Cytoskeletal Protein Phosphorylation in the Murine Macrophage and Its Relationship to Macrophage Activation

83 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1985.To elucidate the molecular processes underlying macrophage activation to cytotoxicity, I embarked upon a search for substrates for protein kinases in the murine macrophage. Based on evidence that the microfilament system participate in macrophage cytotoxicity, I investigated the possibility that substrates for protein kinases (phosphoproteins) may be associated with the microfilament system, and that alteration in these substrates occur with macrophage activation.In C3H/HeN mice, two phosphoproteins, pp66 and pp64, were extractable by a method which also enriched for cellular actin but not other proteins, suggesting that pp66 and pp64 were actin-associated proteins. I detected these two phosphoproteins in macrophages from several other mouse strains, but only pp66 in macrophage from BALB/c mice. In all cells screened that were not of macrophage linage, neither pp66 nor pp64 were detected, suggesting that pp66 and pp64 may be restricted to macrophages, and may perform functions unique to the macrophage.The level at which macrophages from C3H/HeN mice incorporate labeled phosphate into pp66 and pp64 is correlated to the macrophage population's activation state. Within a one hour labeling period, resident macrophages incorporated little label into pp66 and pp64, vaccinia virus-activated macrophages incorporated much label, and proteose peptone-elicited macrophages incorporated label to a level intermediated between resident and activated macrophages. Of all the abilities that the macrophage acquire through activation, I found pp66 and pp64 phosphorylation to be best correlated to the macrophage's ability to spread-out on substratum in vitro. At present, insufficient data exist to determine whether the spreading ability is a prerequisite for macrophage cytotoxicity.Pp66 and pp64 phosphorylation was suppressed by the calmodulin inhibitor trifluoperazine, suggesting that the two proteins were substrates for a calcium/calmodulin-dependent protein kinase. In conjunction with the observation that macrophage cytotoxicity is inhibited by calmodulin inhibitors, this datum is compatible with the notion that pp66 and pp64 may play the role of modulator of macrophage cytotoxicity.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD