The Src-related protein kinase Lyn plays an important role in B-cell activation. However, several lines of evidence suggest that it is also involved in the control of cellular proliferation and the inhibition of apoptosis. We have discovered that Lyn is expressed in normal prostate epithelia, in 95% of primary human prostate cancer (PC) specimens examined, and in all of the PC cell lines that we assayed. Moreover, Lyn knockout mice display abnormal prostate gland morphogenesis, which suggests that Lyn plays an important role in prostate epithelium development and implies that Lyn is a candidate target for specific therapy for PC. Using a drug-design strategy to construct sequence-based peptide inhibitors, a Lyn-specific inhibitor, KRX-123, targeting a unique interaction site within Lyn, was synthesized. KRX-123 was found to inhibit cellular proliferation in three hormone-refractory PC cell lines, DU145, PC3, and TSU-Pr1 with IC 50 values of 2-4 M. In vivo, tumor volume of DU145 explants in nude mice was significantly reduced after once-a-week injections of KRX-123, at a dose of 10 mg/kg, for a period of 5 weeks. Histological analyses of the treated tumors indicated extensive apoptosis. Thus, we suggest that Lyn inhibition may serve as a prime target for the treatment of hormone-refractory PC.
A platform for specifically modulating kinase-dependent signaling using peptides derived from the catalytic domain of the kinase is presented. This technology, termed KinAce TM , utilizes the canonical structure of protein kinases. The targeted regions (subdomain V and subdomains IX and X) are analyzed and their sequence, threedimensional structure, and involvement in proteinprotein interaction are highlighted. Short myristoylated peptides were derived from the target regions of the tyrosine kinases c-Kit and Lyn and the serine/threonine kinases 3-phosphoinositide-dependent kinase-1 (PDK1) and Akt/protein kinase B (PKB). For each kinase an active designer peptide is shown to selectively inhibit the signaling of the kinase from which it is derived, and to inhibit cancer cell proliferation in the micromolar range. This technology emerges as an applicable tool for deriving sequence-based selective inhibitors for a broad range of protein kinases as hits that may be further developed into drugs. Moreover, it enables identification of novel kinase targets for selected therapeutic indications as demonstrated in the KinScreen application.Protein kinases are important drug targets for oncologic, immunologic, and metabolic disorders. The development of selective protein kinase inhibitors is widely considered a promising approach to drug development. The common strategy, which has led to the development of drugs, such as Glivec and Iressa (1), is to target the ATP binding site (1-3). Other approaches include substrate mimicking inhibitors (4 -9), bi-substrate analogs that target both the ATP and the acceptor binding sites (10), and molecules that target the Src homology 2 domain (11). The KinAce TM (12) approach presented in this article is based upon deriving short peptides from specific regions in the catalytic domain of the kinase that are implicated in kinase-substrate interactions. The KinAce TM peptides mimic regions of the kinase and therefore compete with the kinase for binding to the substrate (or to other modulators of the kinase), and subsequently abrogate the kinase-dependent signaling.Several other groups have used peptides to disrupt proteinprotein interactions and thus modulate kinase signaling. In contrast to our technology, which derives the peptides from the catalytic domain of the kinase, others have derived inhibitory peptides from the substrates (7), pseudosubstrate (13), regulators interacting with the kinase (14 -17), or from non-catalytic domains of the kinase participating in substrate binding (11,18). One of the unique characteristics of the KinAce TM technology is that the regions from which the inhibitory peptides are derived share conserved structural patterns in all kinases. Evidence from the literature supports a potential role for these regions in substrate binding (19 -22). Our technology suggests a general recipe for generating inhibitors of kinase-dependent signaling, applicable to any kinase. To enable permeation of peptides into cells researchers conjugated peptides to membrane penetrat...
