Previous studies suggest that the Ca2+-dependent proteases, calpains, participate in remodeling of the actin cytoskeleton during wound healing and are active during cell migration. To directly test the role that calpains play in cell spreading, several NIH-3T3– derived clonal cell lines were isolated that overexpress the biological inhibitor of calpains, calpastatin. These cells stably overexpress calpastatin two- to eightfold relative to controls and differ from both parental and control cell lines in morphology, spreading, cytoskeletal structure, and biochemical characteristics. Morphologic characteristics of the mutant cells include failure to extend lamellipodia, as well as abnormal filopodia, extensions, and retractions. Whereas wild-type cells extend lamellae within 30 min after plating, all of the calpastatin-overexpressing cell lines fail to spread and assemble actin-rich processes. The cells genetically altered to overexpress calpastatin display decreased calpain activity as measured in situ or in vitro. The ERM protein ezrin, but not radixin or moesin, is markedly increased due to calpain inhibition. To confirm that inhibition of calpain activity is related to the defect in spreading, pharmacological inhibitors of calpain were also analyzed. The cell permeant inhibitors calpeptin and MDL 28, 170 cause immediate inhibition of spreading. Failure of the intimately related processes of filopodia formation and lamellar extension indicate that calpain is intimately involved in actin remodeling and cell spreading.
Mevalonate kinase catalyzes the ATP-dependent phosphorylation of mevalonic acid to form mevalonate 5-phosphate, a key intermediate in the pathways of isoprenoids and sterols. Deficiency in mevalonate kinase activity has been linked to mevalonic aciduria and hyperimmunoglobulinemia D/periodic fever syndrome (HIDS). The crystal structure of rat mevalonate kinase in complex with MgATP has been determined at 2.4-Å resolution. Each monomer of this dimeric protein is composed of two domains with its active site located at the domain interface. The enzyme-bound ATP adopts an anti conformation, in contrast to the syn conformation reported for Methanococcus jannaschii homoserine kinase. The Mg 2؉ ion is coordinated to both -and ␥-phosphates of ATP and side chains of Glu 193 and Ser 146 . Asp 204 is making a salt bridge with Lys 13 , which in turn interacts with the ␥-phosphate. A model of mevalonic acid can be placed near the ␥-phosphoryl group of ATP; thus, the C5 hydroxyl is located within 4 Å from Asp 204 , Lys 13 , and the ␥-phosphoryl of ATP. This arrangement of residues strongly suggests: 1) Asp 204 abstracts the proton from C5 hydroxyl of mevalonate; 2) the penta-coordinated ␥-phosphoryl group may be stabilized by Mg 2؉ , Lys 13 , and Glu 193 ; and 3) Lys 13 is likely to influence the pK a of the C5 hydroxyl of the substrate. V377I and I268T are the most common mutations found in patients with HIDS. Val 377 is located over 18 Å away from the active site and a conservative replacement with Ile is unlikely to yield an inactive or unstable protein. Ile-268 is located at the dimer interface, and its Thr substitution may disrupt dimer formation.Mevalonate kinase (MK, ATP:mevalonate 5-phosphotransferase, EC 2.7.1.36) 1 catalyzes the transfer of the ␥-phosphoryl group from ATP to the C5 hydroxyl oxygen of mevalonic acid to form mevalonate 5-phosphate, a key intermediate in the biosynthetic pathway for isoprenoids and sterols from acetate. Although the enzyme was discovered in the late 1950s (1, 2), it suffered over three decades of neglect, as research on the isoprenoid pathway was focused on HMG-CoA reductase, which catalyzes the previous step in the pathway, i.e. the formation of mevalonic acid from HMG-CoA. However, interest in MK has been revived recently, because it was recognized that this enzyme, together with HMG-CoA synthase and HMG-CoA reductase, is involved in coordinate regulation of this pathway and therefore may represent a secondary control point. The recent recognition of the involvement of the diverse non-sterol isoprenoid metabolites in various cellular functions (e.g. protein prenylation, protein glycosylation, and cell cycle regulation) has also increased interest in this enzyme. In addition, the significance of MK has been further highlighted by the implication of the enzyme in human inherited diseases, such as mevalonic aciduria and hyperimmunoglobulinemia D/periodic fever syndrome (HIDS, Mendelian Inheritance in Man 260920).The enzyme is found in eukaryotes, archaebacteria, and some eubacteria. Th...
