition, there are several kinase subspecies immunologically distinct from the 80 kDa enzyme, which differ markedly in their responses to several compounds such as sphingosine and R59022. Thus, further work on each enzyme species is needed to define the function of DGK in stimulated cells.     

Caspase-mediated activation of PAK2 during apoptosis: proteolytic kinase activation as a general mechanism of apoptotic signal transduction?

p21-activated kinase 2 (PAK2) is proteolytically cleaved during apoptosis through the action of DEVD-sensitive caspase(s). This cleavage event causes PAK2 activation, and PAK2 activity is implicated in regulation of the biochemistry and morphology of the apoptotic cell. PAK2 is just one example of a number of identified caspase targets that are protein kinases involved in regulating various aspects of cell function. We hypothesize that this may reflect their important role in regulating the controlled and orderly demise of the dying cell.     

Diacylglycerol kinase γ is a novel anionic phospholipid binding protein with a selective binding preference

There are ten isozymes of diacylglycerol kinase (DGK), and they regulate diverse patho-physiological functions. Here, we investigated the lipid-binding properties of DGK isozymes using protein–lipid overlay and liposome-binding assays. DGKγ showed a strong binding activity compared with other DGK isozymes for phosphatidic acid (PA) among the various glycerophospholipids tested. However, DGKγ failed to interact with DG and lyso-PA. Moreover, the isozyme was capable of binding to ceramide-1-phosphate but not to ceramide or sphingosine-1-phosphate. The isozyme bound more strongly to PA containing unsaturated fatty acid than to PA having only saturated fatty acid. An analysis using a series of deletion mutants of DGKγ revealed that the N-terminal region, which contains a recoverin homology domain and EF-hand motifs, is responsible for the PA binding activity of DGKγ. Taken together, these results indicate that DGKγ is an anionic phospholipid binding protein that preferably interacts with a small highly charged head group that is very close to the glycerol or sphingosine backbone.     

Through scaffolding and catalytic actions nucleoside diphosphate kinase B differentially regulates basal and β-adrenoceptor-stimulated cAMP synthesis

β-adrenoceptors (βAR) play a central role in the regulation of cAMP synthesis and cardiac contractility. Nucleoside diphosphate kinase B (NDPK B) regulates cAMP signalling by complex formation with Gβγ dimers thereby activating and stabilizing heterotrimeric G_s proteins, key transducer of βAR signals into the cell. Here, we explored the requirement of NDPK B for basal and βAR-stimulated cAMP synthesis and analysed the underlying mechanisms by comparing wild-type NDPK B (WT) and its catalytically inactive H118N mutant. Stable overexpression of both WT- and H118N-NDPK B in cardiomyocyte derived H10 cells increased the plasma membrane content of G_s and caveolin-1 and thus enhanced the isoproterenol (ISO)-stimulated cAMP-synthesis by about 2-fold. Conversely, the loss of NDPK B in embryonic fibroblasts from NDPK A/B-depleted mice was associated with a severe reduction in membranous G_s protein and carveolin-1 content causing a marked decrease in basal and ISO-induced cAMP formation. Re-expression of NDPK B, but not of NDPK A, was able to rescue this phenotype. Both, re-expression of WT- and H118N-NDPK B induced the re-appearance of G_s and caveolin-1 at the plasma membrane to a similar extent. Accordingly, WT- and H118N-NDPK B similarly enhanced ISO-induced cAMP formation. In contrast, the catalytically inactive H118N-NDPK B was less potent and less effective in rescuing basal cAMP production. Identical results were obtained in neonatal rat cardiac myocytes after siRNA-induced knockdown and adenoviral re-expression of NDPK B.Our data reveal that NDPK B regulates G_s function by two different mechanisms. The complex formation of NDPK B with G_s is required for the stabilization of the G protein content at the plasma membrane. In addition, the NDPK B-dependent phosphotransfer reaction, which requires the catalytic activity, specifically allows a receptor-independent, basal G_s activation.     

Cyclin B–Cdk1 inhibits protein phosphatase PP2A-B55 via a Greatwall kinase–independent mechanism

Entry into M phase is governed by cyclin B–Cdk1, which undergoes both an initial activation and subsequent autoregulatory activation. A key part of the autoregulatory activation is the cyclin B–Cdk1–dependent inhibition of the protein phosphatase 2A (PP2A)–B55, which antagonizes cyclin B–Cdk1. Greatwall kinase (Gwl) is believed to be essential for the autoregulatory activation because Gwl is activated downstream of cyclin B–Cdk1 to phosphorylate and activate α-endosulfine (Ensa)/Arpp19, an inhibitor of PP2A-B55. However, cyclin B–Cdk1 becomes fully activated in some conditions lacking Gwl, yet how this is accomplished remains unclear. We show here that cyclin B–Cdk1 can directly phosphorylate Arpp19 on a different conserved site, resulting in inhibition of PP2A-B55. Importantly, this novel bypass is sufficient for cyclin B–Cdk1 autoregulatory activation. Gwl-dependent phosphorylation of Arpp19 is nonetheless necessary for downstream mitotic progression because chromosomes fail to segregate properly in the absence of Gwl. Such a biphasic regulation of Arpp19 results in different levels of PP2A-B55 inhibition and hence might govern its different cellular roles.     

Activation segment exchange: a common mechanism of kinase autophosphorylation?

The crystal structure of the kinase domain from human checkpoint kinase 2 (Chk2) has shown, for the first time, the reciprocal exchange of activation segments between two adjacent molecules and provides the molecular basis for understanding the observed mode of Chk2 kinase activation via trans-autophosphorylation. With further examples of activation segment exchanged kinase domains now publicly available (i.e. Ste20-like kinase, Ser/Thr kinase 10 and Death-associated protein kinase 3), we suggest that this phenomenon represents a common mechanism of activation amongst a particular subset of protein kinases, that is, those that are dimeric (either transiently or constitutively), that undergo activation by autophosphorylation and that have activation segment amino acid sequences that do not resemble those of their substrate consensus sequence.     

The β1-adrenergic receptor mediates extracellular signal-regulated kinase activation via Gαs

β-Adrenergic receptors can activate extracellular signal-regulated kinases (ERKs) via different mechanisms. In this study, we investigated the molecular mechanism of β1-adrenergic receptor (β1AR)-mediated ERK activation in African green monkey kidney COS-7 cells. Treatment of cells with isoproterenol (ISO), a β1AR selective agonist, induced phosphorylation of ERK1/2 in a dose-dependent manner. ISO-stimulated ERK phosphorylation was not influenced by the Gβγ inhibitor, βAR kinase carboxyl terminal (βARKct) or by the Gi inhibitor, pertussis toxin (PTX), but it was clearly abolished via inhibition of protein kinase A (PKA) with H89, or of mitogen-activated protein kinase kinase (MEK1) with PD98059, revealing that the Gαs subunit is involved in ERK regulation through the PKA/MEK1 pathway. We also tested the effect of the adenylate cyclase activator forskolin on ERK activation, and the result was identical to that of ISO stimulation. Moreover, pretreatment with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 or with the Src tyrosine kinase inhibitor PP2 did not affect ERK activation. These observations suggest a mechanism of β1AR-mediated ERK activity that involves the Gαs subunit, but not EGFR or Src tyrosine kinase.