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...
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.