How m-calpain is activated in cells has challenged investigators because in vitro activation requires nearmillimolar calcium. Previously, we demonstrated that m-calpain activation by growth factors requires extracellular signal-regulated kinase (ERK); this enables tail deadhesion and allows productive motility. We now show that ERK directly phosphorylates and activates m-calpain both in vitro and in vivo. We identified serine 50 as required for epidermal growth factor (EGF)-induced calpain activation in vitro and in vivo. Replacing the serine with alanine limits activation by EGF and subsequent cell deadhesion and motility. A construct with the serine converted to glutamic acid displays constitutive activity in vivo; expression of an estrogen receptor fusion construct produces a tamoxifen-sensitive enzyme. Interestingly, EGF-induced m-calpain activation occurs in the absence of increased intracellular calcium levels; EGF triggers calpain even in the presence of intracellular calcium chelators and in calcium-free media. These data provide evidence that m-calpain can be activated through the ERK cascade via direct phosphorylation and that this activation may occur in the absence of cytosolic calcium fluxes.The calpain family of intracellular cysteine proteinases includes 13 known members, of which at least 2 are ubiquitously expressed (47, 48). These, m-and -calpain (calpain II and calpain I, respectively), are involved in cell migration and adhesion, being regulated downstream of both integrin and growth factor receptor activation (19). -Calpain has been implicated strongly in cell motility and adhesion primarily driven by integrin-mediated signals: calpains have been shown to be required during both cell spreading and adhesion (3,4,41) and for the release of the rear of migrating cells (26). On the other hand, m-calpain has been observed to be activated downstream of the epidermal growth factor (EGF) receptor (EGFR) and is required for growth factor-induced motility and deadhesion (18,44). This effect is specific to m-calpain, as antisense down-regulation of -calpain did not appreciably affect growth factor-induced motility and as EGF-induced calpain activity and motility were dependent on m-calpain (18). m-Calpain affects the migration of EGF-induced fibroblasts by promoting rear release during active motility (2, 43, 44). In general, functions of calpains in motility and adhesion apparently derive from their ability to cleave components of adhesion complexes in a limited manner, altering their function and leading to increased adhesion turnover (19,29,39). However, the molecular mechanism by which calpain activities are regulated during these events is not understood.The two ubiquitous isoforms, -and m-calpain, are presumed to be activated by intracellular calcium fluxes, since these enzymes require this divalent cation in vitro. Indeed mand -calpain are named for their relative requirement for calcium, with -calpain requiring micromolar, and m-calpain requiring near-millimolar, concentrations of calcium (16)....
Major changes in the mRNA population of murine liver occur after administration of bacterial lipopolysaccharide, an agent that causes increases in the concentrations of acutephase serum proteins. The mRNA for one of these, serum amyloid A, is increased at least 500-fold compared to the normal level. It becomes one of the most abundant hepatic mRNAs, and serum amyloid A synthesis comprises about 2.5% of total hepatic protein synthesis in the acute-phase response. Its synthesis is tissue-specific in that amyloid A mRNA was not detected in the kidney, an important site of amyloid fibril accumulation. The protein synthesized in largest amount by acute-phase liver tissue in culture is cytoplasmic actin. Its relative rate ofsynthesis is increased about 5-fold compared to the normal tissue; that of serum albumin is decreased to about one-third ofits normal rate. The concentration of mRNA for serum albumin is decreased by a similar amount. Starting with induced liver RNA, we have constructed a recombinant plasmid containing most ofthe DNA sequence encoding the serum amyloid A polypeptide.
Previous studies have indicated that the Ca2؉ -dependent protease, calpain, is activated in platelets within 30 -60 s of thrombin stimulation, but specific roles of calpain in platelets remain to be identified. To directly test the functions of calpain during platelet activation, a novel strategy was developed for introducing calpain's specific biological inhibitor, calpastatin, into platelets prior to activation. This method involves treatment of platelets with a fusion peptide, calpastat, consisting of the cell-penetrating signal sequence from Kaposi's fibroblast growth factor connected to a calpain-inhibiting consensus sequence derived from calpastatin. Calpastat specifically inhibits thrombin peptide (SFLLR)-induced ␣-granule secretion (IC 50 ؍ 20 M) during the first 30 s of activation, thrombin-induced platelet aggregation (IC 50 ؍ 50 M), and platelet spreading on glass surfaces (IC 50 ؍ 34 M). Calpastat-Ala, a mutant peptide in which alanine is substituted at conserved calpastatin residues, lacks calpain inhibitory activity and fails to inhibit secretion, aggregation, or spreading. The peptidyl calpain inhibitors calpeptin, MDL 28,170 (MDL) and E64d also inhibit secretion, aggregation and spreading, but require 3-10-fold higher concentrations than calpastat for biological activity. Together, these findings demonstrate that calpain regulates platelet secretion, aggregation, and spreading and indicate that calpain plays an earlier role in platelet activation following thrombin receptor stimulation than had been previously detected.
