Protein phosphatases: possible bisphosphonate binding sites mediating stimulation of osteoblast proliferation

We investigated the existence of a bisphosphonate (BP) target site in osteoblasts. Binding assays using [³H]-olpadronate ([³H]OPD) in whole cells showed the presence of specific, saturable and high affinity binding for OPD (K_d = 1.39 ± 0.33 μM) in osteoblasts. [³H]OPD was displaced from its binding site by micromolar concentrations of lidadronate, alendronate and etidronate (K_d = 1.42 ± 0.15 μM, 2.00 ± 0.2 μM and 2.4 ± 0.4 μM, respectively), and by millimolar concentrations of the non-permeant protein phosphatase (PP) substrates p-nitrophenylphosphate and α-naphtylphosphate. PP inhibitors orthovanadate, NaF or vpb(bipy) did not displace [³H]OPD.As expected, specific OPD binding was detected in the plasma membrane of ROS 17/2.8 cells, although significant BP binding was also found intracellularly. Moreover, OPD increased DNA synthesis in these cells with a temporal profile similar to the protein tyrosine phosphatase (PTP) inhibitors, Na₃VO₄ and vpb(bipy); but different from a general PP inhibitor (NaF). The stimulatory effect of OPD and PTP inhibitors on osteoblast proliferation was inhibited by the protein tyrosine kinase inhibitors genistein and geldanamycin. These results provide new evidence on the existence of a BP target in osteoblastic cells, presumably a PTP, which may be involved in the stimulatory action of BPs on osteoblast proliferation.     

Src family kinases as mediators of endothelial permeability: effects on inflammation and metastasis

Src family kinases (SFKs) are signaling enzymes that have long been recognized to regulate critical cellular processes such as proliferation, survival, migration, and metastasis. Recently, considerable work has elucidated mechanisms by which SFKs regulate normal and pathologic processes in vascular biology, including endothelial cell proliferation and permeability. Further, when inappropriately activated, SFKs promote pathologic inflammatory processes and tumor metastasis, in part through their effects on the regulation of endothelial monolayer permeability. In this review, we discuss the roles of aberrantly activated SFKs in mediating endothelial permeability in the context of inflammatory states and tumor cell metastasis. We further summarize recent efforts to translate Src-specific inhibitors into therapy for systemic inflammatory conditions and numerous solid organ cancers. The authors’ own research was supported in part by NIH U54 CA 090810 and P20 CA101936 (G.E.G) and NIH T32 CA 09599 (M.P.K.)     

Two new secondary metabolites isolated from Avena sativa L. (Oat) seedlings and their effects on osteoblast differentiation

Seedlings of natural crops are valuable sources of pharmacologically active phytochemicals. In this study, we aimed to identify new active secondary metabolites in Avena sativa L. (oat) seedlings. Two new compounds, avenafuranol (1) and diosgenoside (2), along with eight known compounds (3–10) were isolated from the A. sativa L. seedlings. Their chemical structures were elucidated via 1D and 2D NMR spectroscopy, high-resolution ESIMS, IR spectroscopy, optical rotation analysis, and comparisons with the reported literature. The effect of each isolated compound on alkaline phosphatase (ALP) activity for osteoblast differentiation induced by bone morphogenetic protein-2 (BMP-2) was investigated using the C2C12 immortal mouse myoblast cell line. Compounds 1, 4, 6, 8, and 9 induced dose-dependent increases in ALP expression relative to ALP expression in cells treated with only BMP-2, and no cytotoxicity was observed. These results suggest that A. sativa L. seedlings are a natural source of compounds that may be useful for preventing bone disorders.     

