Pp60 Src Research Articles

Evaluation of the antiaggregant activity of ascorbyl phenolic esters with antioxidant properties.

Beneficial effects of the antioxidant L-ascorbic acid (Asc) in human health are well known. Its particular role in hemostasis deserves further consideration, since it has been described a dose-dependent effect of Asc in platelet activity. Contrary, it has been demonstrated that phenolic compounds have inhibitory effects on platelet aggregation stimulated by the physiological agonist thrombin (Thr). Here, we have evaluated the actions of three synthetic phenolic esters of Asc: L-ascorbyl 6-protocatechuate (Prot Asc), L-ascorbyl 6-gallate (Gal Asc), and L-ascorbyl 6-caffeate (Caf Asc). All these Asc derivatives exhibited greater radical scavenging activity than Asc, and in experiments using human platelets from healthy subjects, they do not evoke changes in platelet viability upon their administration. Nevertheless, these compounds altered platelet calcium homeostasis in response to Thr, although Prot Asc induced a smaller effect than Gal Asc, Caf Asc, and Asc. As a consequence, platelet aggregation was also impaired by these compounds, reporting Prot Asc and Caf Asc a weaker antiaggregant action than Gal Asc and Asc. Treatments with Gal Asc and Caf Asc altered in larger extent the phosphorylation pattern of pp60(Src) and mammalian target of rapamycin (mTOR) evoked by stimulating human platelets with Thr. Summarizing, Prot Asc is the ascorbyl phenolic ester with the strongest antioxidant properties and weakest antiaggregant actions, and its use as antioxidant may be safer than the rest of derivatives in order to prevent thrombotic alteration in patients that need treatment with antioxidant therapies.

Lipid Segregation and Membrane Budding Induced by the Peripheral Membrane Binding Protein Annexin A2*

The formation of dynamic membrane microdomains is an important phenomenon in many signal transduction and membrane trafficking events. It is driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Here we analyzed the ability of one peripherally associated membrane protein, annexin A2 (AnxA2), to induce the formation of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-rich domains in giant unilamellar vesicles (GUVs) of complex lipid composition. AnxA2 is a cytosolic protein that can bind PI(4,5)P2 and other acidic phospholipids in a Ca(2+)-dependent manner and that has been implicated in cellular membrane dynamics in endocytosis and exocytosis. We show that AnxA2 binding to GUVs induces lipid phase separation and the recruitment of PI(4,5)P2, cholesterol and glycosphingolipids into larger clusters. This property is observed for the full-length monomeric protein, a mutant derivative comprising the C-terminal protein core domain and for AnxA2 residing in a heterotetrameric complex with its intracellular binding partner S100A10. All AnxA2 derivatives inducing PI(4,5)P2 clustering are also capable of forming interconnections between PI(4,5)P2-rich microdomains of adjacent GUVs. Furthermore, they can induce membrane indentations rich in PI(4,5)P2 and inward budding of these membrane domains into the lumen of GUVs. This inward vesiculation is specific for AnxA2 and not shared with other PI(4,5)P2-binding proteins such as the pleckstrin homology (PH) domain of phospholipase Cδ1. Together our results indicate that annexins such as AnxA2 can efficiently induce membrane deformations after lipid segregation, a mechanism possibly underlying annexin functions in membrane trafficking.

Caveolin 1 is required for the activation of endothelial nitric oxide synthase in response to 17beta-estradiol.

