Hypergravity signal transduction in HeLa cells with concomitant phosphorylation of proteins immunoprecipitated with anti-microtubule-associated protein antibodies

We have shown that hypergravity (35 g) stimulates production of inositol 1,4,5-trisphosphate (IP 3) and decreases adenosine 3′,5′-cyclic monophosphate (cAMP) levels in HeLa cells. IP 3 production rapidly increased 1.5- and 2.1-fold greater ( P < 0.05) than the control after 2- and 5-min exposures to 35 g, respectively. The intracellular cAMP levels, determined in the presence of isobutylmethylxanthine, were decreased by 11% ( P < 0.05) and 16% ( P < 0.01) relative to the control after 10- and 20-min exposures to 35 g, respectively. The phosphorylation of proteins which were immunoprecipitated by antibodies recognizing microtubule-associated proteins (ipMAPs) was also apparent after exposure of these cells to hypergravity. In the detergent-insoluble fraction, phosphorylation of a 115-kDa protein was significantly enhanced compared to the control after a 5-min exposure to 35 g. In the detergent-soluble fraction, phosphorylation of a 200-kDa protein was observed after a 20-min exposure to 35 g. Our study suggests that IP 3 and cAMP may act as second messengers in hypergravity signal transduction. Phosphorylation of ipMAPs in both the detergent-soluble and -insoluble fractions suggests that cytoskeletal structures may be influenced by gravity.