The role of phosphatases in the metabolism of Peridinium cinctum,from Lake Kinneret

The annual algal bloom (February–June) in Lake Kinneret consists almost entirely of the dinoflagellatePeridinium cinctum f.westii (Dinophyceae). To clarify the role of phosphatases in the alga, experiments were carried out using cells from culture or from the lake. In culture, as the external ambient orthophosphate (Pi) concentration decreased, alkaline phosphatase activity increased (and to some extent acid phosphatase activity, as well). Hot water extractable P decreased, although molybdate reactive phosphorus (MRP) appeared to be utilized in preference to the non-MRP component of this pool. Alkaline phosphatase inPeridinium collected from the lake as well as cells grown in culture under a high (3–6 mg l^–1) ambient Pi concentration in both continuous light and a 12:12 light-dark cycle, showed a diurnal fluctuation in activity. These results, together with previous observations suggest that the phosphatases inPeridinium are controlled by changes in intracellular phosphorus levels (other than the hot water extractable pool) and/or by other metabolic processes not directly involved in P nutrition.     

The role of acid phosphatases in plant phosphorus metabolism

Hydrolysis of phosphate esters is a critical process in the energy metabolism and metabolic regulation of plant cells. This review summarizes the characteristics and putative roles of plant acid phosphatase (APase). Although immunologically closely related, plant APases display remarkable heterogeneity with regards to their kinetic and molecular properties, and subcellular location. The secreted APases of roots and cell cultures are relatively non-specific enzymes that appear to be important in the hydrolysis and mobilization of P_i from extracellular phosphomonoesters for plant nutrition. Intracellular APases are undoubtedly involved in the routine utilization of P_i reserves or other P_i-containing compounds. A special class of intracellular APase exists that demonstrate a clear-cut (but generally nonabsolute) substrate selectivity. These APases are hypothesized to have distinct metabolic functions and include: phytase, phosphoglycolate phosphatase, 3-phosphoglycerate phosphatase, phosphoenolpyruvate phosphatase, and phosphotyrosyl-protein phosphatase. APase expression is regulated by a variety of developmental and environmental factors. P_i starvation induces de novo synthesis of extra- and intracellular APases in cell cultures as well as in whole plants. Recommendations are made to achieve uniformity in the analyses of the different APase isoforms normally encountered within and between different plant tissues.     

Essential role of Src suppressed C kinase substrates in endothelial cell adhesion and spreading

Integrin-mediated substrate adhesion of endothelial cells leads to dynamic rearrangement of the actin cytoskeleton. Protein kinase C (PKC) stimulates reorganization of microfilaments and adhesion, but the mechanism by which this occurs is unknown. Src suppressed C kinase substrate (SSeCKS) is a PKC substrate that may play an important role in regulating actin cytoskeleton. We found that SSeCKS was localized to focal adhesion sites soon after cell adhesion and that SSeCKS translocated from the membrane to the cytosol during the process of cell spreading. Using small interfering RNAs specific to SSeCKS, we show that RPMVEC cells in which SSeCKS expression was inhibited reduce adhesion and spread on LN through blocking the formation of actin stress fibers and focal adhesions. These results demonstrated SSeCKS modulate endothelial cells adhesion and spreading by reorganization of the actin cytoskeleton.     

Opposing effects of cyclosporin A and tyrphostin AG-1478 indicate a role for Src protein in the cellular control of mineralization

