Diacylglycerol kinases: at the hub of cell signalling

The AP-2 clathrin adaptor differs fundamentally from the related AP-1, AP-3, and AP-4 sorting complexes because membrane deposition does not depend directly on an Arf family GTPase. Instead phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) appears to act as the principal compartmental cue for AP-2 placement at the plasma membrane as well as for the docking of numerous other important clathrin coat components at the nascent bud site. This PtdIns(4,5)P 2 dependence makes type I phosphatidylinositol 4-phosphate 5-kinases (PIPKIs) lynchpin enzymes in the assembly of clathrin-coated structures at the cell surface. PIPKI␥ is the chief 5-kinase at nerve terminals, and here we show that the 26-amino acid, alternatively spliced C terminus of PIPKI␥661 is an intrinsically unstructured polypeptide that binds directly to the sandwich subdomain of the AP-2 ␤2 subunit appendage. An aromatic side chain-based, extended interaction motif that also includes the two bulky C-terminal residues of the short PIPKI␥635 variant is necessary for ␤2 appendage engagement. The clathrin heavy chain accesses the same contact surface on the AP-2 ␤2 appendage, but because of additional clathrin binding sites located within the unstructured hinge segment of the ␤2 subunit, clathrin binds the ␤2 chain with a higher apparent affinity than PIPKI␥661. A clathrin-regulated interaction with AP-2 could allow PIPKI␥661 to be strategically positioned for regional PtdIns(4,5)P 2 generation during clathrin-coated vesicle assembly at the synapse.The key regulatory activity of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) 2 during clathrin-mediated endocytosis is firmly established (1, 2). The heterotetrameric AP-2 adaptor complex and numerous clathrin-associated sorting proteins (CLASPs) display dedicated surfaces or domains that engage PtdIns(4,5)P 2 with good selectively (3-5). PtdIns(4,5)P 2 , which is localized to the cell surface, thus biases the deposition and assembly of these coat components at the plasma membrane by synergizing with other low affinity interactions in a phenomenon termed coincidence detection (2, 4). Later acting endocytic regulatory proteins also bind to PtdIns(4,5)P 2 . The large GTPase dynamin contains a pleckstrin homology domain, which engages PtdIns(4,5)P 2 and is required for vesicle scission (6). Similarly the clathrin uncoating cofactor, auxilin, has a PTEN homology domain that also binds to phosphoinositides and is necessary for targeting of this J-domain protein to clathrin-coated membranes (7). The lipid binding features of all these endocytic components is in full accord with PtdIns(4,5)P 2 being necessary for both early and late stages of coated vesicle production (8).PtdIns(4,5)P 2 is a general, apparently ubiquitous marker of the plasma membrane, and the concept of functionally autonomous, stable PtdIns(4,5)P 2 -enriched microdomains within the cytosolic leaflet of the membrane has been challenged (9 -11). This raises the question of whether the prevailing PtdIns(4,5)P 2 concentration at the cell surf...

Section: Discussionmentioning

confidence: 99%

Clathrin Regulates the Association of PIPKIγ661 with the AP-2 Adaptor β2 Appendage

Thieman

Mishra

Ling

et al. 2009

“…7A that governs protein stability, catalysis, and membrane interactions. It is generally thought that the membrane binding properties of mammalian DAG kinases is controlled by the presence of structural modules that lie outside the catalytic core that dock the kinases to the membrane (1,2). Although these accessory modules are important in targeting the DAG kinases to selected intracellular membrane systems and integrating their spatiotemporal activation to signaling events, the phospholipid recognition properties and conformation of the catalytic cores of the DAG kinases described in this report are also critical to the function of all members of this protein family regardless of their intracellular location or ancillary activation mechanisms.…”

Section: Discussionmentioning

confidence: 99%

Molecular Determinants for Interfacial Binding and Conformational Change in a Soluble Diacylglycerol Kinase

Jerga

Miller

White

et al. 2009

DgkB is a soluble diacylglycerol (DAG) kinase that is essential for membrane lipid homeostasis in many Gram-positive pathogens. Anionic phospholipids, like phosphatidylglycerol (PtdGro), were required for DgkB to recognize diacylglycerol embedded in a phospholipid bilayer. An activity-independent vesicle binding assay was used to determine the role of specific residues in DgkB-PtdGro interactions. Lys 15 and Lys 165 were required for DgkB to dock with PtdGro vesicles and flank the entrance to the DgkB active site. Mg 2؉ was required for vesicle binding. The compromised vesicle binding by mutants in the key asparate residues forming the structural Mg 2؉ -aspartatewater network within the substrate binding domain revealed that interfacial binding of DgkB required a Mg 2؉ -dependent conformational change. DgkB interaction with phospholipid vesicles was not influenced by the presence of ATP, but anionic vesicles decreased the K m of the enzyme for ATP. Arg 100 and Lys 15 are two surface residues in the ATP binding domain that were necessary for high affinity ATP binding. The key residues responsible for the structural Mg 2؉ binding site, the conformational changes that increase ATP affinity, and interfacial recognition of anionic phospholipids were identical in DgkB and the mammalian diacylglycerol kinase catalytic cores. This sequence conservation suggests that the mammalian enzymes also require a structural divalent cation and surface positively charged residues to bind phospholipid bilayers and trigger conformational changes that accelerate catalysis.The diacylglycerol (DAG) 2 kinase superfamily (Pfam00781) defines a group of soluble lipid kinases that phosphorylate membrane-associated DAG and function in regulating intracellular phospholipid metabolism and signaling. The mammalian family members share a conserved catalytic core domain that is associated with a variety of modules that confer intracellular membrane targeting specificity (for reviews, see Refs. 1 and 2). Staphylococcus aureus DgkB is a prototypical member of Pfam00781 that has the catalytic core of the DAG kinase superfamily, but lacks the ancillary domains characteristic of its mammalian cousins. DgkB proteins are essential in Gram-positive bacteria to re-introduce the large amount of DAG arising from lipoteichoic acid biosynthesis into the phospholipid biosynthetic pathway (3). DAG is exclusively found in the membrane phospholipid bilayer and the soluble DgkB must be capable of interfacial catalysis despite the absence of the membrane targeting modules present in its mammalian counterparts.Interfacial activation is a key feature of soluble proteins that act on substrates embedded in a phospholipid bilayer and has been extensively studied in phospholipid hydrolases (4, 5). These enzymes can utilize monomeric substrates, but their activity increases significantly when substrate is presented in micelles or unilamellar vesicles. Electrostatic effects are crucial to interfacial recognition by the soluble phospholipase A 2 proteins (4), and the structu...

