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.
Transmission of potyviruses by aphids depends on the presence of a virus encoded helper-component protein (HC) that also exhibits protease activity. HC was expressed in E. coli from two types of clones: a full-length cDNA clone of PVY and two 5' end clones containing the first three cistrons (3.6-3.7 kbp). The clones derived from the 5' end of PVY expressed HC of the size of the mature component. Other proteins reacting with antibodies to HC were also observed, and their sizes corresponded with those of expected intermediates resulting from partial protease cleavage of the three-cistron polyprotein. On the other hand, the only detectable HC-related product of the full-length clone was a mature-size HC. The presence of a third PVY protease among the first three cistrons is therefore suggested.
Full-length cDNA of genomic RNA of potato virus Y (PVY) was cloned in one piece into a lambda vector. The order of the EcoRI and SalI fragments of the inserted cDNA was determined. This is the first report of the cloning of a long, expressible, potyvirus genome. The availability of such a clone is a prerequisite for any further study of the molecular biology of this group of viruses, as they are expressed into a self-processed primary polyprotein.
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