Cyclosporin A (CsA) induces osteoporosis but not through direct activation of osteoclasts. CsA also inhibits cell-mediated mineralization in marrow stromal cell culture, whereas the tyrphostin AG-1478 increases mineralization. These antagonistic effects on mineralization were used to discern molecules that underwent phosphorylation changes in association with their opposing effects on mineralization. In parallel, quantitative changes in Src protein were followed. Multiple dexamethasone (DEX)-stimulated stromal cell cultures were grown with and without a mineralization-inhibiting dose (0.1 μM) of CsA and were harvested on different days of DEX stimulation. Immunoblots of gel-fractionated cell extracts showed that the most noticeable changes in tyrosine phosphorylated proteins (TPP) were seen on day 8 of DEX stimulation. At least 15 TPP bands, mostly smaller than 53 kDa, were more prominent in CsA-treated cultures on day 8. Under CsA, Src protein quantity decreased on day 8, but its cleavage product (52/54 kDa) was sixfold more abundant then on day 7. Day 8 was chosen to test the effect of AG-1478 on the CsA-induced TPP changes. Dimethyl sulfoxide (DMSO) alone, the solvent of AG-1478, increased mineralization in CsA-treated versus CsA-untreated cultures and slightly decreased Src and its cleavage product. AG-1478 at 5 μM, in CsA cultures increased the specific alkaline phosphatase activity threefold, with a slight change in mineralization relative to controls grown with DMSO alone. This was accompanied by decreased intensity of several TPP bands smaller than 36 kDa. In contrast, treatment with 50 μM of AG-1478 increased the intensity of TPP bands at the same molecular size range. This high AG-1478 dose decreased cell counts selecting mineralizing cells. The results indicate that increased Src protein cleavage product on day 8 by CsA is associated with mineralization inhibition, which is opposed by DMSO and 50-μM AG-1478, thus antagonizing the effect of CsA on mineralization. Direct or indirect interaction between Src and TPP, antagonistically affected by CsA and AG-1478, is likely to underlay cellular control of mineralization. Changes in p19 and p29 intensity showed association with mineralization that was reflected by a significant direct and inverse correlation, respectively, with calcium precipitation per cell. J. Cell. Biochem. 71:116–126, 1998. © 1998 Wiley-Liss, Inc.     

Approaches to the molecular cloning of protein-tyrosine phosphatases in insulin-sensitive tissues

The intrinsic tyrosyl kinase activity of the insulin receptor is regulated by a balance between insulin-induced receptor autophosphorylation, which stimulates the receptor kinase, and enzymatic dephosphorylation of the receptor, which deactivates its kinase activity. The cellular protein-tyrosine phosphatase (PTPase) enzymes responsible for reversing the activated state of the insulin receptor have not been characterized. Our laboratory is interested in identifying and cloning the specific PTPase(s) that regulate the phosphorylation state of the insulin receptor. This chapter will summarize the design and results of our initial molecular cloning studies to identify specific PTPases in insulin-sensitive tissues that may have a potential physiological role in insulin action and clinical insulin resistance.     

Modulatory effect of omega-3 polyunsaturated fatty acids on osteoblast function and bone metabolism

Recent investigations indicate that the type and amount of polyunsaturated fatty acids (PUFA) influence bone formation in animal models and osteoblastic cell functions in culture. In growing rats, supplementing the diet with omega-3 PUFA results in greater bone formation rates and moderates ex vivo prostaglandin E(2) production in bone organ cultures. A protective effect of omega-3 PUFA on minimizing bone mineral loss in ovariectomized rats has also been reported. The actions of omega-3 fatty acids on bone formation appear to be linked to altering osteoblast functions. Herein we describe experiments with MC3T3-E1 osteoblast-like cells that support findings in vivo where omega-3 PUFA modulated COX-2 protein expression, reduced prostaglandin E(2) production, and increased alkaline phosphatase activity. Other studies indicate that the dietary source of PUFA may affect protein expression of Cbfa1 and nodule formation in fetal rat calvarial cells.     

Amplification and oscillations in the FAK/Src kinase system during integrin signaling

Integrin signaling is a major pathway of cell adhesion to extracellular matrices that regulates many physiological cell behaviors such as cell proliferation, migration or differentiation and is implied in pathologies such as tumor invasion. In this paper, we focused on the molecular system formed by the two kinases FAK (focal adhesion kinase) and Src, which undergo auto- and co-activation during early steps of integrin signaling. The system is modelled using classical kinetic equations and yields a set of three nonlinear ordinary differential equations describing the dynamics of the different phosphorylation forms of FAK. Analytical and numerical analysis of these equations show that this system may in certain cases amplify incoming signals from the integrins. A quantitative condition is obtained, which indicates that the total FAK charge in the system acts as a critical mass that must be exceeded for amplification to be effective. Furthermore, we show that when FAK activity is lower than Src activity, spontaneous oscillations of FAK phosphorylation forms may appear. The oscillatory behavior is studied using bifurcation and stability diagrams. We finally discuss the significance of this behavior with respect to recent experimental results evidencing FAK dynamics.     

