Inhibition of protein kinase C by N-myristoylated peptide substrate analogs

Sphingosine 1-phosphate (S1P) from mononuclear phagocytes and platelets signals T cells predominantly through S1P 1 G protein-coupled receptors (Rs) to enhance survival, stimulate and suppress migration, and inhibit other immunologically relevant responses. Cellular S1P 1 Rs and their signaling functions are rapidly down-regulated by S1P, through a protein kinase C (PKC)-independent mechanism, but characteristics of cell-surface re-expression of down-regulated S1P 1 Rs have not been elucidated. T cell chemotactic responses (CT) to 10 and 100 nM S1P and inhibition of T cell chemotaxis to chemokines (CI) by 1 and 3 M S1P were suppressed after 1 h of preincubation with 100 nM S1P, but recovered fully after 12-24 h of exposure to S1P. Late recovery of down-regulated CT and CI, but not early down-regulation, was suppressed by PKC and PKC⑀-selective inhibitors and was absent in T cells from PKC⑀-null mice. The same PKC⑀ inhibitors blocked S1P-evoked increases in T cell nuclear levels of c-Fos and phosphorylated c-Jun and JunD after 24 h, but not 1 h. A mixture of c-Fos plus c-Jun antisense oligonucleotides prevented late recovery of down-regulated CT and CI, without affecting S1P induction of down-regulation. Similarly, S1P-elicited threonine phosphorylation of S1P 1 Rs was suppressed by a selective inhibitor of PKC⑀ after 24 h, but not 1 h. Biochemical requisites for recovery of down-regulated S1P 1 Rs thus differ from those for S1P induction of down-regulation.The lysophospholipids sphingosine 1-phosphate (S1P) 1 and lysophosphatidic acid (LPA) are generated and secreted by many types of stimulated cells (1, 2). At concentrations of 0.1-1 micromolar found normally in plasma and other extracellular fluids, S1P and LPA evoke cellular proliferation and diverse other functional responses through at least eight members of a family of homologous G protein-coupled receptors (GPCRs) (3, 4). Originally termed endothelial differentiation gene-encoded or Edg receptors (Rs), those specific for S1P now are officially re-named S1P 1 (Edg-1), S1P 2 (Edg-5), S1P 3 (Edg-3), S1P 4 (Edg-6), and S1P 5 (Edg-8), and those for LPA are LPA 1 (Edg-2), LPA 2 (Edg-4), and LPA 3 (Edg-7). Stimulated mononuclear phagocytes and platelets are the predominant sources of S1P and LPA in the immune system. Blood and lymphoid tissue CD4 and CD8 T cells, B cells, and mononuclear phagocytes all express S1P and LPA GPCRs, in cell type-specific patterns, which are regulated distinctively by their respective ligands and by cellular immune activation (5-8).T cells express predominantly S1P 1 and S1P 4 , of which S1P 1 transduces two distinct effects of S1P on T cell migration and also regulates other T cell functional responses (7,8). S1P is chemotactic for T cells at 0.001-0.1 M, enhances chemotactic responses to chemokines at 0.01-0.1 M, and suppresses T cell chemotaxis to numerous stimuli at 0.3-3 M. As T cell antigen receptor-dependent activation of T cells suppresses expression of S1P GPCRs and functional responses to S1P in parallel, the S1P-S1P 1 R ax...

Section: Resultsmentioning

confidence: 91%

Protein Kinase C ϵ Dependence of the Recovery from Down-regulation of S1P1 G Protein-coupled Receptors of T Lymphocytes

Graeler

Kong

Karliner

et al. 2003

“…Therefore, protein myristoylation may be also involved in the protein-protein interaction between PKC and its substrate proteins. In fact, potentiation of PKC inhibitor peptides by N-terminal myristoylation has been reported (39,40), and even the presence of a myristyl binding site in PKC has been speculated (41). Altogether, these results raise the possibility that the protein myristoylation plays direct roles in the protein-protein interaction of various proteins.…”

Section: Discussionmentioning

confidence: 92%

Identification of the Calmodulin-binding Domain of Neuron-specific Protein Kinase C Substrate Protein CAP-22/NAP-22

Takasaki

Hayashi

Matsubara

et al. 1999

Various proteins in the signal transduction pathways as well as those of viral origin have been shown to be myristoylated. Although the modification is often essential for the proper functioning of the modified protein, the mechanism by which the modification exerts its effects is still largely unknown. Brain-specific protein kinase C substrate, CAP-23/NAP-22, which is involved in the synaptogenesis and neuronal plasticity, binds calmodulin, but the protein lacks any canonical calmodulin-binding domain. In the present report, we show that CAP-23/NAP-22 isolated from rat brain is myristoylated and that the modification is directly involved in its interaction with calmodulin. Myristoylated and non-myristoylated recombinant proteins were produced in Escherichia coli, and their calmodulin-binding properties were examined. Only the former bound to calmodulin. Synthetic peptides based on the N-terminal sequence showed similar binding properties to calmodulin, only when they were myristoylated. The calmodulin-binding site narrowed down to the myristoyl moiety together with a nine-amino acid N-terminal basic domain. Phosphorylation of a single serine residue in the N-terminal domain (Ser 5 ) by protein kinase C abolished the binding. Furthermore, phosphorylation of CAP-23/NAP-22 by protein kinase C was also found myristoylation-dependent, suggesting the importance of myristoylation in protein-protein interactions.

