Identification of an extracellular signal-regulated kinase (ERK) docking site in ribosomal S6 kinase, a sequence critical for activation by ERK in vivo.

Glutathione S-transferase (GST)-fusion proteins containing the carboxyl-terminal tails of three p90 ribosomal S6 kinase (RSK) isozymes (RSK1, RSK2, and RSK3) interacted with extracellular signal-regulated kinase (ERK) but not c-Jun-NH2-kinase (JNK) or p38 mitogen-activated protein kinase (MAPK). Within the carboxyl-terminal residues of the RSK isozymes is a region of high conservation corresponding to residues 722LAQRRVRKLPSTTL735 in RSK1. Truncation of the carboxyl-terminal 9 residues, 727VRKLPSTTL735, completely eliminated the interaction of the GST-RSK1 fusion protein with purified recombinant ERK2, whereas the truncation of residues 731PSTTL735 had no effect on the interaction with purified ERK2. ERK1 and ERK2 co-immunoprecipitated with hemagglutinin-tagged wild type RSK2 (HA-RSK2) in BHK cell cytosol. However, ERK did not co-immunoprecipitate with HA-RSK2((1-729)), a mutant missing the carboxyl-terminal 11 amino acids, similar to the minimal truncation that eliminated in vitro interaction of ERK with the GST-RSK1 fusion protein. Kinase activity of HA-RSK2 increased 6-fold in response to insulin. HA-RSK2((1-729)) had a similar basal kinase activity to that of HA-RSK2 but was not affected by insulin treatment. Immunoprecipitated HA-RSK2 and HA-RSK2((1-729)) could be activated to the same extent in vitro by active ERK2, demonstrating that HA-RSK2((1-729)) was properly folded. These data suggest that the conserved region of the RSK isozymes (722LAQRRVRKL730 of RSK1) provides for a specific ERK docking site approximately 150 amino acids carboxyl-terminal to the nearest identified ERK phosphorylation site (Thr573). Complex formation between RSK and ERK is essential for the activation of RSK by ERK in vivo. Comparison of the docking site of RSK with the carboxyl-terminal tails of other MAPK-activated kinases reveals putative docking sites within each of these MAPK-targeted kinases. The number and placement of lysine and arginine residues within the conserved region correlate with specificity for activation by ERK and p38 MAPKs in vivo.

Ribosomal S6 kinase is activated as an early event in preemergence development of encysted embryos of Artemia salina.

Dormant Artemia salina cysts contain desiccated gastrulae that are metabolically inactive, and physiologically arrested. Following rehydration, embryos resume development via alterations in protein expression, in the complete absence of cell division. In mammals, activation of p70 ribosomal S6 kinase (p70S6k) has been implicated in translational control, in particular the selective up-regulation of translation of mRNAs with polypyrimidine tracts at their 5' start sites. We therefore investigated ribosomal S6 kinase activity in preemergence development. We demonstrate that an S6 kinase activity is rapidly stimulated (within < 15 min) following rehydration and coincides with the onset of ribosomal S6 subunit phosphorylation. This S6 kinase activity displays chromatographic and biochemical characteristics that are similar to those of mammalian p70S6k. Partially purified Artemia S6 kinase was inactivated by treatment with protein phosphatase 2A. Activation of S6 kinase activity was shown to be due to an enzymatic step(s), and not simply rehydration of stored, active enzyme. The temporal profile of activation of S6 kinase activity is compatible with a regulatory function for p70S6k in early preemergence development of encysted Artemia. These studies identify activated Artemia cysts as a system for biochemical studies of p70S6k regulation.

Cyclic AMP inhibitors inhibits PDGF-stimulated mitogen-activated protein kinase activity in rat aortic smooth muscle cells via inactivation of c-Raf-1 kinase and induction of MAP kinase phosphatase-1.