Purpose: These studies were designed to determine whether ritonavir inhibits breast cancer in vitro and in vivo and, if so, how. Experimental Design: Ritonavir effects on breast cancer cell growth were studied in the estrogen receptor (ER)^positive lines MCF7 and T47D and in the ER-negative lines MDA-MB-436 and MDA-MB-231. Effects of ritonavir on Rb-regulated and Akt-mediated cell proliferation were studied. Ritonavir was tested for inhibition of a mammary carcinoma xenograft. Results: ER-positive estradiol-dependent lines (IC 50 , 12-24 Amol/L) and ER-negative (IC 50 , 45 Amol/L) lines exhibit ritonavir sensitivity. Ritonavir depletes ER-a levels notably in ER-positive lines. Ritonavir causes G 1 arrest, depletes cyclin-dependent kinases 2, 4, and 6 and cyclin D 1 but not cyclin E, and depletes phosphorylated Rb and Ser 473 Akt. Ritonavir induces apoptosis independent of G 1 arrest, inhibiting growth of cells that have passed the G 1 checkpoint. Myristoyl-Akt, but not activated K-Ras, rescues ritonavir inhibition. Ritonavir inhibited a MDA-MB-231 xenograft and intratumoral Akt activity at a clinically attainable serum C max of 22 F 8 Amol/L. Because heat shock protein 90 (Hsp90) substrates are depleted by ritonavir, ritonavir effects on Hsp90 were tested. Ritonavir binds Hsp90 (K D , 7.8 Amol/L) and partially inhibits its chaperone function. Ritonavir blocks association of Hsp90 with Akt and, with sustained exposure, notably depletes Hsp90. Stably expressed Hsp90a short hairpin RNA also depletes Hsp90, inhibiting proliferation and sensitizing breast cancer cells to low ritonavir concentrations. Conclusions: Ritonavir inhibits breast cancer growth in part by inhibiting Hsp90 substrates, including Akt. Ritonavir may be of interest for breast cancer therapeutics and its efficacy may be increased by sustained exposure or Hsp90 RNA interference.
cDNA encoding human mevalonate kinase has been overexpressed and the recombinant enzyme isolated. This stable enzyme is a dimer of 42-kDa subunits and exhibits a V m ؍ 37 units/mg, K m(ATP) ؍ 74 M, and K m(DL-MVA) ؍ 24 M. The sensitivity of enzyme to watersoluble carbodiimide modification of carboxyl groups prompted evaluation of four invariant acidic amino acids (Glu-19, Glu-193, Asp-204, and Glu-296) by site-directed mutagenesis. Elimination of Glu-19's carboxyl group (E19A, E19Q) destabilizes the enzyme, whereas E19D is stable but exhibits only ϳ2-fold changes in V m and K m values. E296Q is a stable enzyme, which exhibits kinetic parameters comparable to those measured for wild-type enzyme. E193A is a labile protein, whereas E193Q is stable, exhibiting >50-fold diminution in V m and elevated K m values for ATP (ϳ20-fold) and mevalonate (ϳ40-fold). Such effects would be compatible with a role for Biosynthesis of isoprenoids and sterols requires the ATP-dependent phosphorylation of mevalonic acid. In humans, diminished activity of mevalonate kinase (EC 2.7.1.36), the enzyme that catalyzes this reaction (1), results in mevalonic aciduria (2). The enzyme has long been considered to be a cytosolic protein, but recent work on mevalonate kinase has implicated it in peroxisomal metabolism (3). In this context, depressed mevalonate kinase activity has also been correlated with peroxisomal deficiency disorders (4, 5). Although these observations suggest that detailed information on this enzyme would be useful, mevalonate kinase has not received as much attention as other enzymes in the isoprenoid/sterol biosynthetic pathway.Enzymological characterization of mammalian mevalonate kinase has included kinetic work (6), which suggests that the enzyme catalyzes an ordered sequential reaction with mevalonic acid assigned as the first substrate bound and phosphomevalonate as the first product released. Inhibition of the enzyme by geranyl pyrophosphate (7) and farnesyl pyrophosphate (8), metabolites that are formed downstream in the isoprenoid biosynthetic pathway, has been reported, and such inhibition has been suggested to have physiological relevance (9, 10). Although an amino acid substitution that presumably accounts for human mevalonic aciduria has been documented (11), little is known about the active site amino acids that are important to enzyme function. Group-specific reagents have been employed to demonstrate that mevalonate kinase contains reactive cysteine (6) and lysine (12) residues. Recently, the first identification of an active site amino acid was accomplished when protein chemistry and mutagenesis work indicated that lysine-13 influences ATP binding (13).Availability of a recombinant form of human mevalonate kinase would facilitate studies on inherited mutations in this enzyme. Such an enzyme, available in a stable, highly purified form and in substantial amounts, could also be useful for investigation of the structure/function correlations that account for phosphomevalonate production or for feedbac...
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