Two new cloned human cDNAs encode paralogs of the 85-kDa cytosolic phospholipase A 2 (cPLA 2 ). We propose to call these cPLA 2 ␤ (114 kDa) and cPLA 2 ␥ (61 kDa), giving the name cPLA 2 ␣ to the well known 85-kDa enzyme. cPLA 2 ␤ mRNA is expressed more highly in cerebellum and pancreas and cPLA 2 ␥ more highly in cardiac and skeletal muscle. Sequence-tagged site mapping places cPLA 2 ␤ on chromosome 15 in a region near a phosphoinositol bisphosphate phosphatase. The mRNA for cPLA 2 ␤ is spliced only at a very low level, and Northern blots in 24 tissues show exclusively the unspliced form. cPLA 2 ␤ has much lower activity on 2-arachidonoyl-phosphatidylcholine liposomes than either of the other two enzymes. Its sequence contains a histidine motif characteristic of the catalytic center of caspase proteases of the apoptotic cascade but no region characteristic of the catalytic cysteine. Sequence-tagged site mapping places cPLA 2 ␥ on chromosome 19 near calmodulin. cPLA 2 ␥ lacks the C2 domain, which gives cPLA 2 ␣ its Ca 2؉ sensitivity, and accordingly cPLA 2 ␥ has no dependence upon calcium, although cPLA 2 ␤ does. cPLA 2 ␥ contains a prenyl group-binding site motif and appears to be largely membrane-bound. cPLA 2 ␣ residues activated by phosphorylation do not appear to be well conserved in either new enzyme. In contrast, all three previously known catalytic residues, as well as one additional essential arginine, Arg-566 in cPLA 2 ␣, are conserved in both new enzyme sequences. Mutagenesis shows strong dependence on these residues for catalytic activity of all three enzymes.Enzymatic breakdown of glycerophospholipids is carried out by numerous pathways, with the production of various bioactive lipids and fatty acids (1-8). Diverse phospholipase A 2 (PLA 2 ) 1 enzymes hydrolyze the sn-2 bond of phospholipids, releasing lysophospholipids and fatty acids (9 -11). Often the released fatty acid is arachidonic acid, whose further metabolism gives rise to several types of bioactive lipids known as eicosanoids, many of which mediate inflammation. Thus PLA 2 enzymes initiate the production of inflammatory mediators (12, 13), and for that reason they have become targets for the development of anti-inflammatory therapies.The 85-kDa cytosolic phospholipase A 2 (reviewed in Refs. 12-15) has attracted special interest because it is the only one of numerous PLA 2 s that selectively releases arachidonic acid over other fatty acids (16,17). Recent results with transgenic mice ablated for this enzyme (18) have demonstrated its role in allergy and parturition. Several functional regions have been identified within its amino acid sequence, including the C2 or calcium and lipid binding region similar to the C2 regions of other proteins (19) such as the calcium-dependent protein kinase C enzymes; several serine residues capable of activation through phosphorylation (20 -22); a hydrophilic region that may have a structural role (13); and several residues essential for catalysis, identified by mutagenesis (23-25).The cDNA for the 85-kDa cP...