“…Although DGK family enzymes have been known for a long time, their biology has recently gained renewed interest (33,34). At least 10 different DGK isoforms have been identified which in many cell types are essential for proliferative and migratory signaling (34,35).…”

Section: Discussionmentioning

confidence: 99%

“…At least 10 different DGK isoforms have been identified which in many cell types are essential for proliferative and migratory signaling (34,35). In platelets, early works had reported that inhibitors of DGK are able to potentiate agonistinduced secretion and aggregation, but the mechanism of this action has never been investigated in detail (36 -38).…”

Section: Discussionmentioning

confidence: 99%

“…However, it must be considered that all these measurements provide a value of the global amount of these metabolites in the cell, as it results from the comprehensive action of all the expressed isoforms, and they are neither indicative of the contribution of single isoforms nor informative as to the possibly different subcellular distribution of DAG. An increasing number of evidence in nucleated cells has indicated that DKG isoforms undergo complex but crucial spatial regulation (33)(34)(35). This may also be relevant in platelets, and the P2Y12-mediated inhibition of a particular DGK isoform may transiently occur in specific subcellular microdomains, allowing a local increase of DAG sufficient to efficiently trigger cellular responses.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

The Gi-coupled P2Y12 Receptor Regulates Diacylglycerol-mediated Signaling in Human Platelets

Guidetti

Lova

Bernardi

et al. 2008

Stimulation of G q -coupled receptors activates phospholipase C and is supposed to promote both intracellular Ca 2؉ mobilization and protein kinase C (PKC) activation. We found that ADPinduced phosphorylation of pleckstrin, the main platelet substrate for PKC, was completely inhibited not only by an antagonist of the G q -coupled P2Y1 receptor but also upon blockade of the G i -coupled P2Y12 receptor. The role of G i on PKC regulation required stimulation of phosphatidylinositol 3-kinase rather than inhibition of adenylyl cyclase. P2Y12 antagonists also inhibited pleckstrin phosphorylation, Rap1b activation, and platelet aggregation induced upon G q stimulation by the thromboxane A 2 analogue U46619. Importantly, activation of phospholipase C and intracellular Ca 2؉ mobilization occurred normally. Phorbol 12-myristate 13-acetate overcame the inhibitory effect of P2Y12 receptor blockade on PKC activation but not on Rap1b activation and platelet aggregation. By contrast, inhibition of diacylglycerol kinase restored both PKC and Rap1b activity and caused platelet aggregation. Stimulation of P2Y12 receptor or direct inhibition of diacylglycerol kinase potentiated the effect of membrane-permeable sn-1,2-dioctanoylglycerol on platelet aggregation and pleckstrin phosphorylation, in association with inhibition of its phosphorylation to phosphatidic acid. These results reveal a novel and unexpected role of the G i -coupled P2Y12 receptor in the regulation of diacylglycerol-mediated events in activated platelets.It is generally accepted that, with a very few exceptions, platelet response to soluble agonists originates from the convergence of at least two different signal transduction pathways typically initiated by the heterotrimeric G-proteins G q and G i . Several receptors for platelet agonists are coupled to G q , including the P2Y1 receptor for ADP, the TP␣ receptor for the thromboxane A 2 (TxA 2 ), 3 and the PAR1 and PAR4 receptors for thrombin (1, 2). Signaling downstream G q involves the stimulation of members of the ␤ subfamily of phospholipase C (PLC), which generate the second messengers inositol 1,4,5-trisphosphosphate (IP 3 ) and diacylglycerol (DAG), responsible for Ca 2ϩ mobilization from intracellular stores and protein kinase C (PKC) activation, respectively (3). This pathway is necessary but not sufficient to elicit a full platelet response, and the concomitant activation of a G i -coupled receptor is absolutely mandatory (4). To fulfill this requirement, some agonists, like ADP, directly stimulate multiple G-protein coupled receptors, one of which is associated to G i (5, 6), whereas others, such as TxA 2 , rely on the auxiliary action of secondary messengers released by activated platelets to stimulate a G i -coupled receptor (7). Despite their essential role, there are very few G i -coupled receptors on the platelet surface, and these include the ␣2-adrenergic receptor for epinephrine and the P2Y12 receptor for ADP (8, 9). Therefore, ADP, which is also one of the major component of the platelet releas...