Cloning and expression of cystolic phospholipase A2 (cPLA2) and a naturally occurring variant. Phosphorylation of Ser505 of recombinant cPLA2 by p42 mitogen-activated protein kinase results in an increase in specific activity.

Full-length cytosolic phospholipase A2 (cPLA2) was cloned from U937 cells and polymorphonuclear leukocytes (PMNLs) while a naturally occurring variant of cPLA2, which lacks residues Val473-Ala749 but has a C-terminal extension of ILMNLSEYMLWMSKVKRFM (DcPLA2) was cloned from PMNLs and mononuclear leukocytes. We were unable to clone DcPLA2 from U937 cells. When cPLA2 and DcPLA2 were expressed in insect cells, both proteins were detected in cell lysates by SDS/PAGE as single bands of apparent molecular masses 100 kDa and 57 kDa, respectively. Full-length cPLA2 was phosphorylated stoichiometrically by p42 mitogen-activated protein (MAP) kinase in vitro at a similar rate to other physiological substrates of this protein kinase and the major site of phosphorylation was identified by amino acid sequencing as Ser505. [32P]Ser(P)505 in cPLA2 was only dephosphorylated at a slow rate by mammalian tissue homogenates. Protein phosphatases 2A, 2B and 2C all contributed significantly to the overall dephosphorylation of cPLA2. The phosphorylation of cPLA2 by p42 MAP kinase correlated with an approximately 1.5-fold increase in specific enzyme activity which was reversed by dephosphorylation.

Identification of novel phosphorylation sites required for activation of MAPKAP kinase-2.

MAP kinase-activated protein (MAPKAP) kinase-2 is activated in vivo by reactivating kinase (RK), a MAP kinase (MAPK) homologue stimulated by cytokines and cellular stresses. Here we show that in vitro RK phosphorylates human GST-MAPKAP kinase-2 at Thr25 in the proline-rich N-terminal region Thr222 and Ser272 in the catalytic domain and Thr334 in the C-terminal domain. Using novel methodology we demonstrate that activation of MAPKAP kinase-2 requires the phosphorylation of any two of the three residues Thr222, Ser272 and Thr334. Ser9, Thr25, Thr222, Ser272, Thr334 and Thr338 became 32P-labelled in stressed KB cells and labelling was prevented by the specific RK inhibitor SB 203580, establishing that RK phosphorylates Thr25, Thr222, Ser272 and Thr334 in vivo. The 32P-labelling of Thr338 is likely to result from autophosphorylation. GST-MAPKAP kinase-2 lacking the N-terminal domain was inactive, but activated fully when phosphorylated at Thr222, Ser272 and Thr334 by p42 MAPK or RK. In contrast, full-length GST-MAPKAP kinase-2 was phosphorylated at Thr25 (and not activated) by p42 MAPK, suggesting a role for the N-terminal domain in specifying activation by RK in vivo. The mutant Asp222/Asp334 was 20% as active as phosphorylated MAPKAP kinase-2, and this constitutively active form may be useful for studying its physiological roles.

Comparison of the specificities of p70 S6 kinase and MAPKAP kinase-1 identifies a relatively specific substrate for p70 S6 kinase: the N-terminal kinase domain of MAPKAP kinase-1 is essential for peptide phosphorylation.

xxR/KxRxxSxx sequences were phosphorylated with high efficiency by both p70 S6 kinase (p70S6K) and MAPKAP kinase-1. The best substrate for MAPKAP kinase-1 (KKKNRTLSVA) was phosphorylated with a Km of 0.17 microM, and the best substrate for p70S6K (KKRNRTLSVA) with a Km of 1.5 microM. The requirement of both enzymes for Arg/Lys at position n-5 could be partially replaced by inserting basic residues at other positions, especially by an Arg at n-2 or n-4. MAPKAP kinase-1 (but not p70S6K) tolerated lack of any residue at n-5 if Arg was present at n-2 and n-3. p70S6K (but not p90S6K) tolerated Thr at position n and absence of any residue at n + 2. The peptide KKRNRTLTV, which combined these features, was relatively selective for p70S6K having a 50-fold higher Vmax/Km than MAPKAP kinase-1. Inactivation of the N-terminal kinase domain of MAPKAP kinase-1, which is 60% identical to p70S6K, abolished activity towards all peptides tested, but the enzyme retained 30-40% of its activity if the C-terminal kinase domain was inactivated.

Inactivation of glycogen synthase kinase-3 beta by phosphorylation: new kinase connections in insulin and growth-factor signalling.