Evidence suggests that estrogen mediates rapid endothelial nitric oxide synthase (eNOS) activation via estrogen receptor-a (ERalpha) within the plasma membrane of endothelial cells (EC). ERalpha is known to colocalize with caveolin 1, the major structural protein of caveolae, and caveolin 1 stimulates the translocation of ERalpha to the plasma membrane. However, the role played by caveolin 1 in regulating 17beta-estradiol-mediated NO signaling in EC has not been adequately resolved. Thus, the purpose of this study was to explore how 17beta-estradiol stimulates eNOS activity and the role of caveolin 1 in this process. Our data demonstrate that modulation of caveolin 1 expression using small interfering RNA or adenoviral gene delivery alters ERalpha localization to the plasma membrane in EC. Further, before estrogen stimulation ERalpha associates with caveolin 1, whereas stimulation promotes a pp60(Src)-mediated phosphorylation of caveolin 1 at tyrosine 14, increasing ERalpha-PI3 kinase interactions and disrupting caveolin 1-ERalpha interactions. Adenoviral mediated overexpression of a phosphorylation-deficient mutant of caveolin (Y14FCav) attenuated the ERalpha/PI3 kinase interaction and prevented Akt-mediated eNOS activation. Furthermore, Y14FCav overexpression reduced eNOS phosphorylation at serine1177 and decreased NO generation after estrogen exposure. Using a library of overlapping peptides we identified residues 62-73 of caveolin 1 as the ERalpha-binding site. Delivery of a synthetic peptide based on this sequence decreased ERalpha plasma membrane translocation and reduced estrogen-mediated activation of eNOS. In conclusion, caveolin 1 stimulates 17beta-estradiol-induced NO production by promoting ERalpha to the plasma membrane, which facilitates the activation of the PI3 kinase pathway, leading to eNOS activation and NO generation.

SAR of Carbon‐Linked, 2‐Substituted Purines: Synthesis and Characterization of AP23451 as a novel Bone‐Targeted Inhibitor of Src Tyrosine Kinase With In Vivo Anti‐Resorptive Activity

Targeted disruption of the pp60(src) (Src) gene has implicated this tyrosine kinase in osteoclast-mediated bone resorption and as a therapeutic target for the treatment of osteoporosis and other bone-related diseases. Here, we describe structure activity relationships of a novel series of carbon-linked, 2-substituted purines that led to the identification of AP23451 as a potent inhibitor of Src tyrosine kinase with antiresorptive activity in vivo. AP23451 features the use of an arylphosphinylmethylphosphinic acid moiety which confers bone-targeting properties to the molecule, thereby increasing local concentrations of the inhibitor to actively resorbing osteoclasts at the bone interface. AP23451 exhibited an IC50 = 68 nm against Src kinase; an X-ray crystal structure of the molecule complexed with Src detailed the molecular interactions responsible for its Src inhibition. In vivo, AP23451 demonstrated a dose-dependent decrease in PTH-induced hypercalcemia. Moreover, AP23517, a structurally and biochemically similar molecule with comparable activity (IC50 = 73 nm) except devoid of the bone-targeting element, demonstrated significantly reduced in vivo efficacy, suggesting that Src activity was necessary but not sufficient for in vivo activity in this series of compounds.

Bcr interferes with β-catenin–Tcf1 interaction

The β-catenin/Tcf complex is a downstream effector of the Wnt signalling pathway. It is a transcription complex, which activates gene expression and contributes to proliferation and tumor progression. Tcf1 in complex with β-catenin is able to activate β-catenin-dependent gene expression. We demonstrate that expressed Bcr is able to bind the transcription factor Tcf1 to disrupt the Tcf1/β-catenin complex. Phosphorylation of Bcr by the tyrosine kinase pp60 src can lead to dissociation of the transcriptionally inactive Bcr/Tcf1 complex. Thus two independent mechanisms may regulate Tcf/β-catenin-mediated transcription via Bcr: binding to β-catenin as we have previously shown and to Tcf1 as shown here.

Can free energy calculations be fast and accurate at the same time? Binding of low-affinity, non-peptide inhibitors to the SH2 domain of the src protein

The usefulness of free-energy calculations in non-academic environments, in general, and in the pharmaceutical industry, in particular, is a long-time debated issue, often considered from the angle of cost/performance criteria. In the context of the rational drug design of low-affinity, non-peptide inhibitors to the SH2 domain of the (pp60)src tyrosine kinase, the continuing difficulties encountered in an attempt to obtain accurate free-energy estimates are addressed. free-energy calculations can provide a convincing answer, assuming that two key-requirements are fulfilled: (i) thorough sampling of the configurational space is necessary to minimize the statistical error, hence raising the question: to which extent can we sacrifice the computational effort, yet without jeopardizing the precision of the free-energy calculation? (ii) the sensitivity of binding free-energies to the parameters utilized imposes an appropriate parametrization of the potential energy function, especially for non-peptide molecules that are usually poorly described by multipurpose macromolecular force fields. Employing the free-energy perturbation method, accurate ranking, within +/-0.7 kcal/mol, is obtained in the case of four non-peptide mimes of a sequence recognized by the (pp60)src SH2 domain.

High glucose-induced apoptosis through store-operated calcium entry and calcineurin in human umbilical vein endothelial cells

Diabetes mellitus causes multiple cardiovascular complications. Previous studies have shown that prolonged exposure (96 h) of human umbilical vein endothelial cells (HUVECs) to hyperglycemia causes a significant increase in apoptosis. We report here that this increase in apoptosis is associated with an increase in Ca 2+ current (whole cell patch-clamp recorded) resulting from Ca 2+ entry mediated by store-operated channels (SOCs). The number of apoptotic cells after prolonged high glucose (HG, 30 mmol/L) exposure was significantly reduced in the presence of the SOC inhibitor 2-APB or of La 3+. A marked increase (∼80%) in Ca 2+-dependent calcineurin (CN-A) phosphatase activity also occurred after prolonged HG exposure. Prolonged HG exposure-induced increase in CN-A activity was prevented by 2-APB, and selective CN-A phosphatase inhibition by FK506 or calmodulin inhibition by calmidazolium decreased HG-induced apoptosis. Blocking hydrogen peroxide production using catalase or inhibiting the tyrosine kinase pp60 src during prolonged exposure to HG, resulted in a marked decrease in apoptosis and was further associated with a significant reduction in CN-A phosphatase activity. The results demonstrate a significant role for Ca 2+ entry in HG-induced apoptosis in HUVECs, and suggest that this role is mediated via H 2O 2 generation and the action of the Ca 2+-activated protein phosphatase calcineurin.

Atypical Antipsychotics and a Src Kinase Inhibitor (PP1) Prevent Cortical Injury Produced by the Psychomimetic, Noncompetitive NMDA Receptor Antagonist MK-801

Noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists such as phencyclidine, ketamine, and MK-801 produce schizophrenia-like psychosis in humans. The same NMDA antagonists injure retrosplenial cortical neurons in adult rats. We examined the effects of atypical antipsychotics and an inhibitor of nonreceptor tyrosine kinase pp60 (Src) on the cortical injury produced by MK-801. An atypical antipsychotic (either clozapine, ziprasidone, olanzapine, quetiapine, or risperidone) or vehicle was administered to adult female Sprague-Dawley rats. PP1 (Src inhibitor), PP3 (nonfunctional analog of PP1) or vehicle (DMSO) was administered to another group of animals. After pretreatment, animals were injected with MK-801, killed 24 h after the MK-801, and injury to retrosplenial cortex assessed by neuronal Hsp70 protein expression. All atypical antipsychotics examined significantly attenuated MK-801-induced cortical damage. PP1 protected compared to vehicle, whereas PP3 did not protect. The ED50s (decrease injury by 50%) were as follows: PP1 <0.1 mg/kg; olanzapine 0.8 mg/kg; risperdal 1 mg/kg; clozapine 3 mg/kg; ziprasidone 32 mg/kg; and quetiapine 45 mg/kg. The data show that the atypical antipsychotics tested as well as a Src kinase inhibitor prevent the injury produced by the psychomimetic MK-801, and the potency of the atypical antipsychotics for preventing cortical injury was roughly similar to the potency of these drugs for treating psychosis in patients.

Ca 2+-independent activation of Bruton's tyrosine kinase is required for store-mediated Ca 2+ entry in human platelets

Store-mediated Ca 2+ entry (SMCE), which is rapidly activated by depletion of the intracellular Ca 2+ stores, is a major mechanism for Ca 2+ influx. Several studies have involved tyrosine kinases in the activation of SMCE, such as pp60 src, although at present those involved in the early activation steps are unknown. Here we report the involvement of Bruton's tyrosine kinase (Btk) in the early stages of SMCE in human platelets. Cell treatment with thrombin or thapsigargin (TG) plus ionomycin (Iono) results in rapid activation of Btk, which was independent of rise in intracellular Ca 2+ concentration ([Ca 2+] i) but dependent on H 2O 2 generation. Platelet treatment with Btk inhibitors, LFM-A13 or terreic acid, significantly reduced TG+Iono- and thrombin-evoked SMCE. Btk was rapidly activated by addition of low concentrations of H 2O 2, whose effect on Ca 2+ entry was prevented by Btk inhibitors. Our results indicate that pp60 src and Btk co-immunoprecipitate after platelet stimulation with TG+Iono, thrombin or H 2O 2. In addition, we have found that LFM-A13 impaired actin filament reorganization after store depletion and agonist-induced activation of pp60 src, while the inhibitor of pp60 src, a protein that requires actin reorganization for its activation, did not modify Btk activation, suggesting that Btk is upstream of pp60 src. We propose a role for Btk in the early steps of activation of SMCE in human platelets.

Store-operated Ca 2+ entry and tyrosine kinase pp60 src hyperactivity are modulated by hyperglycemia in platelets from patients with non insulin-dependent diabetes mellitus

We have investigated the involvement of store-operated Ca 2+ entry (SOCE) in the abnormal platelet Ca 2+ homeostasis in patients with non insulin-dependent diabetes mellitus (NIDDM). In a medium containing 180 mg/dL glucose, platelets from NIDDM patients showed an increased SOCE compared to controls. We found that tyrosine phosphorylation was elevated in platelets from NIDDM patients. Consistent with this, the activity of the tyrosine kinase pp60 src is enhanced in platelets from diabetic patients. When the experiments were performed in a medium containing 90 mg/dL both, SOCE and pp60 src activity, were similar to those found in control platelets. Our results indicate that SOCE is altered in platelets from NIDDM patients probably due to the increased activity of the tyrosine kinase pp60 src. Both, SOCE and pp60 src activity in platelets from NIDDM patients are more susceptible to the extracellular glucose concentration, which seems to be involved in the dysfunction of these mechanisms.

Expression, localization, and correlation of N-myristoyltransferase and its inhibitor in bovine eye.

N-myristoyltransferase (NMT) is a ubiquitously distributed eukaryotic enzyme that catalyzes myristoylation of proteins. Very little is known about the process of myristoylation, particularly in the eye. In the present study, the distribution, expression, and correlation of NMT and its inhibitor (NMT inhibitor protein, NIP) in the bovine eye were investigated. Whole bovine eyes were either homogenized or regionally dissected to determine the activity and localization of NMT and NIP. Dissected tissues were homogenized, and Western blot analysis was performed using polyclonal anti-NMT and anti-NIP antibodies. The NMT activity was assayed using cAMP-dependent protein kinase or pp60(src) derived peptide as a substrate. Fresh samples were then prepared for immunohistochemical analysis and probed with polyclonal anti-NMT and anti-NIP antibodies. The total bovine eye cytosolic fraction displayed both NMT and NIP expression. NMT was present in all the regions of the eye at various levels of expression. The highest expression of NMT was in the cornea, whereas NIP was present in the retina, optic nerve, sclera, and choroid only. NIP expression was the highest in the optic nerve, sclera, and retina. NMT activity was observed in the cornea, iris, and retina after DEAE-Sepharose CL-6B column chromatography. The inhibitory activity of crude homogenate on recombinant human NMT activity was found to be greater for optic nerve and choroid. Immunohistochemistry results displayed similar findings. The varied expression of NMT in different regions of the eye reveals a regulatory relationship of NMT with NIP. These findings indicate that NMT and NIP are present in various regions of the eye and will lead to further understanding of visual signaling in ocular cells.

Hydrogen Peroxide Generation Induces pp60src Activation in Human Platelets: EVIDENCE FOR THE INVOLVEMENT OF THIS PATHWAY IN STORE-MEDIATED CALCIUM ENTRY

Reactive oxygen species, such as H2O2, have been recognized as intracellular messengers involved in several cell functions. Here we report the activation of the tyrosine kinase pp60(src) by H2O2, a mechanism required for the activation of store-mediated Ca2+ entry (SMCE) in human platelets. Treatment of platelets with H2O2 resulted in a time- and concentration-dependent activation of pp60(src). Incubation with GF 109203X, a protein kinase C (PKC) inhibitor, prevented H2O2-induced pp60(src) activation. In contrast, dimethyl-BAPTA loading did not affect this response, suggesting that activation of pp60(src) by H2O2 is independent of increases in [Ca2+](i). Cytochalasin D, an inhibitor of actin polymerization, significantly reduced H2O2-induced pp60(src) activation. We found that platelet stimulation with thapsigargin (TG) plus ionomycin (Iono) or thrombin induced rapid H2O2 production, a mechanism independent of elevations in [Ca2+](i). Treatment of platelets with catalase attenuated TG plus Iono- and thrombin-induced activation of pp60(src). In addition, catalase as well as the pp60(src) inhibitor, PP1, reduced both the activation of SMCE and the coupling between the hTrp1 and the IP(3)R type II without having any effect on the maintenance of SMCE. Consistent with the role of PKC in the activation of pp60(src) by H2O2, the PKC inhibitors GF 109202X and Ro-31-8220 were found to reduced SMCE in platelets. This study suggests that platelet activation with TG plus Iono or thrombin is associated with H2O2 production, which acts as a second messenger by stimulating pp60(src) by a PKC-dependent pathway and is involved in the activation of SMCE in these cells.

