Synthesis and Binding of Stable Bisubstrate Ligands for Phosphoglycerate Kinase

Stable bisubstrate ligands of phosphoglycerate kinase (PGK) have been synthesised with AMP or ADP conjugated to hydrolytically-stable, symmetrical analogues of 1,3-bisphosphoglycerate and their binding to yeast PGK evaluated. Their Kds decrease with net negative charge, with a penta-anionic analogue 7 showing highest affinity - in accordance with its approximation to the transition state for the reaction catalysed by PGK.

Preproendothelin-1 expression in human mesangial cells: evidence for a p38 mitogen-activated protein kinase/protein kinases- C-dependent mechanism

Endothelin-1 (ET-1) has been implicated in the pathogenesis of renal inflammation. This study investigated the mechanisms underlying the synergistic upregulation of preproET-1 gene expression in human mesangial cells after co-stimulation with thrombin and tumor necrosis factor alpha (TNFalpha). Whereas thrombin induced a moderate upregulation of preproET-1 mRNA, co-stimulation with TNFalpha resulted in a strong and protracted upregulation of this mRNA species. Thrombin+TNFalpha-induced upregulation of preproET-1 expression was found to require p38 mitogen-activated protein kinase and protein kinases C, whereas activation of extracellular signal-regulated kinase, c-Jun-N-terminal kinase, or intracellular Ca(2+) release were not required. Actinomycin D chase experiments suggested that enhanced stability of preproET-1 mRNA did not account for the increase in transcript levels. PreproET-1 promoter analysis demonstrated that the 5'-flanking region of preproET-1 encompassed positive regulatory elements engaged by thrombin. Negative modulation of thrombin-induced activation exerted by the distal 5' portion of preproET-1 promoter (-4.4 kbp to 204 bp) was overcome by co-stimulation with TNFalpha, providing a possible mechanism underlying the synergistic upregulation of preproET-1 expression by these two agonists. In conclusion, human mesangial cell expression of preproET-1 may be increased potently in the presence of two common proinflammatory mediators, thereby providing a potential mechanism for ET-1 production in inflammatory renal disease.

Fgr but not Syk tyrosine kinase is a target for beta2 integrin-induced c-Cbl-mediated ubiquitination in adherent human neutrophils

An early and critical event in beta2 integrin signalling during neutrophil adhesion is activation of Src tyrosine kinases and Syk. In the present study, we report Src kinase-dependent beta2 integrin-induced tyrosine phosphorylation of Cbl occurring in parallel with increased Cbl-associated tyrosine kinase activity. These events concurred with activation of Fgr and, surprisingly, also with dissociation of this Src tyrosine kinase from Cbl. Moreover, the presence of the Src kinase inhibitor PP1 in an in vitro assay had only a limited effect on the Cbl-associated kinase activity. These results suggest that an additional active Src-dependent tyrosine kinase associates with Cbl. The following observations imply that Syk is such a kinase: (i) beta2 integrins activated Syk in a Src-dependent manner, (ii) Syk was associated with Cbl much longer than Fgr was, and (iii) the Syk inhibitor piceatannol (3,4,3´,5´-tetrahydroxy-trans-stilbene) abolished the Cbl-associated kinase activity in an in vitro assay. Effects of the mentioned interactions between these two kinases and Cbl may be related to the finding that Cbl is a ubiquitin E3 ligase. Indeed, we detected beta2 integrin-induced ubiquitination of Fgr that, similar to the phosphorylation of Cbl, was abolished in cells pretreated with PP1. However, the ubiquitination of Fgr did not cause any apparent degradation of the protein. In contrast with Fgr, Syk was not modified by the E3 ligase. Thus Cbl appears to be essential in beta2 integrin signalling, first by serving as a matrix for a subsequent agonist-induced signalling interaction between Fgr and Syk, and then by mediating ubiquitination of Fgr which possibly affects its interaction with Cbl.

Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation

FcRI activation of mast cells is thought to involve Lyn and Syk kinases proximal to the receptor and the signaling complex organized by the linker for activation of T cells (LAT). We report here that FcRI also uses a Fyn kinase-dependent pathway that does not require Lyn kinase or the adapter LAT for its initiation, but is necessary for mast cell degranulation. Lyn-deficiency enhanced Fyn-dependent signals and degranulation, but inhibited the calcium response. Fyn-deficiency impaired degranulation, whereas Lyn-mediated signaling and calcium was normal. Thus, FcRI-dependent mast cell degranulation involves cross-talk between Fyn and Lyn kinases.

Diabetes-induced activation of protein kinase C inhibits store-operated Ca2+ uptake in rat retinal microvascular smooth muscle.