Abstract. Myosin II heavy chain (MHC)-specific protein kinase C (MHC-PKC) isolated from the ameba, Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cAMP (Abu-Elneel et al. 1996. J. Biol. Chem. 271:977-984). Recent studies have indicated that cAMP-induced cGMP accumulation plays a role in the regulation of myosin II phosphorylation and localization (Liu, G., and P. Newell. 1991. J. Cell. Sci. 98: 483-490). This report describes the roles of cAMP and cGMP in the regulation of MHC-PKC membrane association, phosphorylation, and activity (hereafter termed MHC-PKC activities), cAMP stimulation of Dictyostelium cells resulted in translocation of MHC-PKC from the cytosol to the membrane fraction, as well as increasing in MHC-PKC phosphorylation and in its kinase activity. We present evidence that MHC is phosphorylated by MHC-PKC in the cell cortex which leads to myosin II dissociation from the cytoskeleton. Use of Dictyostelium mutants that exhibit aberrant cAMP-induced increases in cGMP accumulation revealed that MHC-PKC activities are regulated by cGMP. Dictyostelium streamer F mutant (stmF), which produces a prolonged peak of cGMP accumulation upon cAMP stimulation, exhibits prolonged increases in MHC-PKC activities. In contrast, Dictyostelium KI-10 mutant that lacks the normal cAMP-induced cGMP response, or KI-4 mutant that shows nearly normal cAMP-induced cGMP response but has aberrant cGMP binding activity, show no changes in MHC-PKC activities. We provide evidence that cGMP may affect MHC-PKC activities via the activation of cGMP-dependent protein kinase which, in turn, phosphorylates MHC-PKC. The results presented here indicate that cAMP-induced cGMP accumulation regulates myosin II phosphorylation and localization via the regulation of MHC-PKC. CAMP stimulation of the ameba Dictyostelium generates a number of responses such as increase in cGMP accumulation (26, 46), influx of Ca 2÷ (1, 6), production of inositol phosphates (13), changes in the amount of filamentous actin (15), changes in the phosphorylation rates of myosin II heavy chain (MHC) 1 and light chains (MLC) (4), and changes in cell movement and spreading (38,43).Studies on mutants lacking normal myosin II have indicated that it is not required for cell motility. It is, however, needed for efficient chemotaxis, and myosin II is thought to be involved in the regulation of cell polarity (45). Several lines of evidence have shown a correlation between myosin II reorganization, phosphorylation, and DictyostelAddress all correspondence to Dr. Shoshana Ravid, Department of Biochemistry, Hadassah Medical School, The Hebrew University, Jerusalem 91120, Israel. Tel.: 972-2-758283. Fax: 972-2-757379. Abbreviations used in this paper:MHC, myosin II heavy chain; MHCK, myosin II heavy chain kinase; MHC-PKC, a protein kinase C that phosphorylates Dictyostelium MHC specifically; MLC, myosin II light chain; MLCK, myosin II light chain kinase.ium chemotaxis (4, 24, 29, 47). In response to cAMP, the myosin II ...
Myosin II heavy chain (MHC)-specific protein kinase C (MHC-PKC) isolated from the ameba, Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cAMP. cAMP stimulation of Dictyostelium cells leads to translocation of MHC-PKC from the cytosol to the membrane fraction, as well as causing an increase in both MHC-PKC phosphorylation and its kinase activity. MHC-PKC undergoes autophosphorylation with each mole of kinase incorporating about 20 mol of phosphate. The MHC-PKC autophosphorylation sites are thought to be located within a domain at the COOH-terminal region of MHC-PKC that contains a cluster of 21 serine and threonine residues. Here we report that deletion of this domain abolished the ability of the enzyme to undergo autophosphorylation in vitro. Furthermore, after this deletion, cAMP-dependent autophosphorylation of MHC-PKC as well as cAMP-dependent increases in kinase activity and subcellular localization were also abolished. These results provide evidence for the role of autophosphorylation in the regulation of MHC-PKC and indicate that this MHC-PKC autophosphorylation is required for the kinase activation in response to cAMP and for subcellular localization.We have previously reported the isolation of a MHC 1 -specific PKC (MHC-PKC) from the ameba, Dictyostelium that phosphorylates Dictyostelium MHC specifically and is homologous to ␣, , and ␥ subtypes of mammalian PKC (1, 2). In vitro phosphorylation of MHC by MHC-PKC results in inhibition of myosin II thick filament formation (1) by inducing the formation of a bent monomer of myosin