Heme stimulates platelet mitochondrial oxidant production via the activation of toll-like receptor 4 signaling to mediate targeted granule secretion

Hemolysis is a pathological component of many diseases and is associated with thrombosis and vascular dysfunction. Hemolytic products, including cell-free hemoglobin and free heme directly activate platelets. However, the effect of hemolysis on platelet degranulation, a central process in not only thrombosis, but also inflammatory and mitogenic signaling, remains less clear. Our group showed that hemoglobin-induced platelet activation involved the production of mitochondrial reactive oxygen species (mtROS). However, the molecular mechanism by which extracellular hemolysis induces platelet mtROS production, and whether the mtROS regulate platelet degranulation remains unknown. Here, we demonstrate using isolated human platelets that cell free heme is a more potent agonist for platelet activation than hemoglobin, and stimulates the release of a specific set of molecules from the α-granule of platelets, including the glycoprotein thrombospondin-1 (TSP-1). We uncover the mechanism of heme-mediated platelet mtROS production which is dependent on the activation of platelet TLR4 signaling and leads to the downstream phosphorylation of complex-V by the serine kinase Akt. Notably, inhibition of platelet TLR4 or Akt, or scavenging mtROS prevents heme-induced granule release in vitro. Further, heme-dependent granule release is significantly attenuated in vivo in mice lacking TLR4 or those treated with the mtROS scavenger MitoTEMPO. These data elucidate a novel mechanism of TLR4-mediated mitochondrial regulation, establish the mechanistic link between hemolysis and platelet degranulation, and begin to define the heme and mtROS-dependent platelet secretome. These data have implications for hemolysis-induced thrombo-inflammatory signaling and for the consideration of platelet mitochondria as a therapeutic target in hemolytic disorders.

Identification and enzymatic analysis of an archaeal ATP-dependent serine kinase from the hyperthermophilic archaeon Staphylothermus marinus

Serine kinase catalyzes the phosphorylation of free serine (Ser) to produce O -phosphoserine (Sep). An ADP-dependent Ser kinase in the hyperthermophilic archaeon Thermococcus kodakarensis ( Tk -SerK) is involved in cysteine (Cys) biosynthesis and most likely Ser assimilation. An ATP-dependent Ser kinase in the mesophilic bacterium Staphylococcus aureus is involved in siderophore biosynthesis. Although proteins displaying various degrees of similarity with Tk -SerK are distributed in a wide range of organisms, it is unclear if they are actually Ser kinases. Here we examined proteins from Desulfurococcales species in Crenarchaeota that display moderate similarity with Tk -SerK from Euryarchaeota (42-45% identical). Tk - serK homologs from Staphylothermus marinus (Smar_0555), Desulfurococcus amylolyticus (DKAM_0858), and Desulfurococcus mucosus (Desmu_0904) were expressed in Escherichia coli . All three partially purified recombinant proteins exhibited Ser kinase activity utilizing ATP rather than ADP as a phosphate donor. Purified Smar_0555 protein displayed activity towards l -Ser, but not with other compounds including d -Ser, l -threonine and l -homoserine. The enzyme utilized ATP, UTP, GTP, CTP, and the inorganic polyphosphates triphosphate and tetraphosphate as the phosphate donor. Kinetic analysis indicated that the Smar_0555 protein preferred nucleoside 5’-triphosphates compared to triphosphate as a phosphate donor. Transcript levels and Ser kinase activity in S. marinus cells grown with or without serine suggested that the Smar_0555 gene is constitutively expressed. The genes encoding Ser kinases examined here form an operon with genes most likely responsible for the conversion between Sep and 3-phosphoglycerate of central sugar metabolism, suggesting that the ATP-dependent Ser kinases from Desulfurococcales play a role in the assimilation of Ser. IMPORTANCE Homologs of the ADP-dependent Ser kinase from the archaeon Thermococcus kodakarensis ( Tk -SerK) include representatives from all three domains of life. The results of this study show that even homologs from the archaeal order Desulfurococcales, which are the most structurally related to the ADP-dependent Ser kinases from the Thermococcales, are Ser kinases that utilize ATP, and in at least some cases inorganic polyphosphates, as the phosphate donor. The differences in properties between the Desulfurococcales and Thermococcales enzymes raise the possibility that Tk -SerK homologs constitute a group of kinases that phosphorylate free serine with a wide range of phosphate donors.

The Mechanism of Raf Activation through Dimerization

Raf, a threonine/serine kinase in the Raf/MEK/ERK pathway, regulates cell proliferation. Raf’s full activation requires dimerization. Aberrant activation through dimerization is an important therapeutic target. Despite its clinical importance, fundamental...

