Crystal structure of the H256A mutant of rat testis fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase. Fructose 6-phosphate in the active site leads to mechanisms for both mutant and wild type bisphosphatase activities

Fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase (Fru-6-P, 2-kinase/Fru-2,6-Pase) is a bifunctional enzyme, catalyzing the interconversion of beta-D-fructose- 6-phosphate (Fru-6-P) and fructose-2,6-bisphosphate (Fru-2,6-P2) at distinct active sites. A mutant rat testis isozyme with an alanine replacement for the catalytic histidine (H256A) in the Fru-2,6-Pase domain retains 17% of the wild type activity (Mizuguchi, H., Cook, P. F., Tai, C-H., Hasemann, C. A., and Uyeda, K. (1998) J. Biol. Chem. 274, 2166-2175). We have solved the crystal structure of H256A to a resolution of 2. 4 A by molecular replacement. Clear electron density for Fru-6-P is found at the Fru-2,6-Pase active site, revealing the important interactions in substrate/product binding. A superposition of the H256A structure with the RT2K-Wo structure reveals no significant reorganization of the active site resulting from the binding of Fru-6-P or the H256A mutation. Using this superposition, we have built a view of the Fru-2,6-P2-bound enzyme and identify the residues responsible for catalysis. This analysis yields distinct catalytic mechanisms for the wild type and mutant proteins. The wild type mechanism would lead to an inefficient transfer of a proton to the leaving group Fru-6-P, which is consistent with a view of this event being rate-limiting, explaining the extremely slow turnover (0. 032 s-1) of the Fru-2,6-Pase in all Fru-6-P,2-kinase/Fru-2,6-Pase isozymes.     

Regulation of connexin-43 gap junctional intercellular communication by mitogen-activated protein kinase

Activation of the Ras/Raf/mitogen-activated protein kinase kinase/mitogen-activated protein (MAP) kinase signaling cascade is initiated by activation of growth factor receptors and regulates many cellular events, including cell cycle control. Our previous studies suggested that the connexin-43 gap junction protein may be a target of activated MAP kinase and that MAP kinase may regulate connexin-43 function. We identified the sites of MAP kinase phosphorylation in in vitro studies as the consensus MAP kinase recognition sites in the cytoplasmic carboxyl tail of connexin-43, Ser255, Ser279, and Ser282. In this study, we demonstrate that activation of MAP kinase by ligand-induced activation of the epidermal growth factor (EGF) or lysophosphatidic acid receptors or by pervanadate-induced inhibition of tyrosine phosphatases results in increased phosphorylation on connexin-43. EGF and lysophosphatidic acid-induced phosphorylation on connexin-43 and the down-regulation of gap junctional communication in EGF-treated cells were blocked by a specific mitogen-activated protein kinase kinase inhibitor (PD98059) that prevented activation of MAP kinase. These studies confirm that connexin-43 is a MAP kinase substrate in vivo and that phosphorylation on Ser255, Ser279, and/or Ser282 initiates the down-regulation of gap junctional communication. Studies with connexin-43 mutants suggest that MAP kinase phosphorylation at one or more of the tandem Ser279/Ser282 sites is sufficient to disrupt gap junctional intercellular communication.     

Reaction mechanism of fructose-2,6-bisphosphatase. A mutation of nucleophilic catalyst, histidine 256, induces an alteration in the reaction pathway

A bifunctional enzyme, fructose-6-phosphate,2-kinase/fructose 2, 6-bisphosphatase (Fru-6-P,2-kinase/Fru-2,6-Pase), catalyzes synthesis and degradation of fructose 2,6-bisphosphate (Fru-2,6-P2). Previously, the rat liver Fru-2,6-Pase reaction (Fru-2,6-P2 --> Fru-6-P + Pi) has been shown to proceed via a phosphoenzyme intermediate with His258 phosphorylated, and mutation of the histidine to alanine resulted in complete loss of activity (Tauler, A., Lin, K., and Pilkis, S. J. (1990) J. Biol. Chem. 265, 15617-15622). In the present study, it is shown that mutation of the corresponding histidine (His256) of the rat testis enzyme decreases activity by less than a factor of 10 with a kcat of 17% compared with the wild type enzyme. Mutation of His390 (in close proximity to His256) to Ala results in a kcat of 12.5% compared with the wild type enzyme. Attempts to detect a phosphohistidine intermediate with the H256A mutant enzyme were unsuccessful, but the phosphoenzyme is detected in the wild type, H390A, R255A, R305S, and E325A mutant enzymes. Data demonstrate that the mutation of His256 induces a change in the phosphatase hydrolytic reaction mechanism. Elimination of the nucleophilic catalyst, H256A, results in a change in mechanism. In the H256A mutant enzyme, His390 likely acts as a general base to activate water for direct hydrolysis of the 2-phosphate of Fru-2,6-P2. Mutation of Arg255 and Arg305 suggests that the arginines probably have a role in neutralizing excess charge on the 2-phosphate and polarizing the phosphoryl for subsequent transfer to either His256 or water. The role of Glu325 is less certain, but it may serve as a general acid, protonating the leaving 2-hydroxyl of Fru-2,6-P2.     