In rat aortic smooth muscle cells (RASMC), pretreatment with forskolin inhibited the activation of p42/44 isoforms of mitogen-activated protein kinase (MAP) kinase stimulated in response to low concentrations of PDGF (10 ng/ml). This correlated with a strong inhibition of PDGF-stimulated MEK and C-Raf-1 kinase activity. However, the effect of forskolin could be surmounted by increasing the concentration of PDGF. Under such conditions forskolin was only effective against prolonged MAP kinase activation. The ability of forskolin to inhibit the late phase of MAP kinase activity was reversed by pretreatment of the cells with cycloheximide, suggesting the involvement of a protein synthesis step. This was not due to effects upstream of MAP kinase since PDGF-stimulated MEK activation was decreased by cycloheximide, an effect potentiated by forskolin. Forskolin stimulated the induction of the dual specific phosphatase MAP kinase phosphatase-1 (MKP-1), although this effect was small relative to levels induced by PDGF and angiotensin II. However, PDGF stimulated induction of MKP-1 was abolished by the protein kinase A inhibitor H89 and this correlated with the reversal of forskolin-mediated inhibition of PDGF-stimulated MAP kinase activity. These studies implicate a role for intracellular cyclic AMP in at least two aspects of MAP kinase signaling, including both the inhibition of Raf-1 activation and the induction of MKP-1.

Role of receptor desensitization, phosphatase induction and intracellular cyclic AMP in the termination of mitogen-activated protein kinase activity in UTP-stimulated EAhy 926 endothelial cells.

We have investigated the mechanisms that bring about the termination of mitogen-activated protein kinase (MAP kinase) activation in response to UTP in EAhy 926 endothelial cells. UTP-stimulated MAP kinase activity was transient, returning to basal values by 60 min. At this time MAP kinase activation was desensitized; re-application of UTP did not further activate MAP kinase, full re-activation of MAP kinase being only apparent after a 1-2 h wash period. However, activation of MAP kinase by UTP could be sustained beyond 60 min by preincubation of the cells with the protein synthesis inhibitor cycloheximide. UTP also stimulated expression of MAP kinase phosphatase-1 and this was abolished after pretreatment with cycloheximide. Pretreatment of cells with forskolin abolished the initial activation of MAP kinase kinase or c-Raf-1 by UTP, but only affected MAP kinase activity during prolonged stimulation. The effect of forskolin on prolonged MAP kinase activation was also prevented by cycloheximide. These results suggest that the termination of MAP kinase activity in response to UTP involves a number of interacting mechanisms including receptor desensitization and the induction of a phosphatase. However, several pieces of evidence do not support a major role for MAP kinase phosphatase-1 in termination of the MAP kinase signal. Raising intracellular cyclic AMP may also be involved but only after an initial protein-synthesis step and by a mechanism that does not involve the inactivation of c-Raf-1 or MAP kinase kinase.

The role of protein kinase C in activation and termination of mitogen-activated protein kinase activity in angiotensin II-stimulated rat aortic smooth-muscle cells.

Mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases activated by both tyrosine kinase and G-protein-linked receptor agonists. In rat aorta vascular smooth-muscle cells (VSMC), vasoconstrictors, angiotension II (AII), and alpha-thrombin (alpha-thr), as well as platelet-derived growth factor beta beta (PDGF) stimulated the tyrosine phosphorylation and activation of MAP kinase in a time- and concentration-dependent manner. Pre-treatment of cells with the protein kinase C (PKC) inhibitor Ro-318220, inhibited the initial increase in tyrosine phosphorylation of MAP kinase in response to vasoconstrictors, suggesting the involvement of PKC. Four isoforms of PKC were identified in VSMC by western blotting: alpha, beta, epsilon, and zeta. Downregulation of PKC alpha and PKC epsilon isoforms following chronic phorbol myristate 12, 13-acetate (PMA) pre-treatment resulted in the abolition of AII-stimulated MAP kinase activation. Selective downregulation of PKC alpha following pre-treatment with bryostatin 1 did not affect AII-stimulated MAP kinase. Preincubation of cells with Ro-318220 enhanced the activation of MAP kinase at later time points. In addition, Ro-318220 pre-treatment inhibited the induction by AII of a novel transcriptionally regulated phosphatase, MAP kinase phosphatase-1 (MKP-1). However, AII-mediated activation of MAP kinase was not prolonged by cycloheximide pre-treatment and was not maintained indefinitely by Ro-318220. These results demonstrate a specific role for the Ca(2+)-independent PKC isoform, PKC epsilon, in the activation of MAP kinase in response to vasoconstrictors, and suggest that PKC-mediated induction of MKP-1 plays no role in the termination of transiently activated MAP kinase.