Here we show that this kinase is identical with or closely related to p38 (the mammalian homolog of HOG1 from yeast), a recently discovered protein kinase typically activated by inflammatory cytokines and environmental stress. Further, we demonstrate that activation of this kinase by thrombin is transient (with maximal stimulation at 1 min), is accompanied by tyrosine phosphorylation, and precedes the activation of the ERK kinases. This is the first report to show that p38 kinase is activated by thrombin and to suggest a role for this MAP kinase in the thrombin-mediated signaling events during platelet activation.We have recently shown that thrombin stimulates the activity of the MAP 1 kinases ERK1 and ERK2 but also activates another proline-directed kinase that is distinguishable from ERK1/2 based on its strong binding to anion exchange resin and the lack of reactivity with anti-ERK1/2 antibodies (1). We further noted that this kinase readily phosphorylates cPLA 2 but not the S505A mutant of cPLA 2 . This observation indicated that the serine residing within the MAP kinase consensus sequence (i.e. Pro-Leu-Ser 505 -Pro) is the target phosphorylation site for the kinase. Significantly, the thrombin receptor agonist peptide SFLLRN also activated this proline-directed kinase but completely failed to stimulate ERK1/2. Nonetheless SFLLRN, like thrombin, mediated activation of cPLA 2 by phosphorylation, and we reasoned that this unidentified kinase could play a role in the signal transduction pathways activated through the thrombin receptor. We therefore further characterized the kinase with the goal to determine its identity and define its role in the thrombin-induced signaling events during platelet activation. EXPERIMENTAL PROCEDURESPlatelet Isolation and Incubation-Fresh human platelets were prepared from platelet-rich plasma of drug-free volunteers in the presence of prostacyclin (10 Ϫ8 M) and apyrase (0.5 units/ml) as described previously (2), suspended at 1.25 ϫ 10 9 /ml in 140 mM NaCl, 27 mM KCl, 1 mM MgCl 2 , 2.2 mM CaCl 2 , 5.5 mM glucose, 0.2 mM EGTA, 10 mM Hepes, pH 7.4, containing 30 M cyclo(S,S)-Mpr(Har)-GDWP-Pen-NH 2 (where Mpr is mercaptopropionyl, Har is homoarginine, and Pen is penicillamine) (3) (kindly provided by Dr. Robert Scarborough, COR Therapeutics), and incubated at 37°C with 5 units/ml ␣-thrombin (ϳ3500 NIH units/mg, Enzyme Research Laboratories). Reactions were terminated by adding (final concentrations) 1% Triton X-100, 5 mM EGTA, 1 mM DTT, 0.2 mM Na 3 VO 4 , 100 nM microcystin (Life Technologies, Inc.), 100 M leupeptin, 0.2 mg/ml aprotinin, 10 M pepstatin A, 1 mM Pefabloc (Centerchem), and 50 mM ␤-glycerophosphate, pH 7.5. The suspension was then briefly sonicated, centrifuged for 30 min at 100,000 ϫ g using a Sorvall RC M120EX microcentrifuge, and diluted with MonoQ buffer as indicated.Partial Purification of p38 and ERK Kinases by MonoQ Chromatography-High speed supernatants were subjected to chromatography on a MonoQ HR 5/5 column (Pharmacia Biotech Inc.) at a flow rate of 1.5 ml/min ...

Fibroblast growth factor 21 is a novel hormonal regulator with the potential to treat a broad variety of metabolic abnormalities, such as type 2 diabetes, obesity, hepatic steatosis, and cardiovascular disease. Human recombinant wild type FGF21 (FGF21) has been shown to ameliorate metabolic disorders in rodents and non-human primates. However, development of FGF21 as a drug is challenging and requires re-engineering of its amino acid sequence to improve protein expression and formulation stability. Here we report the design and characterization of a novel FGF21 variant, LY2405319. To enable the development of a potential drug product with a once-daily dosing profile, in a preserved, multi-use formulation, an additional disulfide bond was introduced in FGF21 through Leu118Cys and Ala134Cys mutations. FGF21 was further optimized by deleting the four N-terminal amino acids, His-Pro-Ile-Pro (HPIP), which was subject to proteolytic cleavage. In addition, to eliminate an O-linked glycosylation site in yeast a Ser167Ala mutation was introduced, thus allowing large-scale, homogenous protein production in Pichia pastoris. Altogether re-engineering of FGF21 led to significant improvements in its biopharmaceutical properties. The impact of these changes was assessed in a panel of in vitro and in vivo assays, which confirmed that biological properties of LY2405319 were essentially identical to FGF21. Specifically, subcutaneous administration of LY2405319 in ob/ob and diet-induced obese (DIO) mice over 7–14 days resulted in a 25–50% lowering of plasma glucose coupled with a 10–30% reduction in body weight. Thus, LY2405319 exhibited all the biopharmaceutical and biological properties required for initiation of a clinical program designed to test the hypothesis that administration of exogenous FGF21 would result in effects on disease-related metabolic parameters in humans.

Cytosolic phospholipase A 2 (cPLA 2 ) hydrolyzes the sn-2-acyl ester bond of phospholipids and shows a preference for arachidonic acid-containing substrates. We found previously that Ser-228 is essential for enzyme activity and is likely to function as a nucleophile in the catalytic center of the enzyme (

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