“…The purified PKC preparation is a mixture of the isozymes ␣, ␤, ␥, ⑀, and (18). A fully active catalytic domain fragment of PKC was generated from the purified PKC preparation by limited trypsinolysis with a yield of Ͼ50%, as described previously (17,28). Where indicated, the catalytic domain fragment was purified from regulatory domain fragment and residual intact PKC by DEAE ionexchange chromatography using a 0.0 -0.4 M NaCl gradient (17,28).…”

Section: Methodsmentioning

confidence: 99%

“…Protein Kinase Assays-The Ca 2ϩ -and PS-dependent histone kinase activity of purified PKC was measured as described previously (28). The histone kinase reaction mixture (120 l) contained 20 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 0.2 mM CaCl 2 , 30 g/ml PS (or none), 6 M [␥- 32 P]ATP (5000 -8000 cpm/pmol), 0.67 mg/ml histone III-S, and 5 ng of purified PKC.…”

Section: Methodsmentioning

confidence: 99%

Irreversible Inactivation of Protein Kinase C by Glutathione

Ward

Pierce

Chung

et al. 1998

Self Cite

The tripeptide glutathione (GSH) is the predominant low molecular weight thiol reductant in mammalian cells. In this report, we show that at concentrations at which GSH is typically present in the intracellular milieu, GSH and the oxidized GSH derivatives GSH disulfide (GSSG) and glutathione sulfonate each irreversibly inactivate up to 100% of the activity of purified Ca 2؉ -and phosphatidylserine (PS)-dependent protein kinase C (PKC) isozymes in a concentration-dependent manner by a novel nonredox mechanism that requires neither glutathiolation of PKC nor the reduction, formation, or isomerization of disulfide bridges within PKC. Our evidence for a nonredox mechanism of PKC inactivation can be summarized as follows. GSSG antagonized the Ca 2؉ -and PS-dependent activity of purified rat brain PKC with the same efficacy (IC 50 ‫؍‬ 3 mM) whether or not the reductant dithiothreitol was present. Glutathione sulfonate, which is distinguished from GSSG and GSH by its inability to undergo disulfide/thiol exchange reactions, was as effective as GSSG in antagonizing Ca 2؉ -and PS-dependent PKC catalysis. The irreversibility of the inactivation mechanism was indicated by the stability of the inactivated form of PKC to dilution and extensive dialysis. The inactivation mechanism did not involve the nonspecific phenomena of denaturation and aggregation of PKC because it obeyed pseudo-first order kinetics and because the hinge region of PKC-␣ remained a preferential target of tryptic attack following GSH inactivation. The selectivity of GSH in the inactivation of PKC was also indicated by the lack of effect of the tripeptides Tyr-Gly-Gly and Gly-Ala-Gly on the activity of PKC. Furthermore, GSH antagonism of the Ser/ Thr kinase casein kinase 2 was by comparison weak (<25%). Inactivation of PKC-␣ was not accompanied by covalent modification of the isozyme by GSH or other irreversible binding interactions between PKC-␣ and the tripeptide, but it was associated with an increase in the susceptibility of PKC-␣ to trypsinolysis. Treatment of cultured rat fibroblast and human breast cancer cell lines with N-acetylcysteine resulted in a substantial loss of Ca 2؉ -and PS-dependent PKC activity in the cells within 30 min. These results suggest that GSH exerts negative regulation over cellular PKC isozymes that may be lost when oxidative stress depletes the cellular GSH pool.The phospholipid-dependent isozyme family protein kinase C (PKC) 1 plays an important role in diverse physiological processes, including cell growth and differentiation, muscle contraction, and neurotransmission, and pathological developments, e.g. tumor promotion and multiple drug resistance in cancer (1-4). The PKC family consists of Ca 2ϩ -dependent, phorbol ester-activated isozymes (␣, ␤ 1 , ␤ 2 , and ␥), Ca 2ϩ -independent, phorbol ester-activated isozymes (␦, ⑀, , ⌰, and ), and Ca 2ϩ -and phorbol ester-independent isozymes ( and ). Phosphatidylserine (PS) dependence is universal among PKC isozymes (1, 5). Both stimulatory and inhibitory endogenous regulators of ...