Requirements for specific binding of low affinity inhibitor fragments to the SH2 domain of (pp60)Src are identical to those for high affinity binding of full length inhibitors.

Results from a novel approach which uses protein crystallography for the screening of a low affinity inhibitor fragment library are analyzed by comparing the X-ray structures with bound fragments to the structures with the corresponding full length inhibitors. The screen for new phospho-tyrosine mimics binding to the SH2 domain of (pp60)src was initiated because of the limited cell penetration of phosphates. Fragments in our library typically had between 6 and 30 atoms and included compounds which had either millimolar activity in a Biacore assay or were suggested by the ab initio design program LUDI but had no measurable affinity. All identified fragments were located in the phospho-tyrosine pocket. The most promising fragments were successfully used to replace the phospho-tyrosine and resulted in novel nonpeptidic high affinity inhibitors. The significant diversity of successful fragments is reflected in the high flexibility of the phospho-tyrosine pocket. Comparison of the X-ray structures shows that the presence of the H-bond acceptors and not their relative position within the pharmacophore are essential for fragment binding and/or high affinity binding of full length inhibitors. The X-ray data show that the fragments are recognized by forming a complex H-bond network within the phospho-tyrosine pocket of SH2. No fragment structure was found in which this H-bond network was incomplete, and any uncompensated H-bond within the H-bond network leads to a significant decrease in the affinity of full length inhibitors. No correlation between affinity and fragment binding was found for these polar fragments and hence affinity-based screening would have overlooked some interesting starting points for inhibitor design. In contrast, we were unable to identify electron density for hydrophobic fragments, confirming that hydrophobic interactions are important for inhibitor affinity but of minor importance for ligand recognition. Our results suggest that a screening approach using protein crystallography is particularly useful to identify universal fragments for the conserved hydrophilic recognition sites found in target families such as SH2 domains, phosphatases, kinases, proteases, and esterases.

Structure-based design of novel nonpeptide inhibitors of the Src SH2 domain:phosphotyrosine mimetics exploiting multifunctional group replacement chemistry.

A series of novel nonpeptide inhibitors of the pp60(c-Src) (Src) SH2 domain is described that exploit multifunctional group replacement of the phenylphosphate moiety of phosphotyrosine (pTyr). Relative to an x-ray structure of citrate complexed to the pTyr binding site of the Src SH2 domain, these nonpeptide ligands illustrate the systematic replacement of the phosphate group by multiple nonhydrolyzable, mono- or dianionic functionalities. Specifically, several phenylalanine (Phe) analogs incorporating key 4' and 3' substituents were synthesized and incorporated into a bicyclic benzamide template previously reported (W. C. Shakespeare et al., Proceedings of the National Academy of Science USA, 2000, Vol. 97, pp. 9373-9378). These pTyr mimetics included 4',3'-diphosphono-Phe (Dpp), 4',3'-dicarboxymethyloxy-Phe (Dcp), and 4'-phosphono-3'-carboxymethyloxy-Phe (Cpp). Noteworthy were nonpeptide inhibitors 8-11 that were 5- to 10-fold more potent than the cognate tetrapeptide ligand Ac-pTyr-Glu-Glu-Ile-NH(2) in binding to the Src SH2 domain.

