The calcium(2+)/CaM-dependent phosphoprotein phosphatase calcineurin: Its role in chemotaxis of axenic cells and analysis of possible substrates in Dictyostelium discoideum.

摘要

Characteristics affecting the chemotactic response were examined in two strains of axenic amoebae derived from wild-type NC-4, the classic axenic strain AX-3 and JH10, a thymidine auxotroph derived from AX-3. Parameters leading to a maximal response to the two major chemoattractants, cAMP and folic acid (FA) were determined. Using the radial bioassay, both AX-3 and JH10 amoebae exhibited maximal chemotaxis to cAMP when harvested in mid-log growth {dollar}(5{lcub}-{rcub}9times10sp6{dollar} cell/mL for AX-3, {dollar}7{lcub}-{rcub}12times10sp6{dollar} cells/mL for JH10), starved for 2 hours and exposed to 1 {dollar}mu{dollar}M extracellular cAMP. Chemotaxis to FA was optimal in unstarved amoebae and declined sharply as starvation time increased. Migration rate was found to peak at 40 {dollar}mu{dollar}M FA. The role of the Ca{dollar}sp{lcub}2+{rcub}{dollar}/CaM dependent phosphatase calcineurin (CN) during chemotaxis to both cAMP and FA was investigated pharmacologically by use of the specific CN inhibitor deltamethrin. Permethrin, similar in structure to deltamethrin but non-inhibitory to CN was employed as a negative control. Chemotactic mobility to cAMP was not significantly affected by deltamethrin or permethrin. Conversely, chemotaxis to FA was increased by 82% in the presence of deltamethrin while permethrin showed no significant effects. Cell extracts of amoebae chemotactically competent to cAMP were immunoblotted with antibodies against phosphoserine in an attempt to identify possible substrates of CN. Sixteen proteins ranging in apparent molecular weight between 42.6-185.6 kD were identified based on increased levels of phosphoserine incorporation in the presence of deltamethrin. These data are discussed in relation to current knowledge and ongoing investigations.