Control of glycogen synthesis in cultured human muscle cells

The regulation of glycogen synthesis and associated enzymes was studied in human myoblasts and myotubes maintained in culture. Both epidermal growth factor (EGF) and insulin stimulated glycogen synthesis approximately 2-fold, this stimulation being accompanied by a rapid and stable activation of the controlling enzyme glycogen synthase (GS). EGF also caused inhibition of glycogen synthase kinase 3 (GSK-3) and activation of the alpha isoform of protein kinase B (PKB) with the time-course and magnitude of its effects being similar to those induced by insulin. An inhibitor of the mitogen-activated protein (MAP) kinase pathway did not prevent stimulation of GS by EGF, suggesting that this pathway is not essential for the effect. A partial decrease in the fold activation of GS was, however, observed when p70(S6k) activation was blocked with rapamycin, suggesting a contribution of this pathway to the control of GS by either hormone. Wortmannin, a selective inhibitor of phosphatidylinositol 3'-kinase (PI-3 kinase) completely blocked the effects of both EGF and insulin in these cells. These results demonstrate that EGF, like insulin, activates glycogen synthesis in muscle, acting principally via the PKB/GSK-3 pathway but with a contribution from a rapamycin-sensitive component that lies downstream of PI-3 kinase.     

Mitochondrial malic enzyme: purification from bovine brain, generation of an antiserum, and immunocytochemical localization in neurons of rat brain

The receptor kinase activity associated with the epidermal growth factor (EGF) receptor and platelet-derived growth factor (PDGF) receptor plays an important role in ligand-induced signaling events. The effect of specific, synthetic chemical inhibitors of PDGF- and EGF-mediated receptor tyrosine autophosphorylation on receptor signaling were examined in NIH 3T3 cells overexpressing PDGF or EGF receptors. Specific inhibition of ligand-dependent receptor autophosphorylation, PI3K activation, mitogen-activated protein kinase (MAPK) activation, cyclin E-associated kinase activity and cell proliferation was measured after treatment of cells with these inhibitors. A synthetic PDGF receptor kinase inhibitor exhibited specific inhibitory properties when tested for PDGF-induced receptor autophosphorylation, MAPK activity, PI3K activation, entry into S phase and cyclin E-associated kinase activity. A synthetic EGF receptor kinase inhibitor showed selective inhibitor properties when tested for EGF-induced receptor autophosphorylation, MAPK activation, PI3K activation, entry into S phase and cyclin E-associated kinase activity. In both cases, these compounds were found to be effective as inducers of growth arrest and accumulation of cells in the G1 phase of the cell cycle after ligand treatment. However, at high concentrations, the EGF receptor kinase inhibitor was observed to exhibit some nonspecific effects as demonstrated by attenuation of PDGF-induced receptor autophosphorylation and cell cycle progression. This demonstrates that it is critical to use the lowest concentration of such an inhibitor that will alter the response under investigation, to have confidence that the conclusions derived from the use of such inhibitor are valid. We conclude that these experimental parameters signify useful end points to measure the relative selectivity of tyrosine kinase inhibitors that affect receptor-mediated signal transduction.     

Differential utilization of ShcA tyrosine residues and functional domains in the transduction of epidermal growth factor-induced mitogen-activated protein kinase activation in 293T cells and nerve growth factor-induced neurite outgrowth in PC12 cells. Identification of a new Grb2.Sos1 binding site

By transient expression of both truncated forms of p52(SHCA) and those with point mutations in 293T cells, it has been shown that, in addition to Tyr-317, Tyr-239/240 is a major site of phosphorylation that serves as a docking site for Grb2.Sos1 complexes. In addition, analysis of epidermal growth factor (EGF)-induced activation of mitogen-activated protein kinase in 293T cells showed that the overexpression Shc SH2 or phosphotyrosine binding (PTB) domains of ShcA alone has a more potent negative effect than the overexpression of the forms of ShcA lacking Tyr-317 or Tyr 239/240 or both. In transiently transfected PC12 cells, the ShcA PTB domain and tyrosine phosphorylation in the CH1 domain, especially on Tyr-239/240, are crucial for mediating nerve growth factor (NGF)-induced neurite outgrowth. These findings suggest that the EGF and NGF (TrkA) receptor can utilize Shc in different ways to promote their activity. For EGF-induced mitogen-activated protein kinase activation in 293T cells, both Shc PTB and SH2 domains are essential for optimal activation, indicating that a mechanism independent of Grb2 engagement with Shc may exist. For NGF-induced neurite outgrowth in PC12 cells, Shc PTB plays an essential role, and phosphorylation on Tyr-239/240, but not on Tyr-317, is required.     