Activated pp60c-Src Leads to Elevated Hypoxia-inducible Factor (HIF)-1α Expression under Normoxia

Hypoxia-inducible factor (HIF)-1 is a master transcription factor, which up-regulates glycolysis, erythropoiesis, and angiogenesis under hypoxia. HIF-1alpha accumulates in normoxic tumor cells, leading to glycolysis under aerobic conditions. This phenomenon, known as the "Warburg effect," is caused by a yet unknown mechanism. Here we show that transformed cells that express constitutively active pp60(c-Src) (Src) express HIF-1alpha protein under normoxia, which results in the expression of multiple HIF-1alpha target genes. We show that this occurrence is due to an enhanced rate of HIF-1alpha protein synthesis and not due to reduced HIF-1alpha degradation. Furthermore, we show that the Src-induced increase in protein synthesis is due to the global increase in the rate of cap-dependent translation and does not involve inhibition of HIF-1alpha degradation.

Low Mr Phosphotyrosine Protein Phosphatase Associates and Dephosphorylates p125 Focal Adhesion Kinase, Interfering with Cell Motility and Spreading

Low M(r) phosphotyrosine protein phosphatase interferes in vivo with the activation of several growth factor receptors and is transiently redistributed, following cell stimulation with platelet-derived growth factor, from the cytosol to the cytoskeleton. We demonstrate here that this phosphatase also participates in the regulation of cell spreading and migration, pointing to its involvement in cytoskeleton organization. Low M(r) phosphotyrosine protein phosphatase-overexpressing fibroblasts are, indeed, less spread than controls and display a significantly decreased number of focal adhesions and increased cell motility. Furthermore, p125 focal adhesion kinase is associated to, and dephosphorylated by, low M(r) phosphotyrosine protein phosphatase both in vitro and in vivo. This event is consistent with an altered association of pp60(src) with focal adhesion kinase. The activation of extracellular signal-regulated kinase, another well known event downstream of the focal adhesion kinase, is also affected. On the other hand, cells overexpressing the dominant-negative form of low M(r) phosphotyrosine protein phosphatase exhibit hyperphosphorylated focal adhesion kinase, reduced motility, and an increased number of focal adhesions, which are distributed all over the ventral cell surface. Taken together, the results reported here are in keeping with low M(r) phosphotyrosine protein phosphatase participation in FAK-mediated focal adhesion remodeling.

Enhanced activity of human N-myristoyltransferase by dimethyl sulfoxide and related solvents in the presence of serine/threonine-containing peptide substrates

Human N-myristoyltransferase (hNMT) activity was found to be stimulated several-fold by DMSO and its analogues in the presence of serine-containing peptide substrates. DMSO caused a concentration-dependent 10-fold stimulation of hNMT activity in the presence of a pp60 src-derived peptide substrate (Gly-Ser-Ser-Lys-Ser-Lys-Pro-Lys-Arg). However, the stimulation of hNMT activity was not observed by DMSO when a cyclic AMP (cAMP)-dependent protein kinase-derived Ser-free peptide substrate (Gly-Asn-Ala-Ala-Ala-Ala-Lys-Lys-Arg-Arg) was used. These findings suggested that the effect of DMSO is on the substrate rather than on the enzyme. When a MARCKS (myristoylated alanine-rich C-kinase substrate)-derived peptide substrate (Gly-Ala-Gln-Phe-Ser-Lys-Thr-Ala-Arg-Arg) and the M2 gene segment of the reovirus type 3 peptide substrate (Gly-Asn-Ala-Ser-Ser-Ile-Lys-Lys-Lys) were used, hNMT activity was increased by approximately 8.5- and 7-fold, respectively. Dimethyl sulfone (20%) increased hNMT activity between 2.5- and 3.5-fold in the presence of pp60 src, MARCKS, and M2 gene segment peptides. Dimethyl formamide (20%) increased the hNMT activity by 8.5-, 8.5-, 5.5- and 3.5-fold when pp60 src, MARCKS, M2, and cAMP-dependent protein kinase-derived peptide substrates were used, respectively. Acetone (20%) also increased the hNMT activity by 20-fold in the presence of the pp60 src peptide substrate. Dimethyl ammonium chloride (20%) caused about 6.5- and 2.5-fold increases in the hNMT activity in the presence of the pp60 src and cAMP-dependent protein kinase-derived peptide substrates, respectively. Infrared spectroscopy showed a decreased intensity in the band at 3500–3600 cm −1 when the infrared spectrum of the pp60 src-derived peptide was determined in the presence of DMSO. These results suggest the involvement of hydrogen bonding between the heteroatoms of the organic molecules and the hydrogen atoms of the free hydroxyl groups of the serine/threonine-containing peptide substrates. Such interactions appear to enhance the activity of hNMT towards its serine-containing substrates.

