The kinase activity of the giant protein projectin of the flight muscle of Locusta migratoria.
Projectin is a member of the functionally and structurally heterogeneous family of myosin light chain kinases associated to myosin of synchronous as well as asynchronous insect muscles. We examined the phosphotransferase activity of projectin from flight muscle of Locusta migratoria. Isolated projectin exhibits an unstimulated autophosphorylation activity in vitro. We observed differences in the formation of synthetic filaments with myosin, and paramyosin depending on if projectin was autophosphorylated in vitro or not. Aggregates of native projectin with myosin and paramyosin (molar ratio 0.08:1:0.5) showed diameters 20-50 nm similar to those of myosin filaments. When in vitro autophosphorylated projectin was used we predominantly obtained, however, subfilament-like structures of only 7-10 nm in diameter. The in vitro autophosphorylation of projectin was suppressed in the presence of either acto-myosin, actin-filaments or myosin, but still seems to exhibit a phosphorylation activity: Projectin added to actomyosin resulted in the phosphorylation of three polypeptides of apparent molecular masses of 200, 165 and 100 kDa, respectively. These data suggest that the autophosphorylation activity of projectin is regulated by its environment. We conclude, therefore, a dual function of its kinase domain: at first, a role of its autophosphorylation in the formation of myosin filaments (association of subfilaments to filaments); secondly, the transphosphorylation activity of projectin modulates the contractile response of the actomyosin system by phosphorylating some of its components. Moreover, we could stimulate in vitro the projectin autophosphorylation 3.4-fold by calmodulin (EC50 = 17.8 nM). However, the transphosphorylations described above were not stimulated by calmodulin.
Fährmann
Michael
M
Institut für Zoophysiologie, Westfälische Wilhelms-Universität Münster, Hindenburgplatz 55, D-48143 Münster, Germany. fahrman@uni-muenster.de
Fonk
Iris
I
Beinbrech
Gernot
G
D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation; D005426:Flight, Animal / Q000502:physiology*; D006110:Grasshoppers / Q000502:physiology*; D007700:Kinetics; D009124:Muscle Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits
D002135:Calcium-Binding Proteins; D009124:Muscle Proteins; C078033:projectin; D009219:Myosin-Light-Chain Kinase