Src controls tumorigenesis via JNK‐dependent regulation of the Hippo pathway in Drosophila

Cell–cell interactions within the tumour microenvironment have crucial roles in epithelial tumorigenesis. Using Drosophila genetics, we show that the oncoprotein Src controls tumour microenvironment by Jun N‐terminal kinase (JNK)‐dependent regulation of the Hippo pathway. Clones of cells with elevated Src expression activate the Rac‐Diaphanous and Ras‐mitogen‐activated protein kinase (MAPK) pathways, which cooperatively induce F‐actin accumulation, thereby leading to activation of the Hippo pathway effector Yorkie (Yki). Simultaneously, Src activates the JNK pathway, which antagonizes the autonomous Yki activity and causes propagation of Yki activity to neighbouring cells, resulting in the overgrowth of surrounding tissue. Our data provide a mechanism to explain how oncogenic mutations regulate tumour microenvironment through cell–cell communication.     

Long-chain polyphosphate in osteoblast matrix vesicles: Enrichment and inhibition of mineralization

Background

Inorganic polyphosphate (polyP) is a fundamental and ubiquitous molecule in prokaryotes and eukaryotes. PolyP has been found in mammalian tissues with particularly high levels of long-chain polyP in bone and cartilage where critical questions remain as to its localization and function. Here, we investigated polyP presence and function in osteoblast-like SaOS-2 cells and cell-derived matrix vesicles (MVs), the initial sites of bone mineral formation.

人Src蛋白N端区段的表达、纯化和体外豆蔻酰化底物活性

利用RT PCR技术 ,从来源于人结肠癌Caco 2细胞总RNA中 ,扩增得到编码人Src蛋白N端 147氨基酸的DNA序列片段。进而构建T7启动子控制下的C端His tag融合的表达质粒pMF SrcHT ,并转化大肠杆菌BL2 1(DE3)。通过SDS PAGE等分析结果显示 ,在 37°C培养条件下经IPTG诱导 ,C端His tag融合的人Src蛋白N端区段 (命名为SrcHT ,2 1kD)得到高效表达 ,并且主要以可溶性形式存在。进一步利用Ni IDA亲和层析分离 ,从表达菌裂解上清液中一步纯化获得重组蛋白SrcHT ,SDS PAGE分析纯度达 95 %以上。在此基础上 ,以 [3H]豆蔻酰 CoA为同位素标记底物进行SrcHT的体外NMT豆蔻酰化反应测定。SDS PAGE分离和放射自显影分析结果表明 ,SrcHT蛋白可被NMT有效豆蔻酰化而具有NMT的底物活性。这些为深入详细研究Src蛋白豆蔻酰化作用和构建以Src蛋白豆蔻酰化为靶标的分子筛药体系等打下了重要基础。     

Evidence for a calpeptin-sensitive protein-tyrosine phosphatase upstream of the small GTPase Rho. A novel role for the calpain inhibitor calpeptin in the inhibition of protein-tyrosine phosphatases.

Activation of the thiol protease calpain results in proteolysis of focal adhesion-associated proteins and severing of cytoskeletal-integrin links. We employed a commonly used inhibitor of calpain, calpeptin, to examine a role for this protease in the reorganization of the cytoskeleton under a variety of conditions. Calpeptin induced stress fiber formation in both forskolin-treated REF-52 fibroblasts and serum-starved Swiss 3T3 fibroblasts. Surprisingly, calpeptin was the only calpain inhibitor of several tested with the ability to induce these effects, suggesting that calpeptin may act on targets besides calpain. Here we show that calpeptin inhibits tyrosine phosphatases, enhancing tyrosine phosphorylation particularly of paxillin. Calpeptin preferentially inhibits membrane-associated phosphatase activity. Consistent with this observation, in vitro phosphatase assays using purified glutathione S-transferase fusion proteins demonstrated a preference for the transmembrane protein-tyrosine phosphatase-alpha over the cytosolic protein-tyrosine phosphatase-1B. Furthermore, unlike wide spectrum inhibitors of tyrosine phosphatases such as pervanadate, calpeptin appeared to inhibit a subset of phosphatases. Calpeptin-induced assembly of stress fibers was inhibited by botulinum toxin C3, indicating that calpeptin is acting on a phosphatase upstream of the small GTPase Rho, a protein that controls stress fiber and focal adhesion assembly. Not only does this work reveal that calpeptin is an inhibitor of protein-tyrosine phosphatases, but it suggests that calpeptin will be a valuable tool to identify the phosphatase activity upstream of Rho.     