AIMS/HYPOTHESIS: To assess the effects of diabetes-induced activation of protein kinase C (PKC) on voltage-dependent and voltage-independent Ca2+ influx pathways in retinal microvascular smooth muscle cells. METHODS: Cytosolic Ca2+ was estimated in freshly isolated rat retinal arterioles from streptozotocin-induced diabetic and non-diabetic rats using fura-2 microfluorimetry. Voltage-dependent Ca2+ influx was tested by measuring rises in [Ca2+]i with KCl (100 mmol/l) and store-operated Ca2+ influx was assessed by depleting [Ca2+]i stores with Ca2+ free medium containing 5 micromol/l cyclopiazonic acid over 10 min and subsequently measuring the rate of rise in Ca2+ on adding 2 mmol/l or 10 mmol/l Ca2+ solution. RESULTS: Ca2+ entry through voltage-dependent L-type Ca2+ channels was unaffected by diabetes. In contrast, store-operated Ca2+ influx was attenuated. In microvessels from non-diabetic rats 20 mmol/l D-mannitol had no effect on store-operated Ca2+ influx. Diabetic rats injected daily with insulin had store-operated Ca2+ influx rates similar to non-diabetic control rats. The reduced Ca2+ entry in diabetic microvessels was reversed by 2-h exposure to 100 nmol/l staurosporine, a non-specific PKC antagonist and was mimicked in microvessels from non-diabetic rats by 10-min exposure to the PKC activator phorbol myristate acetate (100 nmol/l). The specific PKCbeta antagonist LY379196 (100 nmol/l) also reversed the poor Ca2+ influx although its action was less efficacious than staurosporine. CONCLUSION/INTERPRETATION: These results show that store-operated Ca2+ influx is inhibited in retinal arterioles from rats having sustained increased blood glucose and that PKCbeta seems to play a role in mediating this effect.

Glucose-induced phosphatidylinositol 3-kinase and mitogen-activated protein kinase-dependent upregulation of the platelet-derived growth factor-B receptor potentiates vascular smooth muscle cell chemotaxis

The aim of this study was to investigate the effects of elevated D-glucose concentrations on vascular smooth muscle cell (VSMC) expression of the platelet-derived growth factor (PDGF) beta receptor and VSMC migratory behavior. Immunoprecipitation, immunofluorescent staining, and RT-PCR of human VSMCs showed that elevated D-glucose induced an increase in the PDGF beta receptor that was inhibited by phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathway inhibitors. Exposure to 25 mmol/l D-glucose (HG) induced increased phosphorylation of protein kinase B (PKB) and extracellular-regulated kinase (ERK). All HG chemotaxis assays (with either 10 days' preincubation in HG or no preincubation) in a FCS or PDGF-BB gradient showed positive chemotaxis, whereas those in 5 mmol/l D-glucose did not. Assays were also run with concentrations ranging from 5 to 25 mmol/l D-glucose. Chemotaxis was induced at concentrations >9 mmol/l D-glucose. An anti-PDGF beta receptor antibody inhibited glucose-potentiated VSMC chemotaxis, as did the inhibitors for the PI3K and MAPK pathways. This study has shown that small increases in D-glucose concentration, for a short period, increase VSMC expression of the PDGF beta receptor and VSMC sensitivity to chemotactic factors in serum, leading to altered migratory behavior in vitro. It is probable that similar processes occur in vivo with glucose-enhanced chemotaxis of VSMCs, operating through PDGF beta receptor-operated pathways, contributing to the accelerated formation of atheroma in diabetes.

Modification of P13K- and MAPK-dependent chemotaxis in aortic vascular smooth muscle cells by protein kinase CBII

Hyperglycemia increases expression of platelet-derived growth factor (PDGF)-beta receptor and potentiates chemotaxis to PDGF-BB in human aortic vascular smooth muscle cells (VSMCs) via PI3K and ERK/MAPK signaling pathways. The purpose of this study was to determine whether increased activation of protein kinase C (PKC) isoforms had a modulatory effect on the PI3K and ERK/MAPK pathways, control of cell adhesiveness, and movement. All known PKC isoforms were assessed but only PKC alpha and PKC beta II levels were increased in 25 mmol/L glucose. However, only PKC beta II inhibition affected (decreased) PI3K pathway and MAPK pathway activities and inhibited PDGF-beta receptor upregulation in raised glucose, and specific MAPK inhibition was required to completely block the effect of glucose. In raised glucose conditions, activity of the ERK/MAPK pathway, PI3K pathway, and PKC beta II were all sensitive to aldose reductase inhibition. Chemotaxis to PDGF-BB (360 pmol/L), absent in 5 mmol/L glucose, was present in raised glucose and could be blocked by PKC beta II inhibition. Formation of lamellipodia was dependent on PI3K activation and filopodia on MAPK activation; both lamellipodia and filopodia were eliminated when PKC beta II was inhibited. FAK phosphorylation and cell adhesion were reduced by PI3K inhibition, and although MAPK inhibition prevented chemotaxis, it did not affect FAK phosphorylation or cell adhesiveness. In conclusion, chemotaxis to PDGF-BB in 25 mmol/L glucose is PKC beta II-dependent and requires activation of both the PI3K and MAPK pathways. Changes in cell adhesion and migration speed are mediated mainly through the PI3K pathway.