Expression of bone morphogenetic protein 6 in normal mammary tissue and breast cancer cell lines and its regulation by epidermal growth factor

Bone morphogenetic proteins (BMPs) are multifunctional regulators of proliferation, differentiation and apoptosis. BMP-6 is involved in numerous developmental processes. We have demonstrated expression of BMP-6 in breast cancer cell lines by RT-PCR and immuno-histochemistry. The level of BMP-6 mRNA decreased upon serum starvation, whereas epidermal growth factor (EGF) treatment led to elevation of BMP-6 mRNA levels in a dose-dependent manner, with a maximum at 50 ng/ml EGF under serum-free conditions in hormone-sensitive (MCF-7) and in hormone-insensitive (SK-BR-3) breast cancer cell lines. The EGF-like growth factors transforming growth factor-alpha, amphiregulin and betacellulin were also able to elevate the BMP-6 mRNA level after 24 hr. Inhibition of EGF receptor tyrosine kinase with tyrphostine AG 1517 repressed the inductive effect of these growth factors, indicating an EGF receptor-mediated regulation of BMP-6 mRNA. In addition, BMP-6 mRNA was detected in tumor samples from breast carcinoma patients. However, levels were reduced in 18/44 samples compared with tumor-free resection margins. In 12 of these 18 patients, at least a 10-fold reduction of EGF receptor mRNA levels in tumor samples vs. tumor-free samples was observed. This suggests a putative relationship between EGF receptor and BMP-6 mRNA levels in breast cancer.     

Reduction in numerical synapse density in chick (Gallus domesticus) dorsal hippocampus following transient cerebral ischaemia

BACKGROUND: The epidermal growth factor (EGF) signal transduction pathway, frequently activated in pancreatic cancer, is an important regulator of cellular growth and transformation. This study examined whether activation of the cyclic adenosine monophosphate protein kinase A pathway may inhibit the EGF signal transduction pathway in pancreatic cancer cell lines. METHODS: Human pancreatic cancer lines BxPC-3 and AsPC-1 were stimulated with EGF, forskolin, or both. Forskolin is a compound that increases cyclic adenosine monophosphate levels. Assays of cell lines were then obtained for cellular growth (MTT assay), anchorage-independent growth (soft agar), and EGF-induced mitogen-activated protein kinase activation as measured by an in-gel kinase assay. RESULTS: Treatment with forskolin resulted in inhibition of EGF-induced activation of mitogen-activated protein kinase activity (BxPC-3 78% inhibition and AsPC-1 70% inhibition, p < 0.005), diminished cellular proliferation (BxPC-3 92% inhibition and AsPC-1 86% inhibition, p < 0.001), and formation of colonies in soft agar (BxPC-3 98% inhibition and AsPC-1 76% inhibition, p < 0.001). Forskolin did not inhibit EGF receptor autophosphorylation or tyrosine kinase signaling in response to EGF. CONCLUSIONS: Forskolin-induced inhibition of mitogen-activated protein kinase is associated with diminished pancreatic cancer cell proliferation in vitro. Use of strategies to increase cyclic adenosine monophosphate levels may have therapeutic application in pancreatic cancer.     

Regulation of prostaglandin H synthase-2 expression in cerebromicrovascular smooth muscle by serum and epidermal growth factor