Cell density-dependent changes in the insulin action pathway: Evidence for involvement of protein-tyrosine phosphatases

In order to examine alterations in the phosphorylation state of proteins involved in insulin action that might accompany the reduced growth state of density-arrested cells, we measured the insulin-stimulated phosphorylation of the receptor and high M_r cellular substrates of the receptor kinase in rat hepatoma cells at different cell densities. As cell density increased from 2 × 10⁵ to 3.2 × 10⁶ per 35-mm well, the rate of DNA synthesis fell to 22% of control, while insulin-stimulated tyrosine phosphorylation of high M_r receptor substrates (“pp185”) was enhanced to 198% of control, without a change in the abundance of insulin receptor substrate (IRS)-1 protein. In anti-IRS-1 immunoprecipitates, tyrosine phosphorylation was increased by only 30%, suggesting that increased tyrosine phosphorylation of additional high M_r proteins (e.g., IRS-2) accounted for much of the observed increase in tyrosine phosphorylation of the receptor substrates. In spite of increased tyrosine phosphorylation of IRS-1 and total pp185-related proteins, however, cells studied at high growth density exhibited a 25% decrease in IRS-1-associated phosphatidylinositol 3′-kinase activity and only a 39% increase in phosphatidylinositol 3′-kinase activity in antiphosphotyrosine immunoprecipitates. To explore the potential role of hepatic protein-tyrosine phosphatases (PTPases) in the hyperphosphorylation of pp185 proteins, we found by immunoblotting that at high cell density the intracellular PTPase PTP18 and the transmembrane PTPase LAR were reduced in abundance by 49% and 55%, respectively, while the abundance of the SH2-domain containing PTPase SH-PTP2 was increased by 48%. These data demonstrate that the attenuation of post-receptor signaling by insulin in hepatoma cells at increasing growth density involves changes in endogenous substrate phosphorylation which may result from alterations in specific PTPases implicated in the regulation of the insulin action pathway. © 1996 Wiley-Liss, Inc.     

A novel disulfide bond in the SH2 Domain of the C-terminal Src kinase controls catalytic activity

The SH2 domain of the C-terminal Src kinase [Csk] contains a unique disulfide bond that is not present in other known SH2 domains. To investigate whether this unusual disulfide bond serves a novel function, the effects of disulfide bond formation on catalytic activity of the full-length protein and on the structure of the SH2 domain were investigated. The kinase activity of full-length Csk decreases by an order of magnitude upon formation of the disulfide bond in the distal SH2 domain. NMR spectra of the fully oxidized and fully reduced SH2 domains exhibit similar chemical shift patterns and are indicative of similar, well-defined tertiary structures. The solvent-accessible disulfide bond in the isolated SH2 domain is highly stable and far from the small lobe of the kinase domain. However, reduction of this bond results in chemical shift changes of resonances that map to a cluster of residues that extend from the disulfide bond across the molecule to a surface that is in direct contact with the small lobe of the kinase domain in the intact molecule. Normal mode analyses and molecular dynamics calculations suggest that disulfide bond formation has large effects on residues within the kinase domain, most notably within the active-site cleft. Overall, the data indicate that reversible cross-linking of two cysteine residues in the SH2 domain greatly impacts catalytic function and interdomain communication in Csk.