Chronic stress down-regulates growth hormone gene expression in peripheral blood mononuclear cells of older adults.

"Pituitary" peptides are produced in both endocrine and immune cells. Acute and chronic stress can alter pituitary peptide secretion and might also influence neuroendocrine gene expression in human immune cells. We reasoned that, in Alzheimer caregivers, the chronic stress of caregiving would impact on the sympathetic-adrenal-medullary and hypothalamicpituitary-adrenal axis possibly leading to alterations in GH mRNA in their peripheral blood mononuclear cells (PBMCs). Therefore, we evaluated 10 caregivers and 10 controls subjects using a math and speech stress protocol to determine their neuroendocrine profile and to evaluate any relationship with mononuclear cell GH mRNA levels simultaneously acquired and then evaluated by a quantitative competitive RT-PCR technique. We found a significant (p<.0001) decrease 50% in GH mRNA levels in cells from caregivers. Plasma ACTH and norepinephrine levels were negatively correlated with GH mRNA levels, suggesting their possible role in the down-regulation of mononuclear cell GH gene expression. These observations support the hypothesis that experiences associated with caregiving alter the brain's autonomic nervous system and neuroendocrine control of the hypothalamic-pituitary axis. These and perhaps other influences may then produce altered GH gene expression in mononuclear cells of chronically stressed individuals. It is tempting to speculate that the decreased GH mRNA that we found in these chronically stressed caregivers was partially responsible for their poor response to influenza vaccine and their delayed wound healing.

Stimulation by endothelin-1 of mitogen-activated protein kinases and DNA synthesis in bovine tracheal smooth muscle cells.

1. In cultures of bovine tracheal smooth muscle cells, platelet-derived growth factor-BB (PDGF), bradykinin (BK) and endothelin-1 (ET-1) stimulated the tyrosine phosphorylation and activation of both pp42 and pp44 kDa forms of mitogen-activated protein (MAP) kinase. 2. Both ET-1 and PDGF stimulated a sustained activation of MAP kinase whilst the response to BK was transient. 3. Activation of MAP kinase occurred in a concentration-dependent manner (EC50 values: ET-1, 2.3 +/- 1.3 nM; BK, 8.7 +/- 4.1 nM, PDGF, 9.7 +/- 3.2 ng ml-1). 4. Pretreatment with the protein kinase C (PKC) inhibitor Ro-318220, significantly reduced ET-1 activation of MAP kinase at 2 and 5 min but enhanced MAP kinase activation at 60 min. 5. Following chronic phorbol ester pretreatment, BK-stimulated activation of MAP kinase was abolished whilst the responses to PDGF and ET-1 were only partly reduced (80 and 45% inhibition respectively). 6. Pretreatment with pertussis toxin reduced ET-1 stimulated activation of MAP kinase particularly at later times (60 min), but left the responses to both PDGF and BK unaffected. 7. ET-1 also stimulated a 3 fold increase in [3H]-thymidine incorporation which was abolished by pertussis toxin pretreatment. In contrast, PDGF stimulated a 131 fold increase in [3H]-thymidine incorporation which was not affected by pertussis toxin. 8. These results suggest that a pertussis toxin-sensitive activation of MAP kinase may play an important role in ET-1-stimulated DNA synthesis but that activation of MAP kinase alone is not sufficient to induce the magnitude of DNA synthesis observed in response to PDGF.

Regulation of lysophosphatidic acid-stimulated tyrosine phosphorylation of mitogen-activated protein kinase by protein kinase C- and pertussis toxin-dependent pathways in the endothelial cell line EAhy 926.