Growth factors may play a role in the formation of prostaglandins (PG) by cerebral blood vessels during development or reaction to injury. In smooth muscle cultures isolated from murine cerebral microvessels PG production was induced with either serum or epidermal growth factor (EGF). Prostaglandin H synthase (PGHS) activity peaked at 6 h after the addition of 10% serum or 50 ng/ml EGF. Increases in expression of PGHS-1 mRNA were small (7- to 10-fold) compared with PGHS-2 (30- to 120-fold), and the induction patterns were different for serum and EGF. An increase in PGHS-2 message was detected by 0.5 h of adding either agent, but peak induction occurred earlier for EGF than for serum, 1 h vs. 3 h, respectively. The response to either stimulus had returned to prestimulation levels by 12 h. The induction of PGHS-2 protein was also transient, but followed a more delayed time course (peak levels at 6 h). Induction of activity, message, and protein by either agent was blocked by 1 microM dexamethasone and attenuated by genistein (100 microM), a nonspecific tyrosine kinase inhibitor. Tyrphostin 47, a more selective EGF receptor tyrosine kinase inhibitor, dose-dependently inhibited EGF-stimulated PGHS activity, completely abolishing PG production at 100 microM. However, this inhibitor had no effect on serum-stimulated PG production. Curiously, 100 microM tyrphostin 47 enhanced EGF-induced PGHS-2 mRNA and protein expression. These data suggest that EGF induces the expression of PGHS-2 in cerebromicrovascular smooth muscle by a mechanism that requires tyrosine kinase activity and that is distinct from serum.     

The diaphragm and respiratory muscles

A rapid procedure for the purification of the redox-regulated chloroplast fructose-1,6-bisphosphatase [EC 3.1.3.11] from spinach leaf extract to homogeneity is described. No thiol-reducing agents were present during the purification and the enzyme is > 99% in the oxidized form. A rapid procedure to reduce and activate the Fru-1,6-P2ase by dithiothreitol in the absence of thioredoxin is described. Reduction activates the enzyme up to several hundred-fold when assayed at pH 8.0 with 2 mM Mg2+. The activity of the purified oxidized enzyme is unusually sensitive to changes in Mg2+ and H+ concentration. Tenfold changes in Mg2+ or H+ concentration lead to > 100-fold increases in activity. The recoveries of fructose-1,6-bisphosphatase activity as determined by the activity of the oxidized enzyme at pH 8.0/20 mM Mg2+; pH 9.0/2 mM Mg2+; pH 8/2 mM Mg2+ plus 0.1 mM Hg(II) or of the reduced enzyme at pH 8.0/2 mM Mg2+ are similar (approximately 40%) indicating that the major proportion of these activities in a leaf extract is catalyzed by the same enzyme. Moreover, antibodies raised against the purified enzyme inhibit all of the above activities in crude leaf extracts. The kinetic properties of the purified enzyme suggest that the oxidized Mg(2+)-dependent enzyme can play no significant role in photosynthetic carbon assimilation. A survey of some kinetic properties of Fru-1,6-P2ase activity in extracts of various photosynthetic organisms reveals that all 11 species examined possess a redox- and pH/Mg(2+)-stimulated Fru-1,6-P2ase, whereas Fru-1,6-P2ase in extracts of Taxus baccata (a gymnosperm), Chlorella vulgaris (a green alga), and the cyanobacterium Nostoc muscorum were not activated by Hg(II). The heat stability that proved useful in the purification of the spinach enzyme was conserved in both angiosperms and gymnosperms. The oxidized enzyme (which normally has no thiol groups accessible to 5,5'-dithio-bis[2-nitrobenzoic acid]) but not the reduced enzyme can be stimulated many hundred-fold by addition of extraordinarily low concentrations of Hg(II) to a complete assay mixture. With the aid of EDTA as a Hg(II) buffer, half-maximal stimulation was achieved at 2 x 10(-16) M free Hg(II). Methylmercury also stimulates the enzyme many hundred-fold at very low concentrations. The concentration for half-maximal stimulation by methylmercury was determined with a cyanide buffer to be approximately 10(-16) M. This, together with the high affinity of the enzyme for Hg(II), suggests that Hg(II) stimulates the enzyme by binding to an enzyme thiol group that be comes exposed in the catalytically active enzyme, thereby stabilizing the oxidized enzyme in an active conformation. By contrast, in the absence of Fru-1,6-P2 and either Mg2+ or Ca2+, Hg(II) (even at 2 x 10(-16) M) rapidly inactivates the oxidized Fru-1,6-P2ase. This inactivation is similar to the inactivation of Fru-1,6-P2ase that occurred at high pH (> 9) and which is also prevented by Fru-1,6-P2 and either Mg2+ or Ca2+. Although the Hg(II)- and high pH-inactivated oxidized enzyme has no activity, both forms of the enzyme can be activated by reduction. The usefulness of buffers to maintain low, defined Hg(II) and organic mercurial concentrations is discussed.     

Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt

We have found that insulin-like growth factor I (IGF-I) can protect fibroblasts from apoptosis induced by UV-B light. Antiapoptotic signalling by the IGF-I receptor depended on receptor kinase activity, as cells overexpressing kinase-defective receptor mutants could not be protected by IGF-I. Overexpression of a kinase-defective receptor which contained a mutation in the ATP binding loop functioned as a dominant negative and sensitized cells to apoptosis. The antiapoptotic capacity of the IGF-I receptor was not shared by other growth factors tested, including epidermal growth factor (EGF) and thrombin, although the cells expressed functional receptors for all the agonists. However, EGF was antiapoptotic for cells overexpressing the EGF receptor, and expression of activated pp60v-src also was protective. There was no correlation between protection from apoptosis and activation of mitogen-activated protein kinase, p38/HOG1, or p70S6 kinase. On the other hand, protection by any of the tyrosine kinases against UV-induced apoptosis was blocked by wortmannin, implying a role for phosphatidylinositol 3-kinase (PI3 kinase). To test this, we transiently expressed constitutively active or kinase-dead PI3 kinase and found that overexpression of activated phosphatidylinositol 3-kinase (PI3 kinase) was sufficient to provide protection against apoptosis. Because Akt/PKB is believed to be a downstream effector for PI3 kinase, we also examined the role of this serine/threonine protein kinase in antiapoptotic signalling. We found that membrane-targeted Akt was sufficient to protect against apoptosis but that kinase-dead Akt was not. We conclude that the endogenous IGF-I receptor has a specific antiapoptotic signalling capacity, that overexpression of other tyrosine kinases can allow them also to be antiapoptotic, and that activation of PI3 kinase and Akt is sufficient for antiapoptotic signalling.     

MAP kinase mediates epidermal growth factor- and phorbol ester-induced prostacyclin formation in cardiomyocytes

We studied the role of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in epidermal growth factor (EGF)-induced prostacyclin (PGI2) production in cultured, spontaneously-beating neonatal ventricular rat cardiomyocytes. To this purpose, the effect of EGF on cardiomyocyte MAPK phosphorylation, MAPK activity and PGI2-production were investigated, and compared to those induced by the PKC activator 4 beta phorbol 12-myristate 13-acetate (PMA). Both EGF (0.1 microM) and PMA (0.1 microM) induced the rapid and reversible phosphorylation of 42 KDa-MAPK in ventricular cardiomyocytes, responses that were accompanied by transient increases in MAPK activity (190-230% of control values within 5 min), and two- to three-fold increases in PGI2 formation. The tyrosine kinase inhibitors lavendustin (1 microM) and genistein (10 microM) strongly inhibited EGF-induced MAPK activation and PGI2-formation, but had no effect on PMA-stimulated responses. Experiments with the PKC inhibitor CGP 41251 (1 microM) or with PKC-downregulated cells demonstrated that in contrast to the PMA-stimulated responses, EGF-induced MAPK activation and PGI2-production were PKC-independent processes. Investigating the role of MAPK in EGF- and in PMA-promoted PGI2-formation, we found that the MAPK-inhibitor 6-thioguanine (500 microM), as well as the MAPK-kinase-inhibitor PD98059 (50 microM) abolished both EGF- and PMA-stimulated PGI2-production in cardiomyocytes. Our results indicate that MAPK-activation is at the basis of both growth factor receptor and PKC-dependent eicosanoid-formation in ventricular cardiomyocytes, where EGF-induced prostaglandin-production takes place via a PKC-independent pathway.     

Involvement of protein kinase C during activation of the mitogen-activated protein kinase cascade by leukemia inhibitory factor. Evidence for participation of multiple signaling pathways

We show here that treatment of 3T3-L1 cells with leukemia inhibitory factor (LIF) stimulates the activation of mitogen-activated protein kinase kinase (MAPKK), mitogen-activated protein kinase (MAPK), and S6 protein kinase (S6K) activities both in a time- and dose-dependent manner. A single peak of MAPKK activity, four peaks of activity against the S6 synthetic peptide, RRLSSLRA (S6 peptide), and three distinct peaks toward myelin basic protein (MBP) were observed after Mono-Q chromatography of LIF-stimulated cell extracts. Two of the MBP kinase activities correlated with the stimulation of extracellular signal-regulated kinases 1 and 2. Interestingly, down-regulation of protein kinase C (PKC) by chronic treatment of 3T3-L1 cells with phorbol ester was found to attenuate, but not block, the LIF-mediated stimulation of MAPKK, MAPK, and S6K activities in 3T3-L1 cells. Treatment of 3T3-L1 cells with epidermal growth factor increased MAPKK, MAPK, and S6K activities to a similar extent as LIF, but this activation was not attenuated by down-regulation of PKC. Our results suggest that the full activation of the MAPK cascade by LIF may require inputs from multiple signaling pathways, one of which is dependent upon the presence of functional PKC.