II, whose assembly domain is tied up in an intramolecular interaction that precludes the intermolecular interaction necessary for thick filament formation (3).The MHC-PKC which is expressed during Dictyostelium development has been implicated in the increase in MHC phosphorylation observed in response to cAMP stimulation (1). We have recently found that elimination of MHC-PKC abolishes this cAMP-induced MHC phosphorylation, indicating that MHC-PKC is the enzyme which phosphorylates MHC in response to cAMP stimulation (4). MHC-PKC null cells exhibit a substantial myosin II overassembly in vivo, as well as aberrant cell polarization, chemotaxis, and morphological differentiation. Cells that overexpress MHC-PKC contain highly phosphorylated MHC. They show no apparent cell polarization and chemotaxis, and exhibit impaired myosin II localization (4). These findings establish that, in Dictyostelium, the MHC-PKC plays an important role in regulating the cAMP-induced myosin II localization required for cell polarization and, consequently, for efficient chemotaxis.When cells of Dictyostelium are starved, they acquire the ability to bind cAMP to specific cell surface receptors and to respond to this signal by chemotaxis, which requires phosphorylation and reorganization of myosin II (5-9). That is, the myosin II, which exists as thick filaments, translocates to the cortex (9) in response to cAMP stimulation. This translocation is co...
When cells are exposed to a gradient of chemoattractant, activation occurs selectively at the stimulated edge. Such localized activation, transmitted by the recruitment of cytosolic proteins, may be a general mechanism for gradient sensing by G proteinlinked chemotactic systems. Here we show that in Dictyostelium discoideum cells exposed to a cAMP gradient the myosin II heavy chain kinase (MHC-PKC) and myosin II translocate to opposite ends of the cell. We further show that MHC-PKC C1 domain is responsible for the localization of MHC-PKC to the cell leading edge, but it is not sufficient to promote cell polarization. Our findings suggest a mechanism by which MHC-PKC regulates myosin II, allowing cell polarization and movement in the direction of the cAMP source.Chemotaxis in eukaryotic cells is mediated by changes in the organization and function of cytoskeletal structures containing actin and myosin II (6, 7). Studies on the role of myosin II in Dictyostelium chemotaxis suggest that myosin II monomers undergo transient assembly into bipolar filaments that may precede recruitment into the cytoskeleton and that the cycles of myosin II assembly and disassembly may be regulated by phosphorylation of myosin II heavy chain (MHC) 1 (8, 9). Indeed, cAMP stimulation causes myosin II that exists as thick filaments to translocate to the cell cortex. This translocation is correlated with a transient increase in the rate of MHC as well as light chain phosphorylation (8, 9). In addition, a novel protein kinase C (MHC-PKC), which we purified and cloned from chemotactically competent Dictyostelium cells, phosphorylates MHC in response to cAMP stimulation (10 -12). In vitro phosphorylation of MHC by this kinase results in inhibition of myosin II thick filament formation (11, 13).Morphological polarity is necessary for chemotaxis by eukaryotic cells, but it does not require receptor polarization (5). Dictyostelium chemotaxis is accompanied by asymmetric recruitment to the cell surface of signal transduction proteins (2, 4), the cytosolic regulator of adenylyl cyclase (1,14), and the pleckstrin homology domain of the AKT protein kinase (15). Neutrophils also show such asymmetry when exposed to chemoattractant; the pleckstrin homology domain of the AKT protein kinase is recruited selectively to the membrane at the leading edge of the cell (3). In addition, cytoskeletal proteins such as actin and several actin-binding proteins have been shown to transiently accumulate at the leading edge of chemotaxing cells (16 -19).To define the spatiotemporal dynamics of MHC-PKC and myosin II that may lead to cell polarization and directed movement in Dictyostelium we expressed GFP-tagged MHC-PKC and myosin II in mhc-pkc null cells and myosin II null cells, respectively. We found that in cells exposed to a cAMP gradient, the MHC-PKC-GFP localized to the leading edge of the cell, whereas myosin II translocated to its posterior part. These findings indicate a mechanism whereby MHC-PKC and myosin II contribute to cell polarization and chemotaxis. E...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.