The beta-isoform of glycogen synthase kinase-3 (GSK3 beta) isolated from rabbit skeletal muscle was inactivated 90-95% following incubation with MgATP and either MAP kinase-activated protein kinase-1 (MAPKAP kinase-1, also termed RSK-2) or p70 S6 kinase (p70S6K), and re-activated with protein phosphatase 2A. MAPKAP kinase-1 and p70S6K phosphorylated the same tryptic peptide on GSK3 beta, and the site of phosphorylation was identified as the serine located nine residues from the N-terminus of the protein. The inhibitory effect of Ser-9 phosphorylation on GSK3 beta activity was observed with three substrates, (inhibitor-2, c-jun and a synthetic peptide), and also with glycogen synthase provided that 0.15 M KCl was added to the assays. The results suggest that Ser-9 phosphorylation underlies the reported inhibition of GSK3 beta by insulin and that GSK3 may represent a point of convergence of two major growth-factor-stimulated protein kinase cascades.

The phosphorylation of stathmin by MAP kinase.

Stathmin, a ubiquitous cytosolic phosphoprotein which may play a role in integrating the effects of diverse signals regulating proliferation, differentiation and other cell functions, was found to be phosphorylated rapidly and stoichiometrically by mitogen-activated protein (MAP) kinase in vitro. Ser-25 was identified as the major site and Ser-38 as a minor site of phosphorylation, while the p42 and p44 isoforms of MAP kinase were the only significant stathmin kinases detected in PC12 cells after stimulation by nerve growth factor (NGF). The results suggest that MAP kinases are the enzymes responsible for increasing the level of phosphorylation of Ser-25, which has been observed previously in PC12 cells following stimulation by NGF.

Haemolysins and extracellular enzymes of Listeria monocytogenes and L. ivanovii.

Of 120 laboratory-maintained strains of Listeria monocytogenes and two of L. ivanovii examined for haemolytic and lipolytic activity, 62 exhibited haemolytic activity alone, 20 of these showed haemolytic and lipolytic activity and 40 had neither activity. The L. ivanovii strains showed both activities. The results indicated a relationship between haemolysin production and lipolytic activity which was not explained by the serotype of the organism. In addition, the following hydrolytic activities were detected in the cell-free growth media of strains L. monocytogenes Boldy and L. ivanovii (formerly L. monocytogenes) Type 5 (substrates acted upon are given in parentheses): acid phosphate (4-nitrophenylphosphate, naphthyl phosphate, glycerophosphate, phosphorylcholine and GTP); neutral phosphatase (4-nitrophenylphosphate, naphthyl phosphate, phosphorylcholine, NADP and UDPG); phosphodiesterase (bis-4-nitrophenylphosphate, ATP and NADP); NADase (NAD); phospholipase C (4-nitrophenylphosphoryl-choline, phosphatidyl choline and ethanolamine, and sphingomyelin); and lipase and esterase (triacetin, tributyrin, triolein, naphthyl-laurate,-myristate,-caprylate,-palmitate and -oleate, 4-nitrophenyl-acetate-laurate and Tween 80). The preparations also showed weak catalase activity. No evidence was found for the presence of RNAase, DNAase, peptidase/amidase, phosphoamidase, alpha-amylase, glucosidase, galactosidase, pyranosidase or glucose aminidase.

Separation and properties of the haemolysins and extracellular enzymes of Listeria monocytogenes and L. ivanovii.

Desalted ammonium-sulphate (0-65%) precipitates from the cell-free supernates of 16-24-h cultures of Listeria monocytogenes Boldy and L. ivanovii (previously L. monocytogenes) Type 5 were eluted through Sephadex G-200. The enzyme activities gave rise to two main peaks. The first peak (approximate mol. wt of protein 150,000) contained only phosphatase activity (assayed by hydrolysis of 4-nitrophenylphosphate at pH 5.0 and 7.0). The second peak (approximate mol. wts of proteins 40,000-60,000) contained the haemolysin activity and the following hydrolytic activities (assay substrates are given in parentheses): phospholipase C (phosphatidyl choline and 4-nitrophenyl-phosphoryl-choline); phosphodiesterase (bis-4-nitrophenyl-phosphate); acid phosphatase (4-nitrophenylphosphatase); and esterases and lipases (4-nitrophenyl acetate, naphthyl-acetate and -oleate, triacetin and triolein). DEAE-Sephadex chromatography of appropriate fractions from the Sephadex G-200 purification step separated the first peak into two phosphatases and resolved the second peak into its constituent activities. Polyacrylamide gel electrophoresis showed that the individual fractions from the DEAE-Sephadex step consisted of mixtures of protein. The effects of pH and potential activators and inhibitors on the active proteins purified by DEAE-Sephadex chromatography were examined.

Topical treatment of dermatophytoses and candidoses.

In a double-blind trial clotrimazole cream was compared with tolnaftate cream in the treatment of dermatophytoses and with nystatin cream in the treatment of candidoses. Clotrimazole was shown to be effective against both dermatophytoses and candidoses and was as effective and acceptable as tolnaftate and nystatin creams in each condition.