Dictyostelium Myosin II Is Regulated during Chemotaxis by a Novel Protein Kinase C

Abstract

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. The process of chemotaxis involves phosphorylation and reorganization of myosin II (1-5). In response to cAMP stimulation, myosin II, which exists as thick filaments, translocates to the cortex (5). This translocation is correlated with a transient increase in the rate of myosin II heavy chain (MHC) 1 as well as light chain phosphorylation (3, 4, 6). In addition, in vitro studies strongly suggest that MHC phosphorylation plays an important role in the regulation of myosin II filament formation (7-10). The importance of MHC phosphorylation in the regulation of myosin II in vivo was demonstrated by Egelhoff et al. (11). They found that elimination of the MHC phosphorylation sites allows in vivo contractile activity, but this myosin II shows substantial overassembly. Mimicking the negative charge state of phosphorylated myosin II eliminates filament formation in vitro and renders the myosin II unable to drive any tested contractile event in vivo (11).We previously reported the isolation of a MHC-specific PKC (MHC-PKC) from Dictyostelium that phosphorylates Dictyostelium MHC specifically and is homologous to ␣, ␤, and ␥ subtypes of mammalian PKC (9, 12). This kinase, which is membrane-associated and is expressed during Dictyostelium development, was implicated in the increase in MHC phosphorylation during chemotaxis in this species (9). In vitro phosphorylation of MHC by MHC-PKC results in inhibition of myosin II thick filament formation (9), by inducing the formation of a bent monomer of myosin II whose assembly domain is tied up in an intramolecular interaction that precludes intermolecular interaction, which is necessary for thick filament formation (10). The findings that MHC-PKC is a member of the PKC family and that it regulates myosin II assembly suggest a link between the extracellular chemotactic signal and subsequent intracellular events.A considerable amount of information is now available regarding the regulation of myosin II by MHC phosphorylation, and a picture is beginning to emerge in which the molecular changes in myosin II that are involved in MHC phosphorylation are related to cAMP-induced directed cell movement. Nevertheless, the molecular mechanism by which a cAMP signal is transmitted to myosin II, resulting in myosin II localization and chemotaxis, remains unclear. In an attempt to throw some light on this issue, we studied the role of MHC-PKC in vivo by eliminating and by overexpressing the MHC-PKC protein in Dictyostelium cells. Analysis of these cell lines allowed us to address directly the role of MHC-PKC in vivo and consequently the role of MHC phosphorylation in the regulation of myosin II. The results presented here indicate that MHC-PKC is a key modulator in the regulation of myosin II localization in response to the chemoattractant, cAMP.

Regulation of Dictyostelium Protein-tyrosine Phosphatase-3 (PTP3) through Osmotic Shock and Stress Stimulation and Identification of pp130 as a PTP3 Substrate

“…This rise in cGMP is thought to activate a cGMP-dependent protein kinase, which in turn activates a myosin II heavy chain-specific protein kinase C (15,16). Similarly, extracellular cAMP induces guanylyl cyclase activity and myosin II phosphorylation during Dictyostelium aggregation, which mediates chemotaxis (15,17). However, the kinetics of intracellular cGMP accumulation and the signal transduction pathway leading to guanylyl cyclase stimulation are different than after osmotic shock stimulation (2,18).…”

“…Exposure of the cells to 0.3 M glucose leads to an intracellular rise in cGMP (2,14) which is required for the phosphorylation of myosin II (2). This rise in cGMP is thought to activate a cGMP-dependent protein kinase, which in turn activates a myosin II heavy chain-specific protein kinase C (15,16). Similarly, extracellular cAMP induces guanylyl cyclase activity and myosin II phosphorylation during Dictyostelium aggregation, which mediates chemotaxis (15,17).…”

14–3-3 Inhibits theDictyosteliumMyosin II Heavy-Chain-specific Protein Kinase C Activity by a Direct Interaction: Identification of the 14–3-3 Binding Domain

“…MHC-PKC, which is expressed during Dictyostelium development, regulates the myosin II reorganization in response to cAMP stimulation, by phosphorylating its MHC (Ravid and Spudich, 1992;Abu-Elneel et al, 1996). MHC-PKC null cells exhibit a substantial myosin II overassembly in vivo and aberrant cell polarization, chemotaxis, and morphological differentiation.…”

Identification of a Protein Kinase from Dictyosteliumwith Homology to the Novel Catalytic Domain of Myosin Heavy Chain Kinase A

“…This enzyme has a catalytic domain similar to members of the protein kinase C family and also contains a distinct domain with significant similarity to the catalytic domain of diacylglycerol kinases (4). This enzyme is expressed in a developmental-specific manner and seems to be involved in regulating myosin assembly/disassembly during chemotactic cell migration (5,6). The enzyme MHCK A was first purified from growth-phase cells (7) and is expressed during both growth and development.…”