CASK modulates the assembly and function of the Mint1/Munc18-1 complex to regulate insulin secretion

Calcium/calmodulin-dependent protein serine kinase (CASK) is a key player in vesicle transport and release in neurons. However, its precise role, particularly in nonneuronal systems, is incompletely understood. We report that CASK functions as an important regulator of insulin secretion. CASK depletion in mouse islets/β cells substantially reduces insulin secretion and vesicle docking/fusion. CASK forms a ternary complex with Mint1 and Munc18-1, and this event is regulated by glucose stimulation in β cells. The crystal structure of the CASK/Mint1 complex demonstrates that Mint1 exhibits a unique “whip”-like structure that wraps tightly around the CASK-CaMK domain, which contains dual hydrophobic interaction sites. When triggered by CASK binding, Mint1 modulates the assembly of the complex. Further investigation revealed that CASK-Mint1 binding is critical for ternary complex formation, thereby controlling Munc18-1 membrane localization and insulin secretion. Our work illustrates the distinctive molecular basis underlying CASK/Mint1/Munc18-1 complex formation and reveals the importance of the CASK-Mint1-Munc18 signaling axis in insulin secretion.

Testis-specific serine kinase protein family in male fertility and as targets for non-hormonal male contraception†

Male contraception is a very active area of research. Several hormonal agents have entered clinical trials, while potential non-hormonal targets have been brought to light more recently and are at earlier stages of development. The general strategy is to target genes along the molecular pathways of sperm production, maturation, or function, and it is predicted that these novel approaches will hopefully lead to more selective male contraceptive compounds with a decreased side effect burden. Protein kinases are known to play a major role in signaling events associated with sperm differentiation and function. In this review, we focus our analysis on the testis-specific serine kinase (TSSK) protein family. We have previously shown that members of the family of TSSKs are postmeiotically expressed in male germ cells and in mature mammalian sperm. The restricted postmeiotic expression of TSSKs as well as the importance of phosphorylation in signaling processes strongly suggests that TSSKs have an important role in germ cell differentiation and/or sperm function. This prediction has been supported by the reported sterile phenotype of the Tssk6 knockout (KO) mice and of the double Tssk1 and Tssk2 KO mice and by the male subfertile phenotype observed in a Tssk4 KO mouse model.

Glycogen synthase kinase 3 (GSK-3) controls T-cell motility andinteractions with antigen presenting cells

Objective: The threonine/serine kinase glycogen synthase kinase 3 (GSK-3) targets multiple substrates in T-cells and regulates the expression of Tbet and PD-1. However, it has been unclear whether GSK-3 has any effect on T-cell motility or their interactions with antigen presenting cells. Results: Here, we show that GSK-3 controls T-cell motilityand interactions with other cells. Inhibition of GSK-3, using structurally distinct inhibitors, reduced T-cell motility in terms of speed and distance travelled. Furthermore, SB415286 reduced the number of cell to cell contacts, however the duration of these established contacts with other cells did not differ in the presence of SB415286. This inhibition of motility did not affect the ability of GSK-3 inhibitors to enhance cytolytic T-cell (CTL) function in killing tumor targets. These data show that the inhibition of GSK-3 has differential effects on T-cell motility and CTL function where the negative effects on cell-cell interactions is overridden by the increased cytolytic potential of CTLs.

Glycogen synthase kinase 3 (GSK-3) controls T-cell motility andinteractions with antigen presenting cells

Objective:The threonine/serine kinase glycogen synthase kinase 3 (GSK-3) targets multiple substrates in T-cells and regulates the expression of Tbet and PD-1. However, it has been unclear whether GSK-3 has any effect on T-cell motility or their interactions with antigen presenting cells. Results: Here, we show that GSK-3 controls T-cell motilityand interactions with other cells. Inhibition of GSK-3, using structurally distinct inhibitors, reduced T-cell motility in terms of speed and distance travelled. Furthermore, SB415286 reduced the number of cell to cell contacts, however the duration of these established contacts with other cells did not differ in the presence of SB415286. This inhibition of motility did not affect the ability of GSK-3 inhibitors to enhance cytolytic T-cell (CTL) function in killing tumor targets. These data show that the inhibition of GSK-3 has differential effects on T-cell motility and CTL function where the negative effects on cell-cell interactions is overridden by the increased cytolytic potential of CTLs.

Modular bioengineered kinase sensors via scaffold protein-mediated split-luciferase complementation

Phosphorylation is a key regulation event in cellular signaling. To sense the underlying kinase activity, we engineered modular and easy adaptable serine kinase sensors for the exemplary kinases PKA, PKB and CHK1.