In the endothelial cell line EAhy 926, 1-oleoyl-lysophosphatidic acid (LPA) stimulated the tyrosine phosphorylation of the pp42 isoform of mitogen-activated protein (MAP) kinase. Maximum phosphorylation was observed within 5 min of LPA addition, but the response was sustained for up to 120 min. Re-addition of LPA after 60 min stimulated a further sustained increase in the tyrosine phosphorylation of MAP kinase. In cells pretreated with phorbol 12-myristate 13-acetate (PMA; 24 h) or preincubated with the protein kinase C inhibitor Ro-318220, LPA-induced tyrosine phosphorylation of pp42 MAP kinase was substantially reduced at 2 min but potentiated at 60 min. Ro-318220 in combination with either PMA or pertussis toxin pretreatment abolished the LPA response at all time points, suggesting an involvement of protein kinase C in the pertussis toxin-sensitive part of the pathway. Agents which raised intracellular cyclic AMP levels did not affect the initial phase of LPA-stimulated MAP kinase activation, but abolished the late phase. However, this effect was prevented by Ro-318220, implicating a greater role for protein kinase C than protein kinase A in the regulation of sustained MAP kinase responses. LPA stimulated an increase in the tyrosine phosphorylation of focal adhesion kinase pp125 (pp125FAK) in EAhy 926 cells which was both protein kinase C- and pertussis toxin-independent. These results are discussed in terms of the pathways regulating both MAP kinase and pp125FAK in response to LPA in the EAhy 926 endothelial cells line.

The role of tryptophan 97 of cytochrome P450 BM3 from Bacillus megaterium in catalytic function. Evidence against the 'covalent switching' hypothesis of P-450 electron transfer.

The 'Covalent Switching' hypothesis suggests that a strongly conserved tryptophan residue acts as a mediator of electron-transfer flow between redox partners in cytochrome P-450 systems [Baldwin, Morris and Richards (1991) Proc. R. Soc. London B 245, 43-51]. We have investigated the effect of alteration of the conserved tryptophan (Trp-97) in cytochrome P-450 BM3 (P-450 102) from Bacillus megaterium. Replacement of Trp-97 with Ala, Phe or Tyr results in a decrease in the natural haem content and alters the resting spin state of the remaining haem in the purified mutant enzymes. However, kinetic analyses indicate that the mutant enzymes retain high levels of catalytic activity. C.d. and e.p.r. spectroscopy also reveal little alteration in secondary structure or change in the pattern of haem ligation. These findings cast doubt on the covalent switching mechanism of intermolecular electron flow in the P-450s, but indicate that this residue plays a role in the association of the haem prosthetic group.

Regulation of endothelin-1- and lysophosphatidic acid-stimulated tyrosine phosphorylation of focal adhesion kinase (pp125fak) in Rat-1 fibroblasts.

The characteristics of protein tyrosine phosphorylation were examined in Rat-1 fibroblasts in response to endothelin-1 (ET-1) and 1-oleoyl-lysophosphatidic acid (LPA). Both agonists stimulated the biphasic tyrosine phosphorylation of at least three major proteins of approx. 120 kDa (pp116, pp120 and pp130) and two of 80 kDa (pp80 and pp70). Immunoprecipitation experiments indicated that the pp120 protein corresponded to the recently described focal adhesion protein kinase pp125fak. Phorbol 12-myristate 13-acetate, alone or in combination with the calcium ionophore A23187, also stimulated the phosphorylation of pp125fak but to a smaller extent than LPA or ET-1. Removal of both extracellular and intracellular Ca2+ did not significantly reduce LPA- and ET-1-stimulated tyrosine phosphorylation of pp125fak. In cells where protein kinase C activity was down-regulated or inhibited, ET-1-stimulated tyrosine phosphorylation of pp125fak was reduced to a greater extent than phosphorylation in response to LPA. In addition, ET-1-stimulated tyrosine phosphorylation of pp80 was decreased by 50-70% in response to protein kinase C inhibition at both 2 and 60 min whereas LPA-stimulated tyrosine phosphorylation of this protein was only reduced at 2 min. Pretreatment with pertussis toxin reduced the tyrosine phosphorylation of pp42 and pp44 forms of mitogen-activated protein kinase in response to both ET-1 and LPA but reduced the tyrosine phosphorylation of pp125fak only in response to LPA. These results indicate agonist-specific differences in the regulation of pathways mediating the tyrosine phosphorylation of pp125fak and other target proteins.