Protein Kinase C and Guanosine Triphosphate Combine to Potentiate Calcium-dependent Membrane Fusion Driven by Annexin 7

Exocytotic secretion is promoted by the concerted action of calcium, guanine nucleotide, and protein kinase C. We now show that the calcium-dependent membrane fusion activity of annexin 7 in vitro is further potentiated by the combined addition of guanine nucleotide and protein kinase C. The observed increment involves the simultaneous activation of annexin 7 by these two effectors. Guanosine triphosphate (GTP) and its non-hydrolyzable analogues optimally enhance the phosphorylation of annexin 7 by protein kinase C in vitro. Reciprocally, phosphorylation by protein kinase C significantly potentiates the binding and hydrolysis of GTP by annexin 7. Only protein kinase C-dependent phosphorylation has a significant positive effect on annexin 7 GTPase, although other protein kinases, including cAMP-dependent protein kinase, cGMP-dependent protein kinase, and pp60(c-)(src), have been shown to label the protein with high efficiency. In vivo, the ratio of bound GDP/GTP and phosphorylation of annexin 7 change in direct proportion to the extent of catecholamine release from chromaffin cells in response to stimulation by carbachol, or to inhibition by various protein kinase C inhibitors. These results thus lead us to hypothesize that annexin 7 may serve as a common site of action for calcium, guanine nucleotide, and protein kinase C in the exocytotic membrane fusion process in chromaffin cells.

Principles governing the binding of a class of non-peptidic inhibitors to the SH2 domain of src studied by X-ray analysis.

A total of 11 structures of the (pp60)src SH2 domain with non-peptidic inhibitors based on the same two closely related inhibitor scaffolds were determined using X-ray crystallography. Surprisingly, the inhibitors that have an IC(50) value between 4 and 2700 nM bind in three different binding modes. Structure comparisons show that the inhibitors aim to maximize the interaction between the hydrophobic substituent and the hydrophobic pY+3 pocket. This is achieved either by an alternative binding mode of the phenyl phosphate or by including water molecules that mediate the interaction between the inhibitor scaffold and a rigid surface of the SH2 domain. The combination of the rigid pY+3 pocket and the rigid protein surface to which the scaffolds bind results in severe distance and angular restraints for putative scaffolds and their substituents. The X-ray data suggest that these restraints seem to be compensated in our system by including water molecules, thereby increasing the flexibility of the system.

Biological activity of substrate-bound basic fibroblast growth factor (FGF2): recruitment of FGF receptor-1 in endothelial cell adhesion contacts.

Substrate-bound FGF2 promotes endothelial cell adhesion by interacting with alpha(v)beta(3) integrin. Here, endothelial GM7373 cells spread and organize focal adhesion plaques on immobilized FGF2, fibronectin (FN), and vitronectin (VN). alpha(v)beta(3) integrin, paxillin, focal adhesion kinase, vinculin and pp60(src) localize in cell-substratum contact sites on FGF2, FN or VN. However, only immobilized FGF2 induces a long-lasting activation of extracellular signal-regulated kinases(1/2) (ERK(1/2)) and cell proliferation that was inhibited by the ERK(1/2) inhibitor PD 098059 and the tyrosine kinase (TK) inhibitor tyrphostin 23, pointing to the engagement of FGF receptor (FGFR) at the basal side of the cell. To assess this hypothesis, GM7373 cells were transfected with a dominant negative TK(-)-DeltaFGFR1 mutant (GM7373-DeltaFGFR1 cells) or with the full-length receptor (GM7373-FGFR1 cells). Both transfectants adhere and spread on FGF2 but GM7373-DeltaFGFR1 cells do not proliferate. Also, parental and GM7373-FGFR1 cells, but not GM7373-DeltaFGFR1 cells, undergo morphological changes and increased motility on FGF2-coated plastic. Finally, FGFR1, but not TK(-)-DeltaFGFR1, localizes in cell adhesion contacts on immobilized FGF2. In conclusion, substrate-bound FGF2 induces endothelial cell proliferation, motility, and the recruitment of FGFR1 in cell-substratum contacts. This may contribute to the cross talk among intracellular signaling pathways activated by FGFR1 and alpha(v)beta